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    Tutorial for the GRASP Software Package

    Authors:John E. Kerrigan, Ph.D.Vladyslav Kholodovych, Ph.D.

    University of Medicine & Dentistry of New JerseyRobert Wood Johnson Medical School675 Hoes LanePiscataway, NJ 08854 U.S.A.(732) 235-4473 phone(732) 235-3229 phone(732) 235-5252 [email protected]

    [email protected]://www2.umdnj.edu/~kerrigjehttp://www2.umdnj.edu/~kholodvl

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    What is Grasp? GRASP stands for Graphical Representation and Analysis of SurfaceProperties 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 generationcephalosporin) and a carboxypeptidase/transpeptidase. See Kuzin, A.P. et al.Biochemistry1995,34, 9532.

    N

    S

    O CH2OAc

    N H

    H

    O

    NN

    S

    H2N

    OCH3

    Cefotaxime

    OO

    First we must prepare the complex and compute charges on the protein. Enter the Sybylsoftware suite (type sybyl at the wintermprompt then enter). NOTE: You may skip the Sybylportion of this tutorial if the starting cef_pep.pdbfile is available. The Grasp tutorial begins onpage 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)

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    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: > 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 OKagain

    Remove the drug

    Build/Edit > Delete > Atom > Atom Expression > M1: > Sets > UNK_ATOMS > OK > ClickOK 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.

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    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 toreconstruct the drug in its former glory before formation of the covalent complex. In otherwords, 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: (pickthe individual atoms to change with your mouse!)

    Compute > Charges > Gasteiger-Huckel

    Compute > Minimize >

    Method: Conj Grad Initial Optimization: SimplexTermination: Gradient 0.10 kcal/mol*A

    Max Iterations: 1000

    Energy Setup: Force Field: TriposCharge: Use Current

    Minimize hydrogen atoms only for the protein (pep.mol2).

    Compute > Minimize >

    Method: Powell Initial Optimization: NoneTermination: Gradient 0.05 kcal/mol*

    Max Iterations: 1000

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    Energy Setup: Force Field: Tripos

    Charge: Use CurrentUnder Modify > Energy > check Aggregates box

    Define using Atom Expression > Atom Types > check the box by H > OK > Invert > OK

    Name the aggregate NON_HYDComment: Non-hydrogen atoms

    Merge the drug with the protein.

    Build/Edit > Merge > Atom Expression > M2: cefotaxime > All > OKMolecule Area > M1: Peptidase > OK

    Build/Edit > Zap/Delete > Molecule Area > M2:

    Delete all aggregates and minimize the complex.

    Compute > Minimize >

    Method: Powell Initial Optimization: NoneTermination: Gradient 0.50 kcal/mol* (Just to relieve bad van der Waals contacts)

    Max Iterations: 400

    Energy Setup: Force Field: TriposCharge: 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 coursewebpage 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 bethe first two lines of the file!). (Note: These lines are already included in the cef_pep.pdb fileprovided for this tutorial.)

    GRASP PDB FILEFORMAT NUMBER=3

    You have told Grasp to read columns 55-60 and 61-67 and store them general properties 1 and 2.

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    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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    right mouse button. The textport (labeled in picture above) is the white background region at thebottom of the user interface.

    You may find that molecule appeared on a screen as a series of small circles connected to eachother rather than lines. To change the view to the line mode

    click with the right mouse button anywhere in GRASP windowfrom the menu choose Display > Hide ALLto hide all atomsand then againDisplay > Show > Bonds > YES!

    Mouse Controls:

    The structure appears in lines format. You may rotatethe structure using your mouse and the leftmouse button. Be patient, the response may be slow.

    Hold down the left and middle mouse buttons to translatethe structure.

    Hold down the middle mouse button tozoomin 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 theactive site before continue on the tutorial.

    From the Menu under MiscellaneousSelect toggle crosshairs on and off to remove thecrosshairs.

    Make a subset for the protein:

    From the menu select Formal Subsets. Under Formal Subset Operations Select Make aFormal 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 formalsubset name.

    Make a formal subset for the drug:

    From the menu select Formal Subsets. Under Formal Subset Operations Select Make aFormal Subset.

    Make Formal Subset from Atoms > Enter Specification > In the text box type r=cef(meansselect 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 Globalcontrol do the following:

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    Formal Subsets > Fix dials to the World

    Building the surface using Panels: From the menu select Panels. From the Panels menu selectSurfaces > Build a Surface

    Under Type select Accessible then > in Calculate on select A Subset of Atoms Subset > inthe 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 withHide ALLcommand. Turn surfaces on:from the menu chooseDisplay > 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 SurfacesDelete > Surfaces > All Surfaces

    If you dont want to use the panels, just use the following method to construct a surface on theprotein:Build > Accessible Surface > A Formal Subset > m1

    To display ESP charges mapped to the surface: Calculate: Simple Property Math: Map AtomValue to Surface: All Surfaces : All atoms : General Property #2: Potential

    Then go to Panels > Surfacesin Color section: Potentialsin Visualization Stylesection 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 GRASPsignificantly. Use it only when you need to make a nice picture of your complex and then changeeverything 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 graspmenu select Write : RGB/Snapshot File. Give the file a name (cef_pep_sas.rgb) and hit enter.NOTE: Use imgviewin 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

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    Display > Show > Bonds > No, Let me set them > User defined > Lines

    Look at the potential map. Areas in redare regions of negative potential and areas inblueareregions 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 theactive site cavity.

    Delete all surfaces.from the menu choose Delete > Surfaces > All Surfaces

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    View of the drug in the active site. AMBER 95 charges mapped to a molecular surface.

    The molecular surfaceis better for viewing the drug in the active site. The negatively chargedcarboxylate is pointing towards a region of positive charge (blue).

    For generation molecular surface useBuild > Molecular Surface > Formal Subset > m1

    To display ESP charges mapped to the surface: Calculate: Simple Property Math: Map AtomValue to Surface: All Surfaces : All atoms : General Property #2: Potential

    Then visualize ESP mapped surface if neededPanels > Surfaces

    in Color section: Potential (if it is on Potential and you still do not see color click on DiscreteColors 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 togenerate a Surface of a proper type (Molecular or Accessible). After calculations for eachindividual surface have been finished, delete all surfaces to avoid an overlapping with othertypes of surfaces and an accidental summation for double volumes/areas. Perform allcalculations 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 output10647.78 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 output43760.75 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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    Volume of Molecular Surface 2 (drug) ________________3

    How to calculate the Occluded Accessible SA. The occluded SA gives the contact areabetween two individual surfaces.

    Use the following commands in the textport: c=1, c=2,sub=m1, c=2,sub=m2. Commandsin quotes are separate commands. What you have done is represent all atoms as color #1 and theindividual subsets m1 & m2 as color #2. Therefore, the m1 + m2 combo will not be colored 1and 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 andtype 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 tableA_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.pdbfile)

    To change the view to the line modeclick with the right mouse button anywhere in GRASP windowfrom the menu choose Display > Hide > Atomsto hide all atomsand then againDisplay > Show > Bonds > YES!

    Toggle off the crosshairs!

    From the menu select Formal Subsets. Under Formal Subset Operations Select Make aFormal 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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    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 ofthe 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 > AFormal Subset > m1

    to calculate and display a molecular surface color-coded by its curvature.

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    Surface Curvature map for molecular surface. The shading in the curvature map gives a bettersense 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 youcalculated 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 theelectrostatic field about the protein.

    APPENDIX

    Codes used for manipulation of surfaces etc. via the textport.

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

    The more common codes are used as follows:

    a = atoman = atom numberr = residue

    rn = residue numberch = chain name

    an=84selects atom 84 an=(50,80)selects atoms 50,51,...80 rn=(1,7)selects first seven residues ch=aselects chain a a=C5Mselects all atoms named C5M (case INsensitive)

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    a=baselects all backbone atoms a=schselects all sidechain atoms r=proselects all atoms in all the proline residues r=crgselects all charged residues r=hydselects all hydrophobic residues r=polselects all polar residues

    Preceding a selection with - negates it. Listing selections consecutively, separated by commasperforms the operation. E.g., rn=(5,15),a=sch,-r=ala selects all sidechain atoms (a = sch) onresidues from 5 to 15 unless the residue is alanine.

    For our molecular surfaces of our drug and protein, these two sets of individual commandsproduced the following image

    vc=5, sub=m1

    vc=7, sub=m2