GRASP: Graphical Representation and Analysis of Surface Properties

Introduction: Why Grasp became what it is (pgs 1-4)

Features: What Grasp can do and what it cannot do (pgs 5-17)

Color Coding

Surfaces

Electrostatics

Distance Calculations

Maps

Atoms

Bonds

Contours

Colors

Subsetting

Simple Property Mathematics

Objects

Matrix Representations

Stereo / Split Screen Operations

Basic operations: Getting Started (pgs 18-21)

Environment Variables

Paths

Data Files

The Control Keys: Selective Short Cuts (pgs 22-27)

The Grasp A-Z.

The Command Line: Inquiring by property (pgs 28-38)

Coloring Objects

Mapping Atoms Colors to the Surface

Undo and Restore Coloring

Precise Translations and Rotations

Listing Atom Properties

Changing Atom and Surface Information Fields for Mouse Picking

Altering Radii and Charges

Specific Grasp Syntax for Negation, Concatenation and Projection

General Grasp Subsetting Syntax:

Atoms

Surfaces

Bonds

Backbone Boxes

Matrix Strands

The Menus: Doing everything else (pgs 39-67)

Display (or not) Structures in a Particular Form.

Build and/or Calculate Structures and Structural Properties.

Mouse Functions, (and how to alter them).

Read and Write Grasp data files.

Formal Subset Operations.

Other Programs, i.e. Superimpose.

Setting Internal Parameters.

Miscellaneous, Help and Quit.

Worked Examples (pgs 68-73)

Getting a molecular surface color coded by electrostatic potential.

Displaying surface potential and surface curvature side by side.

Surfacing two interacting parts of a molecule and selecting an interface.

Calculate the occluded accessible surface area between these parts.

Reading in atomic B-values from a PDB file, displaying them on the surface.

Calculating and displaying the effective dielectric from a single charge site.

Calculating average surface areas per hydrophobic and hydrophilic residue.

Finding and displaying all residues within 3 Angstroms of an active site.

Finding the common volume between two superimposed molecules.

Forming a six helix bundle from a single helix and surfacing the result.

Planned Developments (pgs 74-82)

General Improvements

Specific Projects:

Intelligent DelPhi

Secondary Structure Display

Docking with Realistic Energies

Appendices A, B and C (pgs 83-89)

Appendix A: File Formats

Appendix B: .init_grasp commands

Appendix C: Grasp data files

Addenda (pgs 90-92)

References (pg 93)

Index

Introduction:

Grasp came about because I wanted to visualize electrostatic potentials at surfaces, in particular the surface of biological molecules. Barry Honig's lab is well known for the program DelPhi which calculates electrostatic potentials from the Poisson-Boltzmann equation, and has the led the way in applying this equation in structural biology. The program can be used to get productive quantitative numbers for a variety of biochemical phenomena but qualitative visualization had been limited to isopotential contouring, typically with a program such as Insight from Biosym Technologies. The limitation of such an approach is that such contours do not capture local topology or shape. They often extend significant distances away from the molecule while one would expect most of the 'action' to be close to the molecule, in fact at the surface of molecules. So I decided it was time to attempt a graphics program which emphasis surfaces and

electrostatics.

I would not have been so confident in starting this project had it not been for considerable groundwork laid by others, in particular Kim Sharp. He and Mike Gilson had devised a novel algorithm some years ago for calculating the molecular volume (i.e. water inaccessible volume) using a cubic lattice. This method, though not efficient when the lattice spacing is small relative to atomic radii, can be made rapid at lower resolution. Given the molecular volume, Kim had also reinvented a technique commonly known as the "Marching Cubes" algorithm to produce a surface tessellation. Putting these together in an optimized form produced a rough and ready surface description which could be easily visualized on a Silicon Graphics Iris (SGI) computer. The potentials at surface points could then be interpolated from the 3-D map produced by Delphi and color coded to indicate the result.

The initial results were surprisingly good, both in terms of aesthetics and usefulness. The large difference in dielectric between water and the interior of proteins modelled by DelPhi means that local electrostatic effects can dominate global ones. So, for instance, an active site can be negative even when the protein total net charge is very positive. This is seldom seen if Coulomb's Law is used to calculate potentials because of the long range nature of (1/r). Grasp was immediately able to display this consequence of electrostatic screening, for instance showing the deeply negative binding site of the catalytic magnesium of RNAse H ,crystallized without that ion by Yang and Hendrickson.

So it was clear that this approach held some merit. The combination of surface shape and electrostatic potential was synergistic. Moreover I began to see that just having a rapid surfacing and visualization algorithm was useful. For instance it was simple using surface connectivity to display only the internal cavities of proteins. Here the initial use was the bacteriorhodopsin structure of Henderson which has numerous "holes" surrounding the retinal moiety.

It was also instructive to "project" properties of the underlying atoms onto the surface and color code them. An example being the B values normally accompanying crystallographic structures. So I began to see the surface itself as useful construct, regardless of electrostatics. This became a central tenet of Grasp, that surfaces and atoms should be treated with equal importance.

There was still a need for other electrostatic representations, such as isopotential contours, projection planes, field lines etc. Also, since typical use of the program was visualization of large molecules with thousands of atoms I devised a simple representation for those atoms that was fast to display and could be colored by property. This led to other representations of atoms and groups of atoms.

The program grew beyond my initial plans. Hopefully, however, the program maintains a coherent philosophy. For instance the use of surfaces both for displaying properties and as objects in their own right, the visualization of electrostatic properties, and more lately the generalization of the idea of an object representing both a set of atoms (as the surface does) and a property (such as electrostatic potential). An example of the latter is the DNA representational project of Rex Bharadwaj. Here DNA bases can be represented as elongated boxes whose width can represent a property associated with that base, such as base twisting, sliding or rolling.

The program has achieved most of the goals I had concerning the development of these ideas. Of course in their actualization they have spawned many more. But hopefully the present version is at least complete enough to be useful.

Comments as always are much appreciated.

Anthony Nicholls

October 1992

Features

What follows is a fairly complete listing of Grasp's capabilities, in some sort of logical ordering.

General

Grasp uses a perspective based view, i.e. things farther away are smaller. To enlarge a view one simply moves it closer to the eye. Manipulations are by mouse or by dials. Molecules are mapped to a "unit box" which can be displayed with the front side removed. There are also embedded cross-hairs to remind the user of any rotations and translations they have applied. The default clipping planes are very close to the "eye" position and very far away. These can be temporarily altered via a slice control tool. The background is either black or that produced by the unit box. The default window size may be changed in the usual window resizing manner. There is also a full screen option where the entire screen is given over to the display. All functions are accessed by hierarchical menus via the right-most mouse button, or via the command line. This has the advantage of leaving the Grasp field of view completely uncluttered. Commands may also be read in from an external "script" file. All display objects (surfaces, atoms etc.) are independent, i.e. they can all appear in view at the same time or can be individually hidden from view.

Color Coding

Grasp supports two different modes of color coding. The first mode, 3 color continuous, requires three numerical values, called "control" values, along with three colors, one color per value (colors are defined by RGB triplets, or by an index into a list of colors, see color). Color coding is then implemented as follows:

If a number is less than the minimum of the three control values it is assigned the color associated with that minimum value. If it is greater than the maximum value it is assigned that value's color. If it is between the minimum and middle values the assigned color is found by linearly interpolating between the minimum and middle colors, and if it is between the middle and maximum values by linearly interpolating between the middle and maximum colors. By linearly interpolating is meant the following: Colors are made up of red, green and blue components, each component having a strength of 0.0 to 1.0. If a number is, for example, halfway between one value and another, then its interpolated color is similarly halfway between the two colors assigned to those values, i.e. its red, blue and green components are half those of one color and half of the other.. Grasp also supports 2 color continuous which is equivalent to 3 color continuous but with the middle value and color set to be the same as that of the minimum value and color.

The second method is zonal coloring, wherein a certain range of values is assigned a certain color. The color boundaries between different zones are sharp. This is also referred to as discrete coloring.

Default colors are provided for all properties, e.g. for electrostatic potential they are red, white and blue, with red for the minimum value (typically negative), white at zero, and blue for the maximum value (typically positive). However these colors may be also set by the user.

Default maximum and minimum control values are taken as the maximum and minimum values of the property being represented (surface potential, atom distances etc), the middle value is set to zero unless the maximum and minimum are both greater than or less than zero, in which case it is set to the average of those two values. These control values can also be explicitly altered.

Continuous color maximum, minimum and middle values may also be adjusted via a mouse activated widget. This is useful in rescaling the color code to bring out particular features on the fly. Independent widgets appear for each continuous property displayed. These also offer access to other features via drop-down menus.

Surfaces

Grasp supports two types of surfaces, molecular and accessible. The molecular surface is defined as the boundary of that volume within any probe sphere (meant to represent a water molecule) of given radius sharing no volume with the hard sphere atoms which make up the molecule). The accessible surface can be defined as the locus of the centers of all possible such probes described above in contact with the hard sphere atoms. Alternatively it can be defined as the hard sphere surface if each atomic radius is increased by the probe radius. The default probe radius for each type of surface is 1.4 †ngstroms, but can be set by the user.

Everything which can be done with molecular surfaces can be done with accessible surfaces. For brevity therefore, except where differentiated, they will both be referred to as molecular surfaces, since both are derived from molecules, to distinguish them from other surfaces, such as isopotential surfaces.

The surfacing resolution, i.e the lattice spacing used to generate the surface, is determined automatically. The lattice spacing used in the process is scaled relative to the largest dimension of the molecule. Hence coarser lattices are used for larger molecules. This scaling has the advantage that the surface of very large molecules can be as easy to manipulate as that of small molecules. Though the surface of larger molecules will then be less accurate one is often interested in courser features of such molecules. Grasp does not currently support surface refinement.

If the molecule has only a few atoms this method can lead to a lattice spacing at which the method Grasp uses is inefficient, hence there is a minimum allowed lattice spacing. This parameter can be set larger by the user to force use of a coarser grid (as might be preferred to improve draw speed, since fewer triangles will comprise the final surface, or to overcome memory limitations).

Surfaces can be constructed for all atoms or for subsets of atoms (see subsetting). The process takes a few seconds at most and results in a smooth tessellation of the surface which is colored white but shaded by the SGI lighting routines. This can be quite slow on some machines as there may typically be 20,000 triangles. To enhance drawing speed there are three other draw modes. The first is a rendered surface (i.e. the triangles are filled in) but the lighting calculations are simplified (pseudo-lit) and done in software rather than by using the SGI hardware calls. The second is a mesh representation, i.e. the triangles are not filled in. The third is a points only representation. The default surface produced by the program upon construction can be preset in an external file (see grasp settings).

All displays of surfaces (and also contours, atoms and bonds) can be depth shaded. This means that the farther away a part of the surface the darker the color. More exactly, its color is interpolated to black based on the distance from the viewer. Grasp uses a dynamic depth range, where the front edge, i.e. where interpolation begins, is determined by the nearest point on the structure to the viewer, and the back edge, i.e. the depth at which the color is set to black, is a fixed distance behind this point. Depth shading makes a dramatic difference to the mesh representation, and to a lesser extent with other representations. A default depth is set for all relevant structures. However, since the optimal values tends to depend upon the size of the structure or feature under consideration, the depth can be altered by the user.

For the rendered and lit, and rendered and pseudo-lit surfaces the direction of the incident light may be varied. At present other material properties of the surface (reflectivity, transparency, etc.) are not user accessible.

Surfaces are colored by assigning colors to each vertex. This can be done according to any value associated with that vertex as described in color coding. Alternatively surfaces can be colored by selecting a subset and assigning a discrete color. As described in subsetting there are many ways of selecting a surface subset based on vertex properties, associated atom properties, or by hand with the surface scribing mode. Any surface or subset can be 'uncolored' i.e. undisplayed. Hence one can remove portions of surfaces to create "windows" into the underlying molecule. Any surface property can be interrogated using the mouse buttons, i.e. placing the mouse at any given point and clicking returns a value or values associated with the nearest surface vertex. Which property values are returned, and by which mouse button, can be set by the user.

Surface properties can be classified as those which can be used in subsetting and displayed, and those which can be used in subsetting but can not be displayed. The latter tend to be intrinsic or assigned properties and include absolute coordinates, relative position, current discrete color, surface construction number, formal subset name (see below), and vertex number. Surface properties which can be displayed are generally calculated within the program and include electrostatic potential, curvature, distance (to another surface or set of atoms), and two general property fields. The latter two can be used in a variety of ways. For instance any atom property can be mapped to the surface and stored (and hence displayed) as general property one or two. Or the user can produce a new quantity out of others by the simple math facility. Or properties can be imported (see below).

Surface potentials are interpolated from potential maps as described in Electrostatics. Distances as a surface property are calculated either from another surface or surface subset, or from a set of atoms, and in each case are the minimum such distances. Surface curvature is as defined in Nicholls et al and is derived from a concept of local hydrophobicity. Briefly, each possible placement of a water "sphere" against the surface of the molecule reduces the accessibility of that water to other waters. Against a concave surface this accessibility is less than against a convex surface, and a formal correspondence can be made between contact to an arbitrarily complicated surface to contact with a sphere of a certain curvature. Curvature thus defined is a property of the accessible surface, but can be uniquely mapped to the molecular surface. When color coded it reinforces the effect of SGI lighting in that surface hollows are made distinct from surface projections, and so is useful in visualizing patterns of surface shape.

Surface data (which means vertex coordinates, connections, and normals), and any properties, calculated or assigned, of any surface or subset of surfaces can be written to a data file. These data files can be read in during program execution or at start up. Surface data can be appended to other files to make surface 'libraries'. Since subsets of surfaces can be saved one could, for instance, make a library of the surfaces of the active sites of different enzymes.

The property data of a surface or subset can also be saved as an ascii file for analysis, or for temporary storage. This file can also be read back in and hence a user generated function mapped to a particular surface.

Surfaces or subsets of surfaces can be inverted, i.e. the surface normals reversed. In this way one can look at molecules from the inside. One can also do like-with-like comparisons of two surfaces which might form complexes by inverting one surface of the pair. Surface properties can also be "inverted", for instance positive turned to negative and vice versa through the simple math utility.

Along with cavity surfaces (which can be thought of as a surface subset) and contour surfaces, one can calculate the area of any molecular surface or subset of a molecular surface, and similarly the volume enclosed (noting that the volume is not meaningful if the surface is not closed). The surface area for an accessible surface gives a measure which has often been associated with hydrophobicity, since it is related to the number of water molecules in contact with the molecule. Grasp also has a more accurate surface area subroutine for atom by atom accessible area.

Any surface or subset can also be attached to the rotation and translation dials alone. This creation of a formal subset, for which a unique name is assigned, can then be manipulated independently (see formal subsets). This allows for parts of surfaces to be removed, compared, docked etc.

Electrostatics

Grasp includes a Poisson-Boltzmann (PB) solver which is a similar but simpler version of that used by DelPhi. The fields calculated by it are for qualitative use only. For quantitative use there is full support for the output from DelPhi, in terms of the potential maps, the dielectric maps, the modified pdb files, charge files, size files, etc. Grasp does not (yet) contain an interface to DelPhi, hence that program has to be run separately.

The Grasp PB solver uses two 33 cubed grids, one nested within the other. The inner grid dimension is set to be larger, by the diameter of one water molecule, than the maximum x, y or z dimension of the collection of atoms used in the calculation. The second grid is twice as big as the first, with the same center. The potentials on the outer grid are solved for first, then interpolated and refined further on the inner grid. Potentials are then interpolated to a 65 cubed grid the same size as the outer grid. This final grid, or 'map' as it is referred to, is then used in all subsequent calculations. It may also be written out in DelPhi form. Typical calculation times are around five to six seconds.

Although there is no choice in the sizing of these grids, the user has control of the inner and outer dielectric constants, the probe radius used to determine water inaccessibility, the salt concentration and ion exclusion radius. There is no support for the nonlinear equation, for periodic boundary conditions, membrane slabs and holes, or any other DelPhi features.

Once a map is calculated it can be evaluated in several ways. Isopotential (also referred to as 'through space') contours may be calculated at any value, given any color, and displayed as solid surfaces, meshes or points. Potentials may be interpolated at any molecular surface, and at any set of atoms. (Trilinear interpolation is used throughout). The electric fields may be calculated at a set of points and represented in magnitude and direction as a three dimensional arrows. Molecular dipoles may be calculated and similarly displayed. Field lines can be calculated from a set of points, colored and displayed in 1-D or 3-D (i.e. lines or tubes). The potential may also be interpolated at a slice plane perpendicular to the Z direction (i.e. parallel with the screen). This latter display is updated as the map/molecule is moved, or alternatively as the position of the slice plane is altered.

Values at surfaces and atoms may be colored by 2 or 3 color continuous or by discrete colors, where as the Z plane may only show the former. Field vectors may have their magnitude encoded in their length. Field lines can be assigned directionality and color when calculated.

Distance Calculations

Grasp will calculate minimal distances from surfaces to surfaces, from surfaces to atoms, from atoms to surfaces and from atoms to atoms. In each of the preceding the ability to define a subset is assumed understood. In the case of atoms there is also the option to subtract the assigned van der Waals radii from the distance.

An example of a novel use of distances in Grasp is to calculate a 'depth' map, i.e. the depth of atoms from the surface to every atom. Distance maps are also useful in defining interfaces between domains, either surface-wise or atom-wise.

Maps

Grasp contains room for two internal 'maps', i.e. 65-cubed, cubic lattices. Internally generated maps are stored in the first of these arrays. Maps read in are put in the second array. Simple operations are allowed on and between maps, such as differences, sums etc. Maps can also be swapped so that both could be internally generated or both externally generated by DelPhi. Difference maps are particularly useful to highlight the effect of changing parameters such as charge, radii, salt concentration, dielectrics, etc.

As one of the three primary external data files supported by Grasp (the other two being protein data bank (pdb) files for atoms and surface representation files (srf) for surfaces) maps may be read at startup, i.e. one can analyse maps without any atom data or surface data. Although maps are usually associated with electric potential they can be used quite generally for any 3-D data, though at present Grasp requires the grid to be 65 cubed. For example, the consensus volume option in Grasp, i.e. finding the common volume between a set of molecules, results in values assigned to a 65 cubed grid which can then be manipulated and displayed as a potential map (e.g. Z-plane projection, isopotential contours). The dielectric boundary map, or salt exclusion map from DelPhi can also be read in as this form.

Atoms

Atomic coordinates are the fundamental data structure from which everything else is derived. Grasp does not contain any 'build' utility and hence is dependent on external files for this data. Primary support is for PDB files, the standard crystallographic format, along with certain variants.

Atoms can be displayed in several ways. The traditional method, which is included in Grasp, is as spheres of given radius. This is often referred to as CPK modelling. In Grasp the surfaces of the spheres are lit. CPK can be demanding on the graphics resources for large molecules. An efficient alternative is to represent the atom as a circle of the correct radius always oriented flat with respect to the viewer. With a little differential coloring these flat circles can be given an apparent three-dimensionality. There is also an option to instead color these circles with patterns. Also, there is a representation which gives small, uniformly sized circles for use with bond representations (see bonds). Finally, spheres may be drawn with lines or dots, i.e. unrendered.

Atoms can be colored discretely, i.e. any subset of atoms can be assigned any color, or continuous color colding may be used, as described in color coding. The properties supported for this are potential, distance, charge and two general property fields. Atoms can be uncolored to remove them from view. Atom colors are depth shaded.

Upon reading a PDB file radii are set to default values from an external file (which the user may edit). This file is in the same format as the control file for atom size used by DelPhi. Similar files may be read (i.e. radii assigned) during program execution. Charges are zero by default when a structure is read, but Grasp can read DelPhi charge files and assign charges based upon the descriptions therein. Some sample files are provided, such as those to assign charges to each ionizable residue of a protein. Radii and charges may also be assigned via the command line by specifying a radius/charge and the subset of atoms to have this radius/charge. The command line also supports intrinsic operations such as multiplication of radii/charges by constants, or addition of constants.

Since charges are only of importance for electrostatic purposes and since charge is also a display property, it may be utilized as a 'dummy' variable, i.e. actually represent another physical property. This becomes particularly useful when combined with the DelPhi control file format. For instance if one is interested in a per residue property, say helix-forming propensity, a control file can be constructed with one line for each residue and its property value. This can then be read into Grasp, the atoms of each residue will be assigned a 'charge' equal to that residues helix-forming propensity, and can then be color coded and displayed.

Grasp also supports three variants on the standard PDB file which involve the fields to the right of atom coordinates. These typically contain occupancy and B-values. One option is to read this information in as general atom properties one and two, which are generic data fields for discretionary use by the user. There is also an option to read these fields in as the radius and charge of each atom because this is what they are used for in DelPhi modified PDB files. Finally, for higher precision, the entire field to the right of the coordinates can be read, in free format, as general properties one and two. Files in the above formats can all be written from within Grasp.

Separate molecules will be recognized from within a single PDB file if separated by "TER" statements. Each will be assigned an index, i.e. a molecule number, which as a property is analogous to "constructed surface number" in Grasp. Molecules can be superimposed using the Kabsch algorithm, which gives the best rotation and translation (RMS-wise) between molecules or parts of molecules. The only restriction is that the same number of atoms from each molecule must be used to determine the minimum RMS difference. (Note Grasp will not yet superimpose surfaces).

Grasp contains algorithms for calculating both the volume and surface area of molecules or subsets of atoms. Surface area can be either accessible area or that of the van der Waals surface. Control is given to the user over the precision of these calculations.

Grasp does not contain methods to alter structures such as torsional rotations, minimization etc. with one key exception. Grasp allows independent rotations and translations of defined subsets, which may then be "fixed" relative to each other. For instance, a substrate may be selected and moved relative to an active site. Upon making the transformations new surfaces, distances, electrostatic fields etc can be calculated based on the new coordinates. There is support for undoing transformations which have not yet been "fixed".

Atom properties can be queried in the same way as surface properties, i.e. point and click. Atom properties can even be queried when covered by surfaces. The "atom picking" function can also be set to report geometric parameters such as distance, angle and torsion angle between picked pairs, triplets and quadruplets of atoms.



Bonds

Bonding patterns are calculated upon the reading in of a PDB file. Bonds may be represented in three ways, as lines, as sticks and as cylinders, in order of increasing graphical complexity. The former are the traditional line drawings used by most programs. Cylinders are just a three dimensional variant of this, the diameter of which can be set by the user. Sticks, however, follow the method of Kuznetsov and Lim in which bonds are represented by quadrilateral tubes. The advantages of this approach are that the bonds are made significantly more three dimensional, inter bond angles are well brought out, and the display is relatively quick to draw.

Bonds can be colored based upon a preset pattern, upon transferring the discrete colors of the underlying atoms, or by subsetting based on the properties of those atoms. They can also be selectively undisplayed by being "uncolored" (see color below). Bond colors are depth shaded. Properties of atoms can be queried by picking at bond ends.

Contours

Isopotential contour surfaces can be constructed at any potential value for either internal map. Contours can be displayed in all the surface modes available to molecular surfaces, i.e. lit, pseudo-lit, mesh and points. They are also independently depth shaded. Contours are not automatically recalculated if the parent map changes, though contours can be deleted and recalculated. Volume and surface areas may be calculated for any contour.

Colors

Grasp supports ninety-nine independent, indexed colors. These can be set during program execution, using a color 'palette', or in an external file as RGB triplets. All changes to colors are automatically saved, thus the user can design their own set of colors, or use those provided. These colors are numerically indexed, i.e. assigned numbers from one to ninety-nine. There is also color zero, which is always equal to black but which is also used as a flag to prevent display of that object, i.e. an atom colored zero is hidden. This 'uncoloring' applies to surfaces, bonds, backbone boxes and matrix strands. Grasp also has undo and restore commands such that undo removes the previous coloring, while restore acts on objects colored zero by giving them the color previously assigned. Once assigned to atoms or surfaces these discrete colors become a property which can be used in subsequent subsetting selections.

Subsetting

A central feature of Grasp is the ability to specify a subset of atoms, surface vertices, bonds, objects, etc., based upon a very wide range of properties. The method of doing this is via a command line which accepts a series of specifications and forms the intersection of each. There is one exception to subsetting being entirely by command line, this being 'surface scribing' as described below.

The properties which can be used for surfaces are coordinates, screen position, potential, distance, curvature, general surface properties one and two, constructed surface index, formal subset name, assigned discrete color and vertex number. For atoms the list is a little longer, namely coordinates, screen position, potential, distance, charge, radius, general atom properties one and two, molecule number, formal subset name, assigned discrete color, atom number as well as atom name, residue number, residue name, chain name, and accessible area. There is a deliberate similarity between these two lists based on the program philosophy of equality between surfaces and atoms.

Variables are either characters or numbers. The latter may be specified as ranges, for instance one can select all atoms which have a residue number between 5 and 10, or a charge greater than zero. Character variables (except formal subset names) may include wild cards, so one can select, for instance, all carbon atoms or all carbon atoms which have the character "1" in the third position of the atom name etc. There are also 'short cut' names for atoms in a protein backbone or in side chains, and for residues which are ionizable, hydrophobic or hydrophilic. All individual specifications can also be negated, i.e. one can subset by NOT a certain quantity.

The purpose of subsetting depends upon the context. One of the simplest uses is to alter the display color of this subset. For instance, one might want to employ a color scheme which displays all positively charged atoms one color and all negatively charged atoms another, or all hydrophobic residues yellow and all hydrophilic purple, etc. Colors are specified by the integer index (1-99) assigned to each within Grasp.

The use of assigned color as a property allows for some quite flexible subsetting. For example it can act as a bridge between atomic and surface properties. Suppose we want to find all vertices of a surface which are concave, i.e. have a calculated surface curvature less than zero, and which are formed by hydrophobic residues. One way to do this is to color all hydrophobic residues one color, then transfer that color to the surface, then select all vertices which have that color AND which have curvature less than zero.

Color can be used to build up subsets based upon disparate properties which could not be specified in a single command line. For instance if we want to select all atoms which are in a region of positive potential and belong to negatively charged residues plus all those atoms which are negative but belong to positively charged residues. This is a way to include an OR statement into Grasp's subsetting vocabulary.

Another use of subsetting is in creating a formal subset. Formal subsets are assigned names, either by the program, in which case a hierarchical naming convention is enforced, or by the user. Formal subsets may be spatially manipulated independently of the rest of the surfaces/atoms. They can also be referred to by name in all subsequent operations. Formal subsets may be sets of atoms or portions or collections of surfaces, or may have mixed character. By mixed is meant that a surface may be associated with a set of atoms, or a set of atoms associated with a surface. For instance, as well as selecting an active site surface one might want to associate all atoms which are in contact with that surface.

Most other uses depend on the action being undertaken. For instance, when the surfacing subprogram is activated the user has the option to enter a subset of atoms. For example, one could surface a single helix in a protein.

Other than with the above method Grasp syntax is exclusively AND based, i.e. subsetting by progressive refinement based upon properties. The reason for this is that one of the most useful applications of Grasp is to use it to look for correlations. So one might want find all residues which are a certain distance from the molecular surface AND charged. Ideally Grasp would support a query language such as SQL based upon the properties intrinsic, calculated or imported to it, but at present subsetting represents a limited form of the ability to explore for meaningful or interesting connections.

Finally, there is one further method of selecting a surface subset. By activating the surface scribing mode the user can "draw" upon the surface the border of a region of interest with the mouse cursor in much the same way in which one might with a Macintosh-like draw program. The border appears as triangles colored bright blue. When the outline is complete the user double clicks inside the border. This causes the triangle clicked upon to be "selected" and colored bright green. The selection then spreads outward from this triangle until it comes up against the border. If desired the border can then be added to the selection. If the border is not "water-tight" this selection will "spill" out. Such mistakes can be undone. The filling process can also be halted by a further mouse click. This also allows the user to make a borderless selection, i.e. just double click and a patch from this point will gradually grow until the user clicks once more. Once the selection is finalized it can be made into a formal subset or used as a choice when a surface subset is required by the program. The selected region can be 'unselected' within the scribing mode and is automatically unselected when the scribing mode is turned off.

Simple Math

Sometimes the properties calculated or imported into Grasp are not exactly the ones one is interested in. For instance one might be interested in not the surface potential interpolated from map one or that from map two, but some average of them (for instance weighted by the average ionization state of two residues). The same might be true of the maps themselves i.e. one might like a combination of the two maps. Simple math addresses this by providing for arithmetic on one or two fields, putting the result in another (or the same) field.

The operations available for maps include addition or division of two maps, along with multiplication by a constant and swapping the two maps. Those for atom and surface properties are more extensive and include addition, subtraction, division and multiplication of two properties, as well as addition or multiplication, by a constant, of a single property. Also supported are special functions on one field including square root, reciprocal, exponentiation, logarithm, cosine, sine and hyperbolic functions.

More useful to some extent are the contraction operations which take a field and return a single value, i.e. maximum, minimum, average and sum. These can be combined with subsetting so that only portions of the selected fields are acted upon. For instance one can find the accessible area of all lysines, or all charged groups, or all charged groups of a particular helix etc.

Simple math can also be used to shift fields about. For instance, one can multiply a field by 1.0 and place the result in a second field. This can be useful if one field is to be used as a dummy field. For instance, if charges were assigned to represent helix forming potential via a DelPhi charge file format they can then be passed on to one of the general property fields.

Another use is property inversion. As well as inverting the normals of a surface to make it appear similar to its complement one might also want to invert its electrostatic potentials since one might expect a complementary surface to also be complementary in potential. This is simply achieved by selecting the surface of interest and multiplying its surface potential by -1.0.

Finally simple math includes a facility to map an atomic parameter directly to to the surface. One advantage of this is that a surface is to some extent simpler than the collection of underlying atoms and as such is often a better vehicle for displaying properties. One further advantage is that by using the accessible surface one can project these properties into space away for the molecule.

Grasp Settings

Some internal parameters for Grasp can be set in an external 'start-up' file. This feature is not well developed at the moment but does allow the user to specify the initial display of a molecule to be as atoms or as bonds, and also whether surfaces are displayed as lit, pseudo-lit, meshes or points. Both these settings address the concerns that arise if one has a low end Iris but want to look at large molecules. In expensive draw modes these can take several seconds to draw, hence it makes more sense to use a more 'rotatable' representation for examining the molecule, such as a bonding representation.

This facility will be expanded to eventually include most, if not all, of Grasp's internal parameters so that it may be more fully user customizable.

Objects

Objects represent both a new direction for Grasp and an extension of the original concept. The latter because surfaces are in a sense an abstraction of the underlying atoms positions, and as such can be thought of as an 'object', i.e. a form which represents in some way both the shape and a property of a set of atoms. In this sense, back-bone ribbons are objects, even bonds could be so classified (the property being the direction of the bonding force).

In Grasp there are currently two objects for proteins and three for DNA. The first protein object is a backbone trace I call a 'worm'. It consists of a set of cylinders forming a smooth spline though alpha carbon positions. See Future Developments for the ideas to be incorporated herein for secondary structure.

The second is a peptide plane representation. As is well known, the peptide bond has double bond character and so is stiff to torsional rotations. As a consequence the set of backbone atoms CA(n)-C(n)-N(n+1)-CA(n+1) lie in a plane. This can be represented as a quadrilateral with corners at carbon alphas (CA) and at the oxygen and hydrogen of carbon (C) and nitrogen (N) respectively. This is given a little width for display purposes to make a quadrilateral box.

These backbone boxes can then be colored in several ways. The default color scheme is white at the alpha carbons, red at the oxygen, blue at the hydrogen. In this mode it is easy to see, for instance, where backbone loops have all carbonyls or all amide hydrogens aligned in one direction (which can be significant electrostatically). It is also makes secondary structures particularly clear. Boxes can also be colored by subsetting based upon the underlying atoms, or 'uncolored' so as to display only parts of chains.

DNA representations have three components: the phosphate backbone, the pentose sugars and the DNA bases themselves. The backbone can be represented as a thick ribbon smoothly splined through backbone phosphates. The pentose sugars are represented either as rings or line pentagons. The latter are color coded by the endo- or exo- nature of the sugar carbon. Finally the bases are represented as rectangular slabs, colored by base type. This latter representation can also be made to width-encode a DNA base pair parameter. Support is provided for the output from the program CURVES which describe 38 such parameters. These can also be mapped to a helical sheet and color coded. This work is still under development towards a more complete 'module' which will support more typical Grasp-type controls.

Pair-Wise, or Matrix, Representation

One quantity which is difficult to represent by conventional means is pair-wise interactions. This is because the variable has both a value and two positions instead of just one position. For this reason this is like attempting to display a matrix, as opposed to a vector. Such variables occur in electrostatics as the interaction energy between each charge in a set of charged sites. Another example is effective inter-residue forces which some have developed to model protein stability. Since both these uses are essentially residue based, the formulation of the pair-wise interaction representation is residue based in Grasp, i.e. it acts between residues, not atoms.

In Grasp these forces are represented by means of lines running between pairs of sites. These lines have as properties the interaction strength and those properties of both interaction residues themselves. Hence lines may be colored (or 'uncolored') by standard subsetting commands.

Interactions also have the property of 'rank' i.e. since sites may have several interactions each such interaction also has a rank amongst those assigned to that site. Thus one can subset by rank, i.e. only show the strongest interaction for each site. Since interactions can be strong by being very attractive or very repulsive, i.e. the interaction strength very negative or very positive, support is provided for ranking by either criteria. This can be useful in determining 'zones' of interactions i.e. patches of residues which interact mostly amongst themselves.

Grasp goes one step further than merely representing forces with lines by expanding the lines into cylinders. As well as being visually striking this allows the width of the cylinder to act as an indicator of the absolute strength of interaction. By default, Grasp sets the maximum width to represent the maximum absolute interaction, but this can be set to be larger or smaller by the user. All other coloring operations still apply. Grasp also allows for the cylinder representation without width encoding.

The ends of these lines or cylinders are by default set to the average position of all atoms in the residue. This can be altered to any subset within the residue. For instance, in some cases the position of the residue charge might be more appropriate, in others the alpha carbon, or center of the side chain etc.

Finally since interactions will be distance dependent, the user can explore this dependence by multiplying or dividing each value by the distance between its two sites. This scaling can help the user determine which interactions are unusually strong or weak.

Stereo/Split Screen

Stereo viewing has traditionally been achieved by duplicating the view, separating the views by a certain distance so that there is no overlap, and then giving the right-most view a twist about the vertical direction of about 8 degrees. Grasp follows this approach and extends it to a 'split-screen' capability.

Firstly the stereo separation and twist are under user control. (Tests within our labs showed conclusively that everyone has their own preferential stereo twist.) The separation and twist are under mouse and dial control. Twist may also be entered explicitly.

Secondly, the duplicate view can be treated as completely independent, i.e. nearly all display possibilities can be used either on the left or the right. This allows the user to display alternate views side by side. For instance one might want to view the surface color coded by potential and also by curvature at the same time. Views can also be superimposed, i.e. the left-right separation eliminated.

Furthermore one can manipulate (i.e. translate or rotate) either view independently. Thus, for instance, one can display front and back of a molecule simultaneously. Formal subsets in each view are also independent, and so different arrangements of such subsets can be portrayed in right and left views.

The Basics

One can start Grasp simply by typing the program name, e.g.

grasp

There are a few things one should check first however. The first is to ensure that one has write privileges in the directory the command is issued from, which can often be a problem if working from someone else's directory. Grasp needs this permission to enable it to write temporary files, which are removed upon exiting the program, and some permanent data files, such as a color map if the user alters those provided, and also "error" files if it detects odd situations (such as finding too many bonds for an atom when reading a pdb file, or fractionally charged residues upon reading a charge control file).

Secondly Grasp reads in a few small data files upon start up. It needs to know what directory these files are in. To do this it reads the environment variable "GRASP" (note capitals) which should be set to the correct directory. For those not familiar with Unix, the command to do this is

setenv GRASP dirnam

where dirnam is the directory name. One should place this command in the file ".login" in ones root directory so it is read and executed when the user logs in. One can check the 'value' of this variable by entering,

echo $GRASP

Grasp also has a directory of "last resort", i.e. if it can not find the directory set in GRASP. The backup directory it looks for is "./aakdat", i.e. in a directory one lower than the user is in called "aakdat". These data files are listed in the appendix and involve such things as default radii for atoms, default charge sets and information used in surfacing molecules.

Thirdly, Grasp will check for a file called ".init_grasp". This file can contain commands which set variables within Grasp, such as default display modes. The form of the commands are listed in the appropriate appendix. Grasp searches for this file in three places. Firstly it checks the directory stored in the environment variable GRASP, secondly it checks the users root directory, and lastly it checks the local directory from whence the command was issued to start the program. The purpose of having it check all three locations is to allow for hierarchical control of grasp settings. For instance one might want to set some parameters for all users, in which case they are set in the GRASP directory. Individual users might want different parameters for their own work, and so alter the file in their root directory. Finally, the individual user might find that for some projects different parameters are better, e.g. small molecules might want one set of display parameters, large molecules others, in which case control should be via the file in each particular directory. The order the files are read is important because if two commands set the same parameter preference is given to the later command.

Once the grasp command is issued, and the appropriate files searched for and read, the default graphics window opens and shows a set of axes or cross-hairs for the X, Y and Z directions. The Z direction is towards the viewer in Grasp, positive nearer the user, negative farther away, the X direction is left to right, right positive, left negative, the Y direction is up and down, with up positive, down negative. The cross hairs run between plus and minus one in Grasp internal coordinates. To help visualize this domain one can view the Bounding Box. This is done by pressing control O (see 'Control Keys'), or via the menu entry under 'Miscellaneous' (see 'main menu' below).

One moves this view by either using the dials on a dial box or use of the mouse. The dials work accordingly:

left row, top to bottom= x rotation, y rotation, z rotation, stereo twist

right row, top to bottom= x translation, y translation, z trans. , stereo separation

The mouse moves the view by depressing the left or middle buttons or both and moving the mouse. Specifically,

Left-most button: Rotations about the axis perpendicular to the direction of mouse motion. Hence there is a sense of "rolling" the molecule as if the molecule where resting on a solid surface in the XY plane and the cursor was the user finger.

Middle Button: Up and down moves the molecule away and towards the viewer respectively. Note that this is NOT a scaling, but an actual motion in the Z-direction and hence corresponds to pulling the molecule towards or pushing it away from the user. Left or right motion rotates the molecule about the Z axis.

Left and Middle Together: Translates the molecule in the XY plane in the direction of mouse movement.

This implementation of dials via the mouse (mouse-dials) is a little different from some programs, i.e. only two mouse buttons are used. This is partly because only two are actually required to allow the six independent rotations and translations, but more importantly because the third button, the right-most button, is reserved exclusively for the menu interface.

One further convention is adhered to in Grasp which is that, where appropriate the middle button adds and the left button subtracts. For instance when adjusting the indexed colors in Grasp the middle button increases a color component, the left button decreases it.

The rate of rotation or translation, i.e. the sensitivity to mouse or dials, can be set via the menus. It is also possible to assign different functions to the mouse, such as surface scribing, or projection plane position. These are accessed via menus and will be detailed later.

Note that the box does not rotate with the the cross hairs. The significance of this is that box represents a space which is invariant with respect to the user. Rotations and translations do not actually affect the molecules coordinates, only the viewing of them. One way to think of this is that it is not the molecule which is moving but actually the user and the box (since both move the box appears stationary). The language used in Grasp is of "absolute" or "internal" coordinates, which "belong" to the molecule, and "box" coordinates which belong to the user (and hence the box). One can change molecules into the users frame of reference, i.e. make rotations "real", via formal subsetting and some options for file export, and which are detailed later.

One can now read in one of three type of data files, atom files, surface files or maps (3-D grids). Note that instead of reading in a file once the program has executed one can give the name of a data file after one types grasp, e.g.

grasp lys.pdb

which will load in the coordinates of lysozyme from the pdb file lys.pdb. Other than pdb files one can also give the names of maps, with the extension .phi, or surface files .srf. If the name of the file does not have one of these three extension then the program will prompt the user as to which type of file it is.

Reading a data file from within Grasp involves using the menu system. This is discussed later in greater detail. Clicking on "Read" and then on one of the primary data file type will prompt the user as to enter the file name or select a default file or see a list of files. The list will be all files in the initial directory which have the correct extension for that type of input.

When character input is requested in Grasp it is via what is called the textport, which is the character based window from which Grasp is initially launched. To enter information to the textport the cursor has to be positioned over this window. If Grasp is expecting information, e.g. expecting a file name, it tries to make this easy for the user both by automatically positioning the textport over all other windows, and by automatically placing the cursor over this window. And when the information input is complete, i.e. return has been hit, the cursor will automatically jump back to the spot on the graphics window it was before the request for information. Similarly if the user wants to type something to the command line interpreter the cursor will automatically place itself over the textport when the user begins to type, jumping back when return is hit.

Note that in both these examples the cursor starts over the graphics window and end there too. The user should NOT attempt to move the cursor onto the textport themselves except in the following two cases. Sometimes the user may have moved the textport, or resized it, and as a consequence the cursor may miss it when made to "jump" by the program. Also possible is that the cursor will come to rest upon the textport when instructions are not being entered. This causes a "change in input focus" for the program i.e. it expects input from the textport rather than from the graphics window. When this happens, for instance, the molecule will not rotate when the dials are twiddled because the program is not "listening". This question of "focus" can often give beginners the most problems in getting started with Grasp, so when in doubt check the cursor position.

Hitting return when the cursor is over the graphics window causes the textport to alternate between background and foreground, i.e. being behind all other windows or on top of them. If the user has resized the textport, for instance made it bigger to review more information, or repositioned it, hitting return will also resize and reposition the textport. Another use of this is that pushing the textport and then bringing it back will usually force a redraw. If for any reason the graphics look funny, for instance 'damaged' by the movement of some other window or some other program, or if the initial view upon starting the program looks strange, this is a simple way to redraw the view.

If one has read in a pdb file or a srf file there should now be something displayed from within grasp, either a molecule or a surface. The default display of either of these can be altered as described later. Upon reading a structure (i.e. atoms or surfaces) a scale is assigned to the unit box, i.e. a width in †ngstroms is calculated (and written to the textport). This is calculated such that the structure will fill up two thirds of the the box in its longest dimension. If the user instead has initially read a potential map the scale is such that the potential map will fill the unit box exactly, i.e. the boundaries of the potential map are at +/-1 in each direction. This scale is now set for the duration of the Grasp session as there is, as yet, no facility for altering global scaling. The view can now be manipulated, quantities calculated, structures built, etc. Operational details follow in the next two sections.

Exiting the program can be done three ways. The first is NOT recommended except in emergencies (i.e. the program has inexplicably locked up) and is to put the cursor over the textport and hit control C. The normal way to exit is either through the main menu or via control Q (see 'Control Keys'). If the program is correctly exited one should notice that the cursor is no longer yellow, which it is during normal Grasps operation, unless, of course, this is the users normal color for the cursor.

Control Keys

Control keys at present in Grasp mean any alphabetical character depressed while the control key is depressed. Grasp does not yet use any "Alt" keys or any function keys. To active control keys the cursor must be on the graphics window. They are included because people (for instance me) find them useful to have as an alternative to menu driven functions. Not all control keys are at present used, and some may be altered in the future if it becomes clear their particular use is so infrequent a more used function should be assigned to them. Most control key functions can also be found in menus. The current list of such functions is given below:

Control A: Toggle Active Subset Rotations On/Off.

If Grasp is manipulating more than one object (defined as "formal" subsets, see later) independently only one such object can be "attached" to the dials or mouse-dials at a time. This can be set via the menus. However if one wants to switch back and forth between different subsets continuously this can be frustratingly slow. Switching to 'Active' rotations means that when the user is using the mouse to enact translation or rotations the object moved is that upon which the cursor lies when the a mouse button is first depressed. Essentially the object is "picked" then moved. If the cursor lies over no object at the beginning of the motion then the mouse-dials are attached to the world, i.e. everything is moved together. If dials are used instead of the mouse then the object moved is either the one last moved by the mouse or that initially "attached" to the dials when the active mode was invoked. Turning the Active mode off (i.e. pressing control A again) leave the dials attached to the last moved object.

Control B: Toggle Color Scale Widgets On/Off.

Color scales should automatically pop up when a continuous coloring scheme is being displayed. If it does not , or if the user wants to remove them from view, the user can turn them on and off with control B. The color scale has two components, the title space, from which a menu may be accessed by the right-most button and the rest, which controls the color coding.

The color coding part should consist mostly of a colored section and a small white section, with the symbol ">-<" on it, at the right-hand end. The colors should be the colors in use for that property and structure, e.g. red, white and blue for electrostatics, and should vary continuously from right to left. If the coloring mode is two color continuous the colors will vary from the first to third colors, three color continuous will vary from the first to second to third color. Printed on this colored strip should be five, equally spaced, numbers which increase from left to right. The left-most, middle and right-most numbers are those used in the color coding as described in the previous section on program features. If the cursor is placed over any of these three numbers and the left-most button pressed and held down that number will decrease, and continue to decrease while the button is held depressed. When the button is released it causes the display to update color based upon this new number. If the middle button is used the number will increase.

If the scale is in three color mode and the value being altered is the lowest value, the rate of increase or decrease depends upon the difference between that value and the middle value, i.e. it increases by a constant fraction (10%) of that difference. Similarly it changes the upper value based upon the difference between that value and the middle value. The middle value changes based on the difference between it and the value towards which it is being altered. In two color mode only the left-most and right-most values change, and they do so based on their difference.

The ">-<" part of this widget allows compression and expansion of the color scale range. Depressing the left-most button and holding it on this symbol is equivalent to increasing the left-most number while simultaneously decreasing the right-most number, i.e. it compresses the range. Similarly the middle button will expand the range by decreasing the lowest number, increasing the highest number. This is often useful in viewing electrostatics since the range of potentials is usually much wider than is useful for distinguishing positive and negative parts of a surface.

The color scale menu allows the user to enter new values for the control (i.e. minimum, middle, maximum) numbers, also RGB triplets for the colors used. It also allows the user to reset the control values to their original (extrema) values. There is also support for changing the draw mode and property displayed. As two color continuous is invoked by setting the middle value to the lowest value, as since values can only be altered by fractions of a difference, one must use this menu to so set the control values, and to reset to three color continuous. The color bar menu also allows the user to alter the colors used for display, the draw mode for the surface or atoms, and finally the property being displayed. The latter two options produce menus identical to those which appear in regular menu use for these features and are described later in more detail.

One color bar should appear for each different quantity displayed, i.e. if the user is displaying atom potentials and surface potentials two should appear. If the user is using the split screen mode and different properties appear in the left view and right view, one scale should appear for each.

Control C: Change Current Map

The current map is the 65 cubed set of grid values which are used to build contours, interpolate potentials at a slice plane, interpolate potentials at a surface etc. There are two such maps in Grasp, the first of which is the default space for all internally generated maps, the second for all maps read into Grasp. Hitting control C produces a menu the user can use to choose which is "current". The user should beware of hitting control C when the cursor is NOT over the graphics window, since if it is over the textport the program will terminate.

Control D:

Control E:

Control F: Toggle Full Screen View On/Off.

The graphics window can be expanded to fill up the entire screen, removing even the window border from view. When this is turned off the window returns to its previous size and position.

Control G:

Control H:

Control I:

Control J:

Control K:

Control L: Free/Fix Light Source

The direction from which the light source will produces lighting affects on rendered surfaces can be altered after hitting Control L. Moving the mouse (without depressing any button) will then move the light source in that direction. (The source is set at infinity and so only the direction of the light source is altered) Hitting control L a second time fixes the light source's new direction. Note that this will affect the lighting of ALL surfaces, including those pseudo-lit and those which make up the surface of "CPK" atoms. The user should experiment since different lighting angles often bring out different features of surfaces.

Control M: Toggle Textport Depth

Control M is the same as 'return' i.e. it will pop the textport to the front if it is lower down, and push it to the back if it is in front.

Control N:

Control O: Toggle Grasp Box On/On/Off

The Grasp Box has been described before. It represents a box of +/-1, in screen coordinates, in each direction. The first Control O produces a box which is depth shaded, i.e. the box sides get darker the further from the viewer. The edges are also outlined in black. One advantage of this view is that because it has simulated depth it can fool the the eye into expecting any other object in view to have depth. Hence it "trains" the eye to see the objects in the box as three dimensional. Pressing Control O again removes the depth shading of the cube, leaving all sides white. This is provided in case the user is capturing pictures to send to a postscript printer (apparently it makes a difference). Hitting Control O once more removes the box from view.

Control P: Bring Up The Color Palette.

The Color Palette is the tool with which users may alter color from within the program. What should appear when Control P is hit are nine colored squares, each one with a quadrangle of white, red, green and blue attached to the lower, left, upper and right sides respectively. The colors inside the squares are the first nine indexed colors of Grasp. The index number of each color is written in the center of each square and is such that one is at the bottom left, two the one above it, three above it in the upper left, four in the middle bottom, five above it, six above that, seven bottom right, etc.

When the tool is first invoked the colors, along with their RGB triplet (i.e. Red, Green and Blue components as integers from 0 to 255) are:

index color red green blue

one white 255 255 255

two red 255 0 0

three green 0 255 0

four blue 0 0 255

five magenta 255 0 255

six cyan 0 255 255

seven yellow 255 255 0

eight grey 125 125 125

nine orange 200 100 50

These values are written in the appropriate quadrangles for each color.

To alter a color the user positions the cursor over one of the side quadrilaterals of that color. Then either the left or middle button is depressed. The middle button increases that component, the left button decreased that component. If the cursor is on the white component it increases/decreases all components. Colors are updated within the squares as the button is held down, as are the red, blue or green component values..

Altered colors have their RGB triplets automatically stored in the file "defcol.dat" in the current directory. This file is automatically read in the next time grasp is started within the same directory.

The next nine colors are accessed by hitting the space bar, and the next nine after that by hitting it once more, and so on. After colors 91 to 99 the screen returns to colors 1 to 9.

Colors 10 to 89 are left intentionally blank for the user to create their own. Colors 91 to 99 repeat color 1 to 9. Whenever the program is restarted colors 91 to 99 are always as listed above for colors one to nine, i.e. when they are altered within the program the changes are not stored in the external color file, unlike changes to all other colors. The user will probably find that uniformly decreasing the red/green/blue components of colors one through nine from their above given values will give richer display colors.

When the user is finished with this tool hitting any key, other than the space bar, will remove it from the screen. Any structure colored by an indexed color which has been altered should automatically update its color.

Control Q: Quit the Program

Control R: Toggle between Single and Double Buffered Mode

Animation is achieved on an Iris by drawing the view into a secondary or "back" buffer, then switching it into the primary or "front" buffer only when the drawing is complete. In this way none of the actual drawing is seen. Iris machines come with a limited amount of hardware for these two buffers. Power series machines have two 24-bit buffers allowing "full" color mode for animation. Most lower machines come with one 24-bit buffer which is split into two 12-bit buffers for animation. Having 12 bits for three colors means that there are only 4 bits per color, rather than 8 in full mode. Hence the same variety of colors are not available for animation. If the user has such a system and wants to get a 24-bit picture they should switch to single buffer mode. Then all 24-bits are used for coloring, but one "sees" the drawing as the view is constructed. The best use of this is then to enhance a view the user is not going to change, e.g. if one is going to take a photograph from the screen. And occasionally it may be instructive to see how a view is drawn.

Control S: Stereo/Split Screen Mode Menu.

This menu is described in the menuing section

Control T:

Control U: Unhook Dials.

If the dials are used extensively the user may find they suddenly cease to work in one direction. They have come to the end of their range. If this happens Control U will remove the dials from their usual functions. The useless dial can then be rewound without moving the view. Pressing Control U again brings the dials on-line once again.

Control V: Repair Surface.

Occasionally upon construction of a molecular surface there main occur "defects" in the structure, i.e. holes in the rendered surface. These can usually be fixed by hitting Control V once or twice. This is best done before another structure is constructed or imported.

Control W: Swap buffers.

Swap the current front buffer for the back buffer (see Control R).

Control X: Toggle Cross-Hairs On/Off.

Sometimes the cross-hairs are not useful in the view and can be turned off by Control X.

Control Y: Toggle Projection Plane:

The projection plane was briefly described in the features section. Essentially it is like having a molecular surface stretched across the Z=0 plane from X=(-1,1) and Y=(-1,1). The potential at any point in this plane is calculated from whichever potential map is current (although, of course, the values in the maps do not have to be potentials) by the usual trilinear interpolation. Because the plane is linked to the Grasp Box it does not move when the view is rotated, hence the map position of each point on the plane will change, since maps are rotated with the view. Hence the potentials are recalculated upon every time the view is moved. The color coding is calculated as if each point in the plane indeed belonged to a molecular surface, i.e. using the same colors and control values. Hence they may be altered the same way, via the color scale menu (see below). One advantage of the projection plane is that by moving the molecule back and forth in the Z direction one can see patterns of potentials within the molecule. It is also possible to move the projection plane rather than the molecule, i.e. alter the Z value from the default of zero. This is achieved via the "Z-Trans. Alternatives" option under the "Mouse Functions" menu option.

Control Z: Unix Shell

In Unix control Z halts a process and return the user to the shell. In Grasp it has a similar purpose. It actually launches a new shell rather than the one from which Grasp arose, but the effect is the same. The user can issue any unix command, e.g. edit files, change current directory etc. Of course the user can always do these things from within another window on the Iris, but sometimes its just more convenient to hit Control Z. To exit the shell type "quit" or "exit" or "logout". Note the user MUST do this before returning to the program, which is "frozen" until the shell is successfully exited. (N.B. this temporary shell does not have access to any of the aliases in ones login in file, nor will it understand the "~" symbol, or other short-cuts which are set up for the users usual shell.)

The Command Line

Entering commands from the keyboard proceeds as outlined above, i.e. the cursor should initially be over the graphics window. The user then just begins to type the command. The cursor should then jump to the textport where the characters should appear. Upon completion of the command hit 'return' and the command will be executed and the cursor return to the graphics screen.

(N.B. The textport may originally not be in the foreground, i.e. has to jump to the front from the background. This usually takes a finite length of time to complete and if the user types particularly fast the second character entered may get lost in the transition, i.e. will not appear in the typed command. If the textport is in the foreground this usually does not occur. Also if the user tries to backspace over the first letter of the command they will find they can not! Simply reenter the command in this case.)

The commands than can be entered via the keyboard are all color assignments for all structures, precise rotations and translations, the setting of radii and charges of atoms, the listing of atom properties to the screen, the type of such information for atoms and surfaces, and subsetting in the context of requests from menu driven functions or the two previous functions. The specific syntaxes for these commands and subsetting is described below. To simplify the description the following notations will be observed for variables:

n or m= any integer

x or y= any real number

a, b, c or d= any character

Note the Grasp is case insensitive where character variables are concerned, e.g. atom names. This is not always true of the subsetting code letters described below, as will be evident.

Coloring

For altering the colors of atoms the construction is:

c=n

where n can be between 0 and 99 and indexes the Grasp color set. Zero is always the color (0,0,0) i.e. black, and ALSO means not to display. Thus typing,

c=0

will cause all atoms to disappear from view. Note there is also a "hide" feature under the menu system which appears to the same thing. The difference is that "hiding" does not alter a property of atoms or any other structure whereas color is a property. Also "hide" may only be applied to all atoms, whereas "c=0" can be applied to any subset.

The command for coloring surfaces, bonds, backbone boxes and pair-wise interaction (matrix) strands are respectively,

vc=n

bc=n

kc=n

ic=n

In each case coloring to zero has the same effect.

Mapping Atoms Colors to the Surface

An exception to n being an integer involves transferring atom colors to associated vertices. By 'associated' is meant the atom to vertex mapping provided by the intermediate accessible surface. See the section on making a molecular surface for further details. The syntax necessary to map atom colors to the surface is,

vc=a

This can be subsetted only by atoms, i.e. one can map the discrete colors of a subset of atoms onto the surface, e.g.

vc=a, ch=b

only maps atoms colors onto those parts of the surface which are associated with chain 'b'. Note that this is the only exception to a general "like-with-like" rule for atoms and surfaces, i.e. that only atom properties can subset atoms and only surface properties surfaces.

Undo and Restore

There are two further exceptions to n being an integer for 'vc' and 'c'. The first is

c=u(ndo), or vc=u(ndo)

(The brackets indicate that it is not necessary to complete the word 'undo'). The 'undo' command will change the color of each atom/vertex to what it was before the last coloring command. In other words, whenever atom colors are changed a backup copy of the original array is made and this will then replace the current colors if an 'undo' is entered, and similarly for surface vertices. The most common use of this function is to correct a mistakenly typed command.

The second exception is,

c=r(estore), vc=r(estore)

This command only acts upon atoms or vertices respectively which have been assigned color zero, restoring the color to what it was before it was so set. Note that while 'undo' is only one color command "deep" restore persists until colors are changed from zero. The primary use of this command is usually to "unhide" parts of a molecule or surface.

Note that although these commands may often be entered without specification of a subset they can both be used, with subset commands i.e. one can 'restore' only certain atoms, or 'undo' a color command on only part of a surface.

Precise Rotations and Translations

Sometimes is is necessary to move objects by exact distances or angles of rotation. Grasp makes this possible through the command line via the following commands,

xt=x i.e. translate in the x direction by the x Angstroms

yt=x i.e. translate in the y direction by the x Angstroms

zt=x i.e. translate in the z direction by the x Angstroms

xr=x i.e. rotate about the x axis by x degrees

yr=x i.e. rotate about the y axis by x degrees

zr=x i.e. rotate about the z axis by x degrees

Xt=x i.e. translate in the x direction by x Box Units

Yt=x i.e. translate in the y direction by x Box Units

Zt=x i.e. translate in the z direction by x Box Units

N.B. These commands can be supplemented only by formal subset specifications, as described below. For example,

xr=90, sub=m1:a

will only rotate the atoms and/or vertices of formal subset "m1:a"

List

By entering the keyword 'list' Grasp will print atom information to the textport on each atom. The type of information is controlled by the atom information parameter list as described below. Without any restriction this command will list this data for all atoms. More often the user will want information on a certain subset of atoms, for instance,

list, r=lys,a=nz

will list the information only on the terminal zeta nitrogen of lysines.

If there are more than four atoms selected Grasp will automatically expand the textport to list up to twenty atoms at a time. If there are more than twenty atoms in the list, hitting the space bar will give the next twenty atoms, while hitting 'return' lists the next atom. Hence the output is controlled like that supplied by the Unix command 'more', except that the output can be terminated by hitting any key other than 'space bar' or 'return'.

Upon completion of the listing hit 'return' to reduce the textport to its original size and position.

Changing atom/surface information parameters

Grasp maintains a list as to which atom or surface properties are written to the textport by a list command (atoms only) or by "picking" with the mouse (both). The default property for the surface vertices is potential, while for atoms it is the atom name, residue name, residue number and chain name. (In fact for atoms it is simply the character field (12:26) in the pdb file). Each possible property for surfaces and atoms is assigned a single letter code, thus:

Atoms Surfaces

default atom information: a absolute coordinates:x

absolute coordinates: x surface potential: p

box coordinates: X curvature:C

distance: d box coordinates: X

radius: r distance: d

charge: q constructed surface number: s

potential: p subset name: b

molecule number: m cavity number: V

general property #1: 1 general property #1: 1

general property #2: 2 general property #2: 2

subset name: b

One can alter the list in one of three ways, namely adding properties, removing properties, or resetting the whole list. Examples of the commands to do each are listed below,

si=pC , reset surface list to ONLY potential and curvature.

ai=qr , reset the atom list to charges and radii, BUT do not remove default atom information if it is on the list already.

ai=+b , add subset name to the atom list.

ai=-a , remove default atom information from atom list.

si=+xd , add absolute coordinates and distance to the surface list.

Note that the order of the parameter entered does not change the order in which the parameters are written to the textport. That order is determined by the position in the above lists of properties from top to bottom which corresponds to left to right output.

Alter

The command to alter a radius or a charge is:

alt(r=x)

and

alt(q=x)

respectively, the x being the new value. Also supported are the following:

alt(r=r+x)

alt(r=r*x)

alt(q=q+x)

alt(q=q*x)

i.e. implicit modifications of each. Typically these commands will be followed by subsetting command. For instance,

alt(q=1.0), a=nz, r=lys

will assign a plus one charge to all zeta nitrogens of lysine.

Note that charge is a display property for atoms and so if the charge distribution is altered via the command line it will cause a recalculation of the maximum and minimum values used for color scaling.

Note also that one can use these commands in an external file as an alternative method of assigning charges and radii to the DelPhi control files. Simply edit a file to contain the list of commands to assign charges or radii, or both, and then read in the file as a Grasp Script File (see section on Menu Operations).

Finally, note that atoms of zero radii are not displayed, or used in calculations of curvature, accessible area, or low dielectric volume.

Negation, Concatenation and Projection

All subsetting commands listed below can be negated. This is achieved by putting a minus sign immediately after the equals sign. For instance,

c=2, r=-lys

will color red all residues which are NOT lysine. If the selection variable is a number not a character then one has to consider whether confusion would result, e.g.

c=3, p=-(1,2)

will clearly color all atoms whose potential is NOT in the range 1.0 kt to 2.0 kt, but

c=1, q=-1.0

will color all atoms with a charge of minus one, i.e. will not color all atoms which do NOT have a charge of one. If such an ambiguity exist one should wrap the value in brackets, i.e.

c=1, q=-(1.0)

will color all atoms which do NOT have a charge of one.

All subsetting commands can be combined on the command line by separating them by commas. For instance,

c=2,r=lys,q=-(0.0), X=<0.0

will color all atoms which are in lysines, have non-zero charge AND which are in the left hand side of the screen. In other words a comma, when not inside of brackets, .e.g. specifying a range of values, act as a logical AND.

Projection allows the user to select groups of atoms based upon a different, usually smaller, selection. The major use of this at the moment is to select a residue based upon whether an atom of that residue has been selected. For instance, suppose one wants to know which residues are within five angstroms of a certain cofactor which has a residue label of CYT. First one calculates a distance map for all atoms to this cofactor. One would then select all atoms which are within five †ngstroms of the cofactor. Finally one wants to project this onto the 'parent' residues, i.e. find all residues which have at least one atom within five †ngstroms of the cofactor. The command to do these last two operations and to color all atoms in such residues red is,

c=2, d=<5.0 , r=-cyt >r

i.e. the symbols '>r' will cause projection to the appropriate residues.

The only other current use for projection is when coloring bonds. Usually one colors bonds via the command line by selecting a color and a subset of atoms. Those bonds which originate from those atoms are then so colored. If, however, the user wants to color based upon which atoms the bond is ending on the correct syntax is ">p". For example,

bc=0, a=sch >p

This will 'uncolor' i.e. hide all bonds made to side chain atoms.

The concept of "projection" will be expanded in future versions.

Subsetting Syntax

The above command or functions, e.g. coloring, altering and listing, are usually are followed by subsetting specifications. One can subset by many properties of surface and atoms and other objects. There follows a complete listing of the current key letters or words for such properties, along with examples of how each should be used.

Atoms:

atom name, a=abcd

e.g. a=oe1 will select all atoms with the name "oe1" or "OE1" or Oe1" or "oE1". Notice case INsensitivity.

N.B. one can use question marks to act as wild cards

e.g. a=oe? will pick atoms with names "OE1" and "OE2". One can use "_" to indicate an intentional blank,

e.g. a=o_1 will pick only the field "o 1" or "O 1"

There are four characters read for each atom name.

There are a couple of short cut names for backbone atoms and for side chain atoms (defined as NOT backbone atoms). They are,

a=ba which selects atoms C, CA, N, O, HA, and HN.

a=sch which is equivalent to a=-ba.

residue name, r=abc

e.g. r=lys , all atoms in all lysines.

N.B. As above for atom name, except there are only three characters for residue name.

There are several short cut names for residues based upon their hydrophilicity. They are:

r=crg, which selects all residues which are normally assigned formal charges, i.e.

lysine, arginine, glutamate, aspartate

r=pol, which selects all the residues formally polar, i.e.

serine, threonine, tyrosine, histidine, cystine, asparagine, glutamine, and tryptophan.

r=hyd, which selects all hydrophobic residues, i.e.

alanine, valine, leucine, isoleucine, methionine, phenylalanine and proline.

The only residue not included above is glycine, which can of course be selected by 'r=gly'.

atom number, an=n, an=(n,m), an=>n, an=<m

e.g. an=(1,500) ,the first five hundred atoms.

N.B. atom number refers to the internal numbering scheme, i.e. atom 256 is the 256th atom to be read in, NOT an atom assigned an atom number in the pdb file. (The field reserved in the pdb specification for atom number is not interpreted by Grasp.)

residue number, rn=n, rn=(n,m), rn=>n, rn=<m

e.g. rn=(1,25) , all residues which have residue numbers between 1 and 25.

N.B. residue numbers ARE as described in the pdb file, i.e. unlike atom numbers. A consequence of this is that while every atom has a unique number different residues (e.g. on different chains) could have the same number. If a residue number is not assigned in the pdb file it is assigned a value of one for internal purposes.

chain name, ch=a

e.g. ch=-B , all atoms NOT in chain B

N.B. if there is no chain specifier in the pdb file it is assigned the letter "A".

Assigned charge, q=x, q=(x,y), q=>x, q=<y

e.g. q=>0.0 , all positively charged atoms.

Assigned Radius, R=x, R=(x,y), R=>x, R=<y

e.g. R=-(0.0) all atoms which have been assigned a non-zero radius.

Calculated potential, p=x, p=(x,y), p=>x, p=<y

e.q. p=>10.0 , all atoms at whose center the calculated potential is less than 10.

N.B. potentials are in kt, (1 kt = 0.6 kcals).

Original Coordinates, x=x, y=(x,y), z=>x, z=<y

e.g. x=(50.0 , 60.0), y=>45.0

N.B. these are the coordinates as they appear in the pdb file, in †ngstroms.

These are unchanged by any user applied rotations or translations.

Screen Coordinates, X=x, Y=(x,y), Z=>x, Z=<y

e.g. X=>0.5

N.B. these coordinates are relative to the screen and in box coordinates. They alter as the molecule is moved. The coordinates here are relative to the unit box, i.e. which runs from plus to minus one, so the example given above will pick all atoms which are in the right most quarter of the screen.

General Property One, p1=x, p1=(x,y), p1=>x, p1=<y

General Property Two, p2=x, p2=(x,y), p2=>x, p2=<y

e.g. p1=(5.5,6.5)

molecule number, m=n, m=(n,m), m=>n, m=<m

e.g. m=1, the first imported molecule.

N.B. molecule number refers to the order in which molecules are read in, i.e. the first structure read in is assigned the number one, the second two and so on. If more than one molecule is read from one file the molecule numbers are sequentially assigned.

accessible area, S=x, S=(x,y), S=>x, S=<y

e.g. S=0.0 , all buried atoms.

N.B. one should first done an accessible area calculation to be able to select based upon it. See later under Calculation for the appropriate menu entry.

distance, d=x, d=(x,y), d=>x, d=<y

e.g. d=<3.0 , all atoms three †ngstroms or less from some set of points.

N.B. as above one must have calculated a distance map to sensibly select based on this property.

assigned discrete color, cd=n

e.g. cd=7

N.B. The user can select against color zero, i.e. select for atoms not displayed. The default color for atoms is one.

formal subset name, sub=abcd.....

e.g. sub=m1:a

N.B. if you choose subset names, rather than accept those produced for you by Grasp, try not to start the name with a "-", since this will be interpreted as a NOT symbol.

N.B. subset numbers can also be substituted for subset names, i.e. if m1:a is the first subset created then,

sub=1

will have the same affect as sub=m1:a

Surfaces:

Many of the letter codes for surface properties are the same as those for atoms listed above. Since the context should be there is no danger of non-uniqueness, i.e. when one is using any of the following with the vertex coloring command 'vc=n' it is clear the properties being specified belong to the vertices not atoms. Similarly any requests for subsetting via the menus will entail a similar understanding. This said, the following commands are identical for both atoms and vertices:

p=

x=

y=

z=

X=

Y=

Z=

d=

sub=

p1=

p2=

i.e. they both refer to the same property but that for atoms in one case and for vertices in the other.

Commands which are specific for surface vertices are:

assigned discrete color, vcd=n

e.g. vcd=1 , all parts of the surface assigned color index one

N.B. The default discrete color for surfaces is 91. The index color one is not used because it is usually altered to be less than pure white, which although is okay visually for atoms, looks awful on surfaces, whereas color ninety one is always unaltered white..

vertex number, vn=n, vn=(n,m), vn=>n, vn=<m

e.g. vn=15677

N.B. vertex number is assigned like atom number, i.e. serially upon being imported or constructed. Vertex number might not seem as useful a variable, but, for instance, if one calculates the maximum potential on a surface, or portion of a surface, the vertex number of that point is also returned. Hence 'vn' along with a 'vc' command can be used to locate this point.

calculated surface curvature, C=x, C=(x,y), C=>x, C=<y

e.g. C=>0.0 , all concave parts of the surface.

N.B. must first calculate the surface curvature.

constructed surface number, m=n, m=(n,m), m=>n, m=<m

e.g. s=(1,3) , the first three surfaces read in or constructed.

N.B. analogous to molecule number except that surfaces are also constructed as well as imported.

Bonds:

One selects bonds on the basis of the atoms they originate from (except see above on Projections). Note that although bonds conceptually go between pairs of atoms, in Grasp they go half-way, i.e. each bond 'object' goes from the center of an atom half way to the other atom of the bond pair. Each 'bond' is then uniquely associated with an atom. And so when a bond is colored actually only half the bond is acted upon.

All atom commands may be used in assigning bond colors, for example

bc=4,a=n???

will assign the color 4, normally blue, to all half-bonds originating from any nitrogen atom.

In Grasp the only property bonds possess other than those of their atoms is the color they have been assigned, the default value of which is one. This can be accessed via the command,

bcd=n

e.g. bc=2,r=pro,bcd=3 ,

will color red all those bonds in any proline which are currently colored green.

There is no undo or restore for bonds at present.

Note that bonds colors can be automatically mapped from underlying atoms via the appropriate menu command. There is also an internal color scheme for bonds which can be selected from this menu.

Backbone Boxes:

One selects backbone boxes some what similarly to how one selects bond colors, i.e. by specifying a subset of atoms. The backbone boxes are constructed out of four atoms, the alpha carbon of the first residue, the carboxyl oxygen of that residue, the amine nitrogen of the next residue and the alpha carbon of that next residue. If any of these atoms are selected the backbone box that uses that atom is assigned that color.

As has also been mentioned there is an alternative color scheme for the boxes where the corners are colored white (alpha carbon), red (oxygen) or blue (nitrogen) to indicate the electrostatic character of the peptide plane. This is the default color scheme for backbone boxes but it can be explicitly invoked by using the color command,

kc=d(efault)

This command can be followed by any subsetting commands for atoms, just like the regular 'kc' command.

Like bonds, backbone boxes also have a single unique property assigned to them, that being the color assigned to them. This can be invoked by the command 'kcd', for instance,

kd=4, rn=(8,33), kcd=3 , i.e. color blue (=4) all backbone boxes which are a part of residues 8 to 33 which are currently colored green (=3).

Matrix Strands:

Matrix values are not calculated at present by any part of Grasp. They must be imported via a data file. There are two formats for such files which are described in appendix A on file formats.

There are some special commands and syntaxes for pair-wise interactions. These include:

strand strength, ip=x, ip=(x,y), ip=>x, ip=<y

e.g. ip=> 0.0 , all positive interactions.

strand strength rank, ip=n

e.g. ip=2 , select all those strands which are the most positive, or second most positive for that residue.

e.g. ip=-1, select only those strands which are the most negative for that residue.

Both the above commands can be subsetted by using any atom commands. This is implemented by adding commands to an 'ic' command which would select a set of atoms. If then any atom of a residue is selected then that residue becomes selected. For instance,

ic=2, ip=>0.0, a=oe1

will color all strands which have positive strength and originate (or end) on a glutamate or glutamine since these are the only residues with atoms labelled 'oe1'. Similarly,

ic=2, ip=1, r=lys

will select all strands which are the most positive strand coming out of either residue it connects, and for which either of these residues is a lysine.

This latter point is worth repeating, i.e. that the program will check both ends of the strand for selection and uses OR to determine selection. (As such there is no direct way to select on the basis of both ends of the strand, e.g. to only strands which go between lysines and glutamate. However see the 'icd' command below.) This is a notable exception of the usual Grasp philosophy of commands always being AND based, and reflects the two-site nature of matrix strands.

Like all other structures the assigned color becomes a property and can be used in subsetting, i.e. the command 'icd' will select on current color. For instance,

ic=0, icd=2 , i.e. uncolor (=hide) all strands currently colored red.

Note that this command can be used to do the "double" selection mentioned above, i.e. suppose we want to select all strands running between lysines and glutamates. The following set of commands will color these, and only these, red.

ic=0 , uncolor all strands

ic=3, r=lys , color all strands originating or ending on lysines green.

ic= 2, icd=3,r=glu , color all strands originating or ending on glutamates, which are already colored green

ic=0, icd=3 , uncolor, i.e. hide, all those strands which are still green.

There is no undo or restore for strand colors.

Finally, one can find the connectedness of sites via strands using the following variant of the 'ic' command. Typing,

ic=c

causes Grasp to work out the connectedness of all visible strands. That is to say if two strands will be assigned to the same group if they are connected to the same residue, or if it is possible to go from one to the other via other visible strands. Grasp will then report the number of such patches and color each differently. (Only nine different colors are used so if there are more than nine patches the colors repeat).

Note that there is no restriction applicable after this command i.e. one can not restrict this command to certain residues. Instead the proper usage would be, for instance, to first color all strands which are greater than a certain strength, then issue the 'ic=c' command. This will then show up how far 'webs' of that interaction threshold spread.

All other matrix strand operations, such as scaling by distance, deciding upon the display mode, and the maximum interactions strengths used in width encoding, are under menu command and are described in that section.

Grasp Menus

Menus carry most of the functionality of Grasp. All menus are accessed via the right-most button. All selections within a menu must also be chosen with the right-most button. Menus appear when this button is depressed. It remains when the button is released. Note this first release of the right-most button does NOT select an item. The program is essentially frozen until the right-most button is depressed and released AGAIN. If the cursor lies over a menu entry when the button is released this second time that menu item is chosen. If the user 'clicks away' i.e. releases the button when the cursor does not lie on an entry either the function will abort, or in some circumstances it will continue with a default value. For instance when the user is altering the molecular surface they first get a menu for the quantity displayed on the surface and secondly for the draw mode (e.g. mesh, lit, points..) of the surface. If one does not want to change the quantity displayed one clicks away from the first menu and continues on to the menu for the draw mode. On the other hand, if the user were to choose the Build entry in the root menu and then click away the program exits from the menuing system.

Many menu entries will produce another menu. This should appear positioned so that the upper left hand corner is at the same position as the previous menu. The root menus should appear such that the upper left hand corner coincides with the cursor position when the right-most button is initially depressed. Upon completion of menuing the cursor should return to this same position.

Some parts of some widgets, such as the color scale, are sensitive to the right-most button. This means that if that button is depressed the normal root menu will not appear. Instead the user will get a menu associated with that widget. Clicking anywhere else in the graphics window will get the user the "root" menu for Grasp.

The description below of Grasp functions will follow the order of the root menu, with detours where appropriate.

The Root Menu

Display

Build

Calculate

Mouse Functions

Read

Write

Formal Subsets

Programs

Set Parameters

Miscellaneous

Help

Quit

Display

This menus controls what is displayed and how it is displayed. With the exception of the "stereo/split screen" option, submenus of this menu are ordered in the following manner,

operation : structure : options.

The operations are to show a structure, alter it, hide it, hide everything, followed by the stereo/ split screen option. Alter differs from show only in that the user is not prompted as to whether a 'default' display is required for the structure chosen. Hide causes that structure to disappear from view, but retains all characteristics. Note this is different from using the 'uncolor' option for some structures. If a hidden structure is shown it returns to view as it was before hidden. Hide everything is just a shortcut to clearing the view completely.

The structure list contains SURFACES, ATOMS, BONDS, CAVITIES, OBJECTS, CONTOURS, VECTORS and interaction MATRICES. All have been mentioned before in some detail except vectors, which consists of field related items such as dipole vectors, field lines and field vectors. The object entry comes with a submenu listing the possible objects, i.e. backbone worm and boxes, DNA bases, backbone, sugars and axis. For some structures the menu entries just set flags to tell the program to draw this structure, or hide it, depending on the previous menu choice. However most have options associated, especially those in the more developed parts of Grasp.

Options for atoms and surfaces are the property to be displayed, e.g. potential, distance, discrete color, etc., and the drawing mode for that structure, e.g. for surfaces whether lit, pseudo-lit, mesh or points, for atoms, flat spheres, full spheres (CPK), flat but patterned, small "bond" atoms, line spheres or point spheres. For bonds the user gets the option of three coloring schemes, namely to let the user set them (default color=1), adopt the underlying atom colors, or apply an internal color scheme, followed by the bond draw mode, i.e. lines, sticks and cylinders. (N.B. cylinders are very slow to draw). For cavities one has the option of displaying the surfaces as the molecular surfaces are displayed or individually colored. Backbone objects come with no menu options, although see the command line entry for backbone boxes as to how to individually color them.

DNA objects come with several possible options. Most of these are only appropriate if data in the form of a Curves file has been read in, because the options refer to which of the many Curves parameters are to be displayed.

Contour draw modes can be altered to the usual types for surfaces, i.e. mesh, points, lit, pseudo-lit. Control of depth shading for contours is via the depth shading entry in the miscellaneous menu. Colors of contours as well as their values are set when calculated, as later described.

The vectors possible are for molecular dipole (a large 3-D arrow of length 0.3 box units), electric field lines which can be drawn as lines or tubes, or electric field vectors which can be drawn of constant length or with length dependent on the field strength. In the latter case the user can specify the maximum vector length and the field strength this should correspond to, but only when these quantities are calculated, as described later. Note that it is necessary to calculate a vector quantity before displaying it and that the cylinder mode for field lines can be very slow to draw.

The interaction matrix has three draw modes, i.e. ways to draw the interaction strands. These are as lines, fixed width cylinders and variable width cylinders. The first two are self explanatory, the third means that the width of each cylinder will depend upon the absolute value of the interaction. This value is divided by some maximum strength value, rounded off to unity if greater than one and then multiplied by an internal width to give the resultant cylinder thickness. Of these parameters the "maximum strength" value may be altered by the user. This is achieved via the "Extras.." menu option at the bottom of the matrix draw mode menu.

The "Extras.." menu also includes options to alter the exact coordinates of the point within each residue each strand emanates from. Upon choosing this option the user is prompted for a selection on the command line. For instance the user could then input the command "a=ca" where upon the strands would begin on each residues alpha carbon. If more than one atom is selected for a residue then the strand begins at the average position of those atoms.

Also included are options to multiply or divide the interactions by the distance between interaction sites. The new maximum and minimum values are written to the textport upon each use of this option. Note that if the draw mode is set to variable widths then this can affect the relative widths of the strand cylinders. To maintain a similar spread of widths the following process of rescaling the maximum cylinder width value is enacted. The ratio of the largest (absolute) interaction value (before distance scaling) to the value set for the maximum width is found. Then the largest (absolute) value is found after distance scaling, and the value for maximum cylinder width set so the ratio just calculated is maintained.

Finally we describe the stereo or split screen option (note that his option can also be accessed via Control S). When selected the user will be presented with the following menu,

Dials to Both

Dials to Right

Dials to Left

Right-Hand Twist

Stereo Parameters

Stereo/Split Off

The first three entries decide which side is going to be "attached" to the rotations and translations as entered by the mouse or dialbox. The default entry for this menu is the first, i.e. both views are moved equally. Note that in this default mode the two views differ only by an imposed separation (of 0.5 box units) in the X direction, although the views may not appear identical due to the perspective automatically included in all Grasp views. Choosing the second option means the left view can be spatially manipulated independently of the right. To move the right hand view the user has to reinvoke this menu and select the third menu option. Note that if the user has defined one or more formal subsets (see later) then only the views of the subsets on the side for which the dials are attached can be moved.

The next two entries involve the "stereo" part of the "stereo/split screen" functionality. The first of these causes the right hand view to be twisted in an axis running through the center of its world coordinate system in the Y direction (vertical) by a certain angle This twist is set to eight degrees by default. This value can be altered in several ways. If the user has a dialbox then the left bottom dial will alter the stereo twist. The user can also fix the Z-rotation function to stereo twist via the "Mouse Functions: Alternative Z-translation" menu combination. Finally, if the user chooses the fifth menu option in this menu then the second option of this submenu allow the user to enter the value explicitly. This submenu also allows the user to return to the default value or to remove the twist all together AND remove the stereo separation, i.e. to superimpose the left and right views.

Even if the user is in "twist" mode the user can still independently manipulate the two views spatially. This is not recommended since the purpose of twist mode is to allow stereo viewing which requires the two views to be essentially identical except for the vertical twist.

If whilst in split screen mode the user attempts to change display properties Grasp will prompt the user as to which "side" the changes should be made. For instance, if the user attempts to hide the bonding display the choice is presented of "left", "right" or "both", the implications of which should be clear. Thus the display on the left can be representing one facet of a molecule (e.g. electrostatic potential on a surface) while the right represents another (e.g. atomic B-value).

When the user quits the "stereo/split screen" option the left-hand side orientations (world and subset) are the ones retained for the single screen view. Also all differential display characteristics the user may have applied to the right hand view are, at present, lost. The stereo twist value is however still stored and may be retained throughout a session.

Build

The "build" and the "calculate" menu options are sometimes easy to confuse. For instance, does one build a contour or calculate a contour. Or are field lines built or calculated. In general, build deals with the calculating the data intrinsic to a display structure, such as a surface, or a backbone representation, or internal cavities, where as calculate provides numbers which may or may not be related to such structures, such as potential maps, volumes of surfaces etc. The list is,

Molecular Surface

Accessible Surface

Backbone Worm

Backbone Boxes

Cavities

DNA Boxes

Contours

Consensus Volume

Molecular and accessible surfaces are made by essentially the same algorithm. On choosing to construct one or the other the user has to consider two options. The first is which atoms to use in forming the surface, the second is whether to add this surface to previously constructed surfaces, or to overwrite them, i.e. delete previous surfaces (Note that this is the only way to delete a surface from within Grasp). The menu for selecting a subset of atoms is one which occurs quite frequently within Grasp. It gives the user the choice of:

All Atoms

A Molecule

A Format Subset

Enter String

The first is obvious, the second will will result in a menu containing all molecule numbers, e.g. molecules one through five, the third will give a menu with the names of all atom based format subsets, and the fourth will cause the cursor to jump to the textport and wait for the user to enter a subsetting command. If no atoms are chosen at the end of this procedure the routine aborts.

The process of constructing the molecular surface occurs via the construction of a temporary accessible surface. A correspondence between the vertices of this intermediate surface and the underlying atoms improves the accuracy of the final surface. It also allows for a unique mapping between atoms and molecular surface, i.e. each accessible surface vertex is assigned an underlying atom, and each molecular surface point is assigned an accessible surface point. The combination of these two assignments leads to the association of each molecular surface point with an atom, an association which is termed "contact" within the program.

The process of surface formation will cause plenty of information to be written to the screen, including the scale at which the surface is constructed and the number of vertices and triangles in the completed product. The information here can be useful in debugging (for example the total number of vertices might exceed the maximum allowed number).

Surfaces should appear automatically after being calculated. They will not be colored however. This must be done by the user via whatever method chosen, i.e. calculate potentials at the surface, calculate the surface curvature etc.

Backbone Worm and Boxes are built for all current data, i.e. all molecules. There are no options as yet associated with these objects. If they fail to appear after construction go through the "Display" menu explicitly. The backbone worm can be slow to display. Sometimes this can be put to good use. For instance if the user switches to single buffer mode (see above, Control R) while the worm is being drawn the path of the chain from N terminus to C terminus is nicely illustrated. The backbone worm only requires the carbon alpha positions to be correctly produced, while the backbone boxes require also the carbonyl oxygen and amide nitrogen positions to successfully complete construction

Building Cavities causes the program to check all vertices for connectivity. The first point, or seed point, is chosen at an extrema, and so can not belong to a cavity. All points associated with it, i.e. which can be reached by travelling along triangle edges, are deemed the "non-cavity surface". All others belong to cavities. Note that this will give an incorrect assessment if there are more than one disconnected constructed convex surfaces. For this reason the user should calculate cavities on the surface of the whole molecule, not subsets. Printed to the screen are the number of triangles that make up each cavity so found. Cavities are automatically displayed in the same display mode as the molecular surface. Alternatively, as described above, they can be sequentially colored.

Building DNA boxes requires that a DNA pdb file has been reading. (Note that mixed files i.e. ones containing DNA and protein are fine.) Display should be automatic. Grasp can handle structures with up to four independent backbone strands.

Making contours requires two inputs by the user, the isopotential value and the color to be assigned to that contour. The latter should be a color index, i.e. an integer between 1 and 99. One can enter more than one isopotential value to create more than one contour at a time, as long as one enters the same number of colors. For instance,

>> Enter Contour Value (written to screen)

>> 1.0,2.0,3.0 (user enters)

>> Enter Color(s) (written to screen)

>>2,3,4 (user enters)

will create the contours at one, two and three kt, and give them colors red, green and blue.

To delete a contour (which may be necessary to make room for new ones) one goes through the same procedure as making a contour of the same isopotential value, except one gives it color zero, i.e.

>> Enter Contour Value (written to screen)

>> 1.0 (user enters)

>> Enter Color(s) (written to screen)

>>0 (user enters)

will remove the contour at 1.o kt. Note that this remove is NOT the same as hide or uncolor, it actually removes the contour data from the program.

Although the usual use of "Build Contour" will be isopotential contours it can be used for more varied purposes since the actual contents of the map are irrelevant to the contour facility. For example, one can contour a DelPhi "eps" map if one has read one in, or one can contour a consensus volume map (see below) if one is calculated.

Finally, one should remember that there are two internal maps in Grasp. The contouring proceeds on which ever map is "current". This is usually map one, but will be map two, for example, if one has just read in a map. To be sure of which map is current, set it via the menus "Miscellaneous: Change Current Map" or with Control C (Note, be careful not to hit Control C while the cursor lies over the textport or the program will abruptly terminate).

Consensus Volume produces a map, i.e. a 3D lattice of values. It is calculated by adding the value 1.0 to each grid point which lies within the Van der Waals volume of each molecule. Thus if there are five molecules and a grid point lies within all five it will be assigned a value of 5.0. Some points will fall within only some molecules. This map can then be contoured at any level desired. For instance contouring at the level of the number of molecules will give the surface of the volume common to all molecules. At present there are no options to this facility and all atoms are included in the calculation. The map is stored as internal map two.

Calculate

The calculate option does much that is unique in Grasp. It allows the user to quickly calculate electrostatic quantities like maps, fields, site potentials, as well as surface curvatures, distances and volumes, plus manipulate fields of information previously calculated or imported to the program.

The Calculate menu is as follows:

New Potential Map

Pot. via Map at Surfaces/Atoms

Surface Curvature (+Display)

Simple Property Math

Dipole Moment

Field Lines

Field Vectors

Volume of a Surface/Molecule

Area of a Surface/Molecule

Distance Map

Calculating a New Potential Map causes the program to execute its internal Poisson Boltzmann solver. The size (in Angstroms) of the map produced is automatically determined. Only the linearized Poisson-Boltzmann equation is solved. Such parameters as the probe radius, ion exclusion radius, salt concentration and inner and outer dielectric values can all be set via the menus "Set Parameter :Electrostatic Parameters". The map produced is stored in internal map one. If no charges are assigned then the procedure will inform the user and then abort rather than calculate a null map.

It is important to realize that by default all atoms and therefore all charges are used in the calculation. If the user wants to perform a calculation on a subset of atoms then those atoms not required must have their radii and charges set to zero. The algorithm will ignore any atoms with radius zero in so much as they will not contribute to the water exclusion (=low dielectric) volume. However it will NOT ignore the charges on these atoms. Therefore one needs to neutralize the charges on the unwanted atoms as well. Since changing radii to zero also causes atoms not to be displayed, the user ought to remember to reset those atoms to their correct radius after the calculation, for instance by reading in a default size file.

A typical use of the above procedure for removing some atoms from an electrostatics calculation is where the original crystallographic coordinates are included for several water molecules. It is always an interesting question as to whether such waters should be treated as low dielectric, i.e. as constrained in their motion, or high dielectric, i.e. as bulk water. A good rule to use is that if a water is highly coordinated to the protein, i.e. makes two or more hydrogen bonds a case can be made for low dielectric, otherwise set it as high, i.e. set its radius to zero in the calculation and turn off any assigned charges.

As with surface creation much information is written to the screen during the calculation, some of which is useful to the user in verifying the accuracy of the calculation, i.e. that it has converged, that the correct total charge has been assigned, that the scale of the final map is approximately correct etc. A typical calculation should take about five seconds on a Personal Iris.

The next menu item "Pot. via Map at Surfaces/Atoms" calculates the potential at all surface vertices and all atoms from the current map. If one has just calculated a new potential map this is map one. The algorithm uses trilinear interpolation from the eight grid points which make up the map grid cube an atom center or surface vertex falls within. If it lies outside the map a zero value is assigned. Note that this process will overwrite any previous potentials. If this is a problem the user should first store the previous array in one of the other variables, as described below in the "Simple Property Math" option.

The third menu item "Surface Curvature (+Display)" will cause the program to calculate the surface curvature, as defined in Nicholls et al., for a set of surface points and a set of atoms. As our definition of curvature is related to accessibility of water to a single water placed in contact with a particular surface point, the set of atoms chosen is crucial, since the hard sphere radii of these atoms will determine this accessibility (Note that a zero radius atom does not affect accessibility). Thus, for instance, if one want to compare the curvature of two surfaces which make up an interface one should choose surface one and the atoms which made surface one for the first calculation of surface curvature, then choose surface two and its atoms for a second calculation.

Curvature calculations can sometimes take a long time (i.e. over 10 seconds). This is due to the choice of test sphere used to determine accessibility, i.e. more points take a longer time. The choice of this density is made automatically relative to the scale employed in the surface creation. Because Grasp only a fixed number of test densities the time taken in calculation will vary considerably.

Upon completion of a curvature calculation the display should automatically switch to displaying this quantity. The values are scaled to +/- 100, such that 100 would imply that the surface point is completely accessible, which will never happen since this would imply that one could put a water molecule anywhere in contact with the original water molecule touching the surface. However -100 implies that the surface water is completely isolated from other water molecules, which is quite possible, for instance in a deep cleft or if the surface is enclosed inside the molecule, i.e. is part of a molecular cavity.

"Simple Property Math" is one the most useful options in Grasp. Its submenu gives the user four choices namely maps, surface properties, atom properties and atom to surface projections. The "maps" option allows the user to swap maps, i.e. put map one into map two and map two into map one, to add map two to map one, the result going to map one, to subtract map two from map one, again putting the result in map one, to multiply either map by a constant, and to multiply or divide map one by map two, putting the result in map one. There is one further operation which is to apply a "convex" correction. It was noticed that potential maps generated on the the Convex used for most of our DelPhi calculations where misread if transferred onto an iris. To be more precise all real numbers (but not integers) were exactly four times too large. This includes the potential map center and scale as well as the potential values. This option then will rescale all those values to their correct value. In addition, if the Grasp box scale had been derived from a potential map then the user is prompted as whether to reduce the box scale by one quarter as well.

One should note in the "map math" that no checking is done to see if the grids actually have the same center and scale, i.e. whether they refer to the same part of physical space. The numbers of corresponding grid points are just added, multiplied etc. The inclusion of a division operation was prompted by the desire to calculate effective dielectrics, i.e. the ratio of the potential calculated with Coulombs Law to that via the Poisson Boltzmann equation. If a zero value is found in the denominator of a division then the new grid value is set to zero.

Math on surface and atom properties are similar in that the same basic operations can be applied to either. There are four operations which require three properties, i.e. a first and second property and a third or target property for the result. These operations are adding, subtracting, multiplying and dividing two properties.

Then there are operations which require two properties, namely multiplying by a constant, adding a constant and special functions. The first property is that acted upon and the second is the target property for the result. Note that this set of operations gives the user a way to put one array into another, for instance by multiplying property one by 1.0 and storing the result in property two. Special functions causes a further menu to appear which lists functions which can be applied to the first array and the result stored in the second. These functions are square root, reciprocal, raise to a power, exponentiation, absolute value, natural log, plus cosine, sine and their hyperbolic equivalents. Illegal operations such as negative square roots are not performed.

Finally there are operations which take but one property and return a single value printed to the textport, namely minimum value, maximum value, average and sum. The first two of these also return the atom or vertex number to which the extrema value is associated.

The properties for each step of the math calculation is selected from a property list for either atoms or surfaces. The user is also prompted for a s