GRASP Tutorial

Tutorial for the GRASP Software Package

Authors:
John E. Kerrigan, Ph.D.
Vladyslav Kholodovych, Ph.D.
University of Medicine & Dentistry of New Jersey
Robert Wood Johnson Medical School
675 Hoes Lane
Piscataway, NJ 08854 U.S.A.
(732) 235-4473 phone
(732) 235-3229 phone
(732) 235-5252 fax
kerrigje@umdnj.edu
kholodvl@umdnj.edu
http://www2.umdnj.edu/~kerrigje
http://www2.umdnj.edu/~kholodvl

GRASP Tutorial

What is Grasp? GRASP stands for Graphical Representation and Analysis of Surface
Properties GRASP was developed by Barry Honigs research group at Columbia University.
You should cite the following paper whenever you use GRASP in a publication: Nicholls, A.;
Sharp, K.A.; Honig, B. Proteins, Struct. Funct. Gen. 1991, 11, 282.
For more information about GRASP visit http://trantor.bioc.columbia.edu/grasp/
What GRASP can do for you:
1. Provides high resolution display of solvent accessible surfaces.
2. Provides high resolution display of electrostatic potential mapped to surfaces (input from
esp charges in pdb file generated from sybyl or from Delphi files).
3. Reads pdb files, Delphi potential maps, Delphi charge files, and Delphi radius files.
4. Computes surfaces, surface areas and volumes.
In this tutorial, you will study the interaction between cefotaxime (a third generation
cephalosporin) and a carboxypeptidase/transpeptidase. See Kuzin, A.P. et al. Biochemistry 1995,
34, 9532.
OCH3
N

H2N

H
N

O
O

N
O

CH2OAc
O

Cefotaxime

First we must prepare the complex and compute charges on the protein. Enter the Sybyl
software suite (type sybyl at the winterm prompt then enter). NOTE: You may skip the Sybyl
portion of this tutorial if the starting cef_pep.pdb file is available. The Grasp tutorial begins on
page 5.
Biopolymer > Monomer Dictinoary > Open
Option > Amber95protein > OK
Biopolymer > PDB File > Get from FTP
STRING > 1CEF > OK
winterm > Password: Name (ftp.rcsb.org:your_username): Just hit enter here
You will see a message stating pdb1cef.ent retrieved successfully (Open this file)

GRASP Tutorial
You will also see an errant message in the sybyl window stating that the ftp was unsuccessful
(not correct!).
File > Read > Read File > Files: pdb1cef.ent > OK
Yes or No > Center the molecule > Yes
Extract the drug in to molecule area 2.
Build/Edit > Extract > Atom Expression > Sets > UNK_ATOMS > OK > OK
Molecule Area > M2:<empty> > OK
Name the molecules.
Build/Edit > Name Molecule > Molecule Area > M1: > OK
Name Molecule > Peptidase > OK
Build/Edit > Name Molecule > Molecule Area > M2: > OK
Name Molecule > cefotaxime > OK
Cleanup the protein: Remove water and the drug, add hydrogen atoms and charges.
Remove the water.
Build/Edit > Delete > Atom > Atom Expression > M1: > Sets > WATER > OK > Click OK
again
Remove the drug
Build/Edit > Delete > Atom > Atom Expression > M1: > Sets > UNK_ATOMS > OK > Click
OK again
Load the hydrogen atoms.
Biopolymer > Add Hydrogens > Sequence Expression > M1: > All > OK > Option > ALL >
OK
Load the charges.
Biopolymer > Load Charges > Atom Expression > M1: > All > OK > Option >
AMBER95_ALL > OK
Save the protein.

GRASP Tutorial

File > Save As > Save Molecule > m1: > Format: MOL2; File: pep > OK
Hide the protein and work on the drug.
View > Undisplay Atoms > Atom Expression > M1: > All > OK
M2:cefotaxime should be visible as one colour (red?).
Color the drug in CPK coloring (i.e. BY ATOM)
View > Color > BY_ATOM_TYPE > Molecule Expression > M2: > OK
Being that this was a structure of a covalent complex, we must do a little bit of work to
reconstruct the drug in its former glory before formation of the covalent complex. In other
words, our goal is to eventually build a model of the noncovalent complex.
Correct the existing atom types on the drug.
View > Label > Atom Type > Atom Expression > M2: > All > OK
To modify atom types.
Use Build/Edit > Modify Atom > Option > ONLY_TYPE > OK > Atom Expression > M2: (pick
the individual atoms to change with your mouse!)
Compute > Charges > Gasteiger-Huckel
Compute > Minimize >
Method: Conj Grad
Initial Optimization: Simplex
Termination: Gradient 0.10 kcal/mol*A
Max Iterations: 1000
Energy Setup: Force Field: Tripos
Charge: Use Current
Minimize hydrogen atoms only for the protein (pep.mol2).
Compute > Minimize >
Method: Powell
Initial Optimization: None
Termination: Gradient 0.05 kcal/mol*
Max Iterations: 1000

GRASP Tutorial

Energy Setup: Force Field: Tripos

Charge: Use Current
Under Modify > Energy > check Aggregates box
Define using Atom Expression > Atom Types > check the box by H > OK > Invert > OK
Name the aggregate NON_HYD
Comment: Non-hydrogen atoms
Merge the drug with the protein.
Build/Edit > Merge > Atom Expression > M2: cefotaxime > All > OK
Molecule Area > M1: Peptidase > OK
Build/Edit > Zap/Delete > Molecule Area > M2:
Delete all aggregates and minimize the complex.
Compute > Minimize >
Method: Powell
Initial Optimization: None
Termination: Gradient 0.50 kcal/mol* (Just to relieve bad van der Waals contacts)
Max Iterations: 400
Energy Setup: Force Field: Tripos
Charge: Use Current
Save the file as a mol2 file and call it cef_pep.mol2.
In addition, save the file as a Brookhaven (PDB) file and call it cef_pep.pdb.
We will use the pdb file for analysis with GRASP. Just download cef_pep.pdb from the course
webpage if you do not want to go through the setup and minimization in Sybyl.
************ Here is where the actual GRASP Tutorial Begins *******************
Before you begin, you must add the following lines to the PDB file (Note: These lines must be
the first two lines of the file!). (Note: These lines are already included in the cef_pep.pdb file
provided for this tutorial.)
GRASP PDB FILE
FORMAT NUMBER=3
You have told Grasp to read columns 55-60 and 61-67 and store them general properties 1 and 2.

GRASP Tutorial
In addition, note that the drug residue name is CEF.
At the unix prompt in the winterm, type grasp and enter.
A blank screen appears with a blue (horizontal) and green (vertical) crosshair.
Right click anywhere in the grasp screen with the mouse. A menu will appear.
Right click on Read in the menu > then right click on PDB File > right click on Show List >
right click on cef_pep.pdb

The Grasp user interface. The Grasp main menu only displays when you click the viewing area
(black background) with the right mouse button. Make all selections from this menu using the
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GRASP Tutorial
right mouse button. The textport (labeled in picture above) is the white background region at the
bottom of the user interface.
You may find that molecule appeared on a screen as a series of small circles connected to each
other rather than lines. To change the view to the line mode
click with the right mouse button anywhere in GRASP window
from the menu choose Display > Hide ALL to hide all atoms
and then again
Display > Show > Bonds > YES!

Mouse Controls:
The structure appears in lines format. You may rotate the structure using your mouse and the left
mouse button. Be patient, the response may be slow.
Hold down the left and middle mouse buttons to translate the structure.
Hold down the middle mouse button to zoom in and out. Use a vertical up and down motion.
IMPORTANT! Rotate/translate the molecule so as to view the active site and zoom in to the
active site before continue on the tutorial.
From the Menu under Miscellaneous Select toggle crosshairs on and off to remove the
crosshairs.
Make a subset for the protein:
From the menu select Formal Subsets. Under Formal Subset Operations Select Make a
Formal Subset.
Make Formal Subset from Atoms > Enter Specification > In the textport type an=(1,5161)
(means select atom numbers 1 through 5161) then hit the enter key. Accept M1 as the formal
subset name.
Make a formal subset for the drug:
From the menu select Formal Subsets. Under Formal Subset Operations Select Make a
Formal Subset.
Make Formal Subset from Atoms > Enter Specification > In the text box type r=cef (means
select residue name cef) then hit the enter key. Accept M2 as the formal subset name.
What you have done is designate M2 as the formal subset for the drug. To maintain Global
control do the following:

GRASP Tutorial
Formal Subsets > Fix dials to the World
Building the surface using Panels: From the menu select Panels. From the Panels menu select
Surfaces > Build a Surface
Under Type select Accessible then > in Calculate on select A Subset of Atoms Subset > in
the Formal Subset section type M1 (hit Enter) then click on Build a Surface in the Subset menu.
(Quit out of the menus)
If you do not see any surfaces on a screen it is because we hid everything at the beginning with
Hide ALL command. Turn surfaces on:
from the menu choose
Display > Show > Molecular Surfaces > YES!
As you can notice a graphical interface in GRASP is very slow. Try to avoid it where possible.
You can build a surface with the command instead.
Delete all Surfaces
Delete > Surfaces > All Surfaces
If you dont want to use the panels, just use the following method to construct a surface on the
protein:
Build > Accessible Surface > A Formal Subset > m1
To display ESP charges mapped to the surface: Calculate: Simple Property Math: Map Atom
Value to Surface: All Surfaces : All atoms : General Property #2: Potential
Then go to Panels > Surfaces
in Color section: Potentials
in Visualization Style section choose Rendered (Quit all panels)
To Display the drug neatly as Rods, Go to Display > Show > Bonds > No, Let me set them >
User defined > Rods
You will notice that the display of the small molecule is broken in places. This is normal.
Unfortunately Grasp is not very good at displaying small molecules.
CAUTION! Changing the mode of presenting bonds to the Rods will slow down GRASP
significantly. Use it only when you need to make a nice picture of your complex and then change
everything back to the Lines mode for further work on the tutorial!!!
To save the picture as an RGB Snapshot file: Right click on the grasp screen and from the grasp
menu select Write : RGB/Snapshot File. Give the file a name (cef_pep_sas.rgb) and hit enter.
NOTE: Use imgview in the SGI MediaTools to save the rgb file in a format (*.bmp, *.tif, *.png,
etc.) that is preferred by programs like photoshop or macromedia fireworks on your PC.
Change bonds back to Lines mode

GRASP Tutorial
Display > Show > Bonds > No, Let me set them > User defined > Lines
Look at the potential map. Areas in red are regions of negative potential and areas in blue are
regions of positive potential.

AMBER 95 esp charges mapped to Water (probe radius = 1.4 ) Accessible Surface area.
Notice that solvent accessible surfaces are not necessarily the best for viewing a drug in the
active site cavity.
Delete all surfaces.
from the menu choose Delete > Surfaces > All Surfaces

GRASP Tutorial

View of the drug in the active site. AMBER 95 charges mapped to a molecular surface.
The molecular surface is better for viewing the drug in the active site. The negatively charged
carboxylate is pointing towards a region of positive charge (blue).
For generation molecular surface use
Build > Molecular Surface > Formal Subset > m1
To display ESP charges mapped to the surface: Calculate: Simple Property Math: Map Atom
Value to Surface: All Surfaces : All atoms : General Property #2: Potential
Then visualize ESP mapped surface if needed
Panels > Surfaces
in Color section: Potential (if it is on Potential and you still do not see color click on Discrete
Colors and then on Potential again)
in Visualization Style section choose Rendered
Build surfaces for the drug using the same procedure (select m2).
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Delete all surfaces: Delete > Surfaces > All Surfaces

Compute areas and volumes:
Remember every time when you calculate an Area or a Volume of the Surface you have to
generate a Surface of a proper type (Molecular or Accessible). After calculations for each
individual surface have been finished, delete all surfaces to avoid an overlapping with other
types of surfaces and an accidental summation for double volumes/areas. Perform all
calculations separately for the protein and for the drug.
Compute the molecular area for the protein:
Build > Molecular Surface > Formal Subset > m1
Calculate > Area of a Surface/Molecule > Molecular Surface > A Surface Formal Subset
>m1
In the console window of GRASP read the output
10647.78 Square Angstroms
Calculate the volume of this Surface
Calculate > Volume of a Surface/Molecule > Molecular Surface > A Surface Formal Subset
> m1
In the console window of GRASP read the output
43760.75 Cubic Angstroms
Repeat for the drug and perform the similar calculations for Accessible Surfaces.
Fill in the following:
Area of Accessible Surface 1 (protein)

__________________2

Area of Accessible Surface 2 (drug)

__________________2

Area of Molecular Surface 1 (protein)

________________2

Area of Molecular Surface 2 (drug)

________________2

Volume of Accessible Surface 1 (protein)

________________3

Volume of Accessible Surface 2 (drug)

________________3

Volume of Molecular Surface 1 (protein)

________________3

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GRASP Tutorial
Volume of Molecular Surface 2 (drug)

________________3

How to calculate the Occluded Accessible SA. The occluded SA gives the contact area
between two individual surfaces.
Use the following commands in the textport: c=1, c=2,sub=m1, c=2,sub=m2. Commands
in quotes are separate commands. What you have done is represent all atoms as color #1 and the
individual subsets m1 & m2 as color #2. Therefore, the m1 + m2 combo will not be colored 1
and is represented by c=-1 (the minus sign means not). See the Grasp Manual.
Calculate > Area of a Surface/Molecule > Molecule > Accessible Area > Enter String and
type c=-1.
Output in the console window of GRASP is 12757.73 2 A_(m1+m2)
From here
Occluded SA = SUM (A_m1 + A_m2) A_(m1+m2)
A_m1 Accessible area of the protein from your table
A_m2 - Accessible area of the drug from your table
Occluded Accessible SA

________________2

How to analyze regular PDB files using a Delphi charge file. Lets start from scratch.
Read > PDB (read in the cef_pep.pdb file)
To change the view to the line mode
click with the right mouse button anywhere in GRASP window
from the menu choose Display > Hide > Atoms to hide all atoms
and then again
Display > Show > Bonds > YES!
Toggle off the crosshairs!
From the menu select Formal Subsets. Under Formal Subset Operations Select Make a
Formal Subset.
Make Formal Subset from Atoms > Enter Specification > In the textport type an=(1,5161)
(means select atom numbers 1 through 5161). Accept M1 as the formal subset name.
Make the formal subset for the drug as you did previously.
Formal Subset > Atoms >Enter Specification > In the textport type r=cef

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GRASP Tutorial

Formal Subsets > Fix Dials to the World

Read > Radius/Charge File (+Assign) > Let me enter the file name > In the textport type
amber.crg (Hit Enter)
Select amber.crg from the drop down list. This assigns AMBER charges to all amino acids of
the protein. The drug is ignored.
Build a Surface and map the curvature
Build > Molecular Surface > Formal Subset > m1
Display > Show > Molecular Surfaces > YES!
Use Calculate > Surface Curvature (+Display) > A Constructed Surface > Surface 1 > A
Formal Subset > m1
to calculate and display a molecular surface color-coded by its curvature.

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GRASP Tutorial
Surface Curvature map for molecular surface. The shading in the curvature map gives a better
sense of depth and surface curvature (hence its name). Good for visualizing pockets.
Build a Potential Map
Calculate > New Potential Map
Now map to the surface
Calculate > Pot. Via Map at Surfaces/Atoms > A Formal Subset > m1 > A Constructed
Surface > Surface 1
Note the difference between the potential map and the partial atomic charge map that you
calculated earlier
Now display the electrostatic field
Calculate > Field lines > Surface > All surfaces > Bidirectional > Specify > Red Enter 200
in the textport (for line density) and hit Enter
You will see lines (red) extending out from the protein. These lines represent the extent of the
electrostatic field about the protein.
APPENDIX
Codes used for manipulation of surfaces etc. via the textport.

c=2 will set all atoms to color No 2 (red). If your display shows only atoms or bonds,
nothing visible happens.
bc=2 colors all bonds (in this case to red)
vc=2 colors all surfaces (i.e., vertices)
kc=2 colors all backbone boxes

The more common codes are used as follows:

a = atom
an = atom number
r = residue
rn = residue number
ch = chain name

an=84 selects atom 84

an=(50,80) selects atoms 50,51,...80
rn=(1,7) selects first seven residues
ch=a selects chain a
a=C5M selects all atoms named C5M (case INsensitive)
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a=ba selects all backbone atoms

a=sch selects all sidechain atoms
r=pro selects all atoms in all the proline residues
r=crg selects all charged residues
r=hyd selects all hydrophobic residues
r=pol selects all polar residues

Preceding a selection with - negates it. Listing selections consecutively, separated by commas
performs the operation. E.g., rn=(5,15),a=sch,-r=ala selects all sidechain atoms (a = sch) on
residues from 5 to 15 unless the residue is alanine.
For our molecular surfaces of our drug and protein, these two sets of individual commands
produced the following image
vc=5, sub=m1
vc=7, sub=m2

15