autodock2_4_userguide

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AutoDock

Automated Docking of Flexible Ligands to Receptors

Version 2.4

Garrett M. Morris, David S. Goodsell, & Ruth Huey

Arthur J. Olson

User Guide

2.4


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June 3, 1996

Constructive comments, suggestions, feedback and bug reports are welcome; please e-mail me. -Garrett Morris.

E-mail addresses:

Arthur J. Olson, Ph.D.: [email protected] Graber,Administrative Assistant: [email protected] M. Morris, M.A., D.Phil. [email protected] S. Goodsell, Ph.D. [email protected]

Ruth Huey, Ph.D. [email protected]

FAX: (619) 554-6860

AutoDock, Copyright 1994, 1995

The Scripps Research Institute,

Department of Molecular Biology,Mail Drop MB-5,

10666 North Torrey Pines Road,

La Jolla,

CA 92037-5025, USA.

Modification date: June 3, 1996 12:45 pm


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June 3, 1996

1. Contents

Introduction to AutoDock 4

Introduction 4Overview of the Method 4Applications 6

AutoDock, AutoGrid and AutoTors 8

Background 8

Electrostatic Potential Grid Maps 14Simulated Annealing in AutoDock 15Setting Up AutoGrid and AutoDock Jobs 16Preparing the Small Molecule 16Modelling Hydrogen Bonds 17Small Molecule Flexibility and Constraints 17Input for AutoTors 19Running AutoTors 21Adding Polar Hydrogens to the Macromolecule 21Getting Started... 22AutoGrid Parameter File Format 22

Running AutoGrid 26AutoDock Parameter File Format 27Running AutoDock 36Using the Command Mode in AutoDock 37Trajectory Files 39Evaluating the Results of a Docking 40Visualizing Grid Maps and Trajectories Using AVS 41Shell Scripts and Auxilliary Tools 42Parameters from AutoDock Version 1 48


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AutoDock 2.4 User Guide

4

Introduction to AutoDock

1. Introduction

The program AutoDock was developed to provide a procedure for predicting the interaction ofsmall molecules with macromolecular targets. The motivation for this work arises from problemsin the design of bioactive compounds, and in particular the field of computer aided drug design.Progress in biomolecular x-ray crystallography has provided a number of important protein andnucleic acid structures that could be targets for bioactive agents in the control of disease, or asagricultural agents. The precise interaction of such agents or candidates is important in the devel-opment process. Our goal has been to provide a computational tool to aid in this process.

In any docking scheme two conflicting requirements must be balanced: the desire for a robust and

accurate procedure, and the desire to keep the computational demands at a reasonable level. Theideal procedure would find the global minimum in the interaction energy between the substrateand the target protein, exploring all available degrees of freedom for the system. However, itwould also run on a laboratory workstation within an amount of time comparable to other compu-tations that a structural researcher may undertake, such as a crystallographic refinement. In orderto meet these demands a number of docking techniques simplify the docking procedure. Probablythe most common technique in use today is manually assisted docking. Here, the internal and ori-entational degrees of freedom in the substrate are under interactive control. While the energy eval-uation for such techniques can be sophisticated, the exploration of configurational space islimited. At the other end of the spectrum are automated methods such as exhaustive search anddistance geometry. These methods can explore configurational space, but at the cost of a much

simplified model for the energetic evaluation. The procedure developed for AutoDock uses aMonte Carlo simulated annealing technique for configurational exploration with a rapid energyevaluation using grid based molecular affinity potentials, thus combining the advantages of a largesearch space and a robust energy evaluation. This has proven to be a powerful approach to theproblem of docking a flexible substrate into the binding site of a static protein. Input to the proce-dure is minimal. The researcher specifies a volume around the protein, the rotatable bonds for thesubstrate, and an arbitrary starting configuration, and the procedure produces a relatively unbiaseddocking.

2. Overview of the Method

Rapid energy evaluation is achieved by precalculating atomic affinity potentials for each atomtype in the substrate molecule in the manner described by Goodford 1. In the AutoGrid procedurethe protein is embedded in a three-dimensional grid and a probe atom is placed at each grid point.

1. Goodford, P.J. (1985) A Computational Procedure for Determining Energetically Favorable BindingSites on Biologically Important MacromoleculesJ. Med. Chem., 28, 849-857.


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The energy of interaction of this single atom with the protein is assigned to the grid point. Anaffinity grid is calculated for each type of atom in the substrate, typically carbon, oxygen, nitrogenand hydrogen, as well as a grid of electrostatic potential, either using a point charge of +1 as theprobe, or using a Poisson-Boltzmann finite difference method, such as DELPHI 2,3. The energeticsof a particular substrate configuration is then found by tri-linear interpolation of affinity values ofthe eight grid points surrounding each of the atoms in the substrate. The electrostatic interaction isevaluated similarly, by interpolating the values of the electrostatic potential and multiplying bythe charge on the atom (the electrostatic term is evaluated separately to allow finer control of thesubstrate atomic charges). The energy calculation using the grids is proportional only to the num-ber of atoms in the substrate, and not to any function of the number of atoms in the protein.

The docking simulation is carried out using theMetropolis method, also known asMonte Carlosimulated annealing. With the protein static throughout the simulation, the substrate moleculeperforms a random walk in the space around the protein. At each step in the simulation, a smallrandom displacement is applied to each of the degrees of freedom of the substrate: translation ofits center of gravity; orientation; and rotation around each of its flexible internal dihedral angles.

This displacement results in a new configuration, whose energy is evaluated using the grid inter-polation procedure described above. This new energy is compared to the energy of the precedingstep. If the new energy is lower, the new configuration is immediately accepted. If the new energyis higher, then the configuration is accepted or rejected based upon a probability expressiondependent on a user defined temperature, T. The probability of acceptance is given by:

where Eis the difference in energy from the previous step, and kB is the Boltzmann constant. Athigh enough temperatures, almost all steps are accepted. At lower temperatures, fewer high

energy structures are accepted.

The simulation proceeds as a series of cycles, each at a specified temperature. Each cycle containsa large number of individual steps, accepting or rejecting the steps based upon the current temper-ature. After a specified number of acceptances or rejections, the next cycle begins with a tempera-ture lowered by a specified schedule such as:

where Ti is the temperature at cycle i, and g is a constant between 0 and 1.

Simulated annealing allows an efficient exploration of the complex configurational space withmultiple minima that is typical of a docking problem. The separation of the calculation of themolecular affinity grids from the docking simulation provides a modularity to the procedure,allowing the exploration of a range of representations of molecular interactions, from constantdielectrics to finite difference methods and from standard 12-6 potential functions to distributions

2. Sharp, K., Fine, R. & Honig, B. (1987) Science, 236, 1460-1463.3. Allison, S.A., Bacquet, R.J., & McCammon, J. (1988)Biopolymers, 27, 251-269.

P E( ) e

E

kBT---------

=

Ti

gTi 1=


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based on observed binding sites.

3. Applications

AutoDock was initially tested on a number of protein-substrate complexes which had been char-acterized by x-ray crystallography 4. These tests included phosphocholine binding in a antibodycombining site, N-formyltryptophan binding to chymotrypsin and N-acetylglucosamine bindingto Lysozyme. In almost all cases the results of the AutoDock simulations functionally reproducedthe crystallographic complexes. In further applications AutoDock was used to predict interactionsof substrates with aconitase prior to any crystallographic structures for complexes. In this workwe not only predicted the binding mode of isocitrate, but we demonstrated the utility ofAutoDock in generating substrate models during the early stages of crystallographic proteinsstructure refinement 5. Citrate docking experiments showed two binding modes, one of whichapproximated the experimental electron density determined for an aconitase-nitrocitrate complex.

The docking simulation results provided insight into the proposed reaction mechanism of theenzyme.

The initial version ofAutoDock has been distributed to over 35 sites around the world, and hasbegun to be used in universities and research labs to help predict and design bioactive agents. Onenovel and intriguing use of the software was reported from Koshlands laboratory6. These investi-

gators used the known structures of the maltose-binding protein (MBP) and the ligand bindingdomain of the aspartate receptor to predict the structure of the receptor-protein complex (see dia-

4. Goodsell, D.S. & Olson, A.J. (1990) Automated Docking of Substrates to Proteins by Simulated Anneal-ing Proteins: Str. Func. Genet., 8, 195-202.5. Goodsell, D.S., Lauble, H., Stout, C.D & Olson, A.J. (1993) Automated Docking in Crystallography:Analysis of the Substrates of Aconitase Proteins: Str. Func. Genet., 17, 1-10.

6. Stoddard, B.L. & Koshland, D.E. (1992) Prediction of a receptor protein complex using abinary docking method.Nature, 358 (6389), 774-776.

MBPreceptor

?


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gram below). They used knowledge from mutational studies on MBP to select two octapeptides

on the protein known to be involved in the binding to the aspartate receptor, which they docked

independently to the model of the receptor using our automated docking code (the backbones ofthe peptides were fixed, but the side-chain conformations and overall orientations were unre-strained).

The distance and orientation of the two peptides as docked to the receptor corresponded to that inthe intact MBP, thus enabling a reasonable prediction of the protein-receptor complex. This tech-nique could be generally useful in situations where there are data on multi-site interactions.

MBPMBP octapeptides

receptor AutoDockreceptor

AutoDock

receptor

superimpose

MBP MBP+

MBP/receptor complex

receptorMBP +superimpose


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8

AutoDock, AutoGrid and AutoTors

This User Guide describes how to prepare, run and analyze an automated docking of a small mol-ecule to a macromolecule, such as a protein, enzyme or oligomeric DNA, using AutoDock.

1. Background

AutoDock requires grid maps for each atom type present in the small molecule being docked.They are calculated and produced by the AutoGrid program. A grid map consists of a threedimensional array of regularly spaced points, centered (usually) on the active site of the protein ormacromolecule under study. Each point within the grid map stores the potential energy of aprobe atom or functional group at that particular position:

The user must specify an even number for nx, ny and nz, since AutoGrid adds a central point, andAutoDock requires an odd number of grid points. The probes energy at each grid point is deter-mined by the set of parameters supplied for that particular atom type, summed over all atoms ofthe macromolecule, within a non-bonded radius. This potential energy, V(r), can be expressed as a

ny+1

nx+1

nz+1

grid spacing /

grid point

probe atom


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function of internuclear separation, r, as follows,

Graphically, ifreqm is the equilibrium internuclear separation, and is the well depth at reqm, then:

The exchange energy is often approximated thus,

Hence pairwise-atomic interaction energies can be approximated by the following general equa-tion,

V r( )Ae

br

r------------

C6

r6

------=

r

V(r)

+Ae-br/rrepulsive, exchange energy

attractive, dispersion energy -C6/r6

reqm

0

reqm

A

r---e br

C12

r12

--------


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where m and n are integers, and Cn and Cm are constants whose values depend on the depth of the

energy well and the equilibrium separation of the two atoms nuclei. Typically the 12-6 Lennard-Jones parameters (n=12, m=6) are used to model the Van der Waals forces1 experienced betweentwo instantaneous dipoles. However, the 12-10 form of this expression (n=12, m=10) can be usedto model hydrogen bonds (see Modelling Hydrogen Bonds below). Appendix 21 gives theparameters which were distributed with the first (FORTRAN-77) version of AutoDock, and whichhave been used in numerous published articles.

A revised set of parameters has been calculated, which are self-consistent. Here, the sum of theVan der Waals radii of any given pair of atoms is consistent with that of any other pair. Likwise,the well-depths are consistently related. Let reqm, XXbe the equilibrium separation between two likeatomsX, and XX be their potential energy in this configuration. The combining rules for the well

depth, , and the Van der Waals radius, reqm, for two different atomsXand Yare:

A derivation for the Lennard-Jones potential sometimes seen in text books invokes the parameter,, thus,

Then the Lennard-Jones 12-6 potential becomes:

Hence, the coefficients C12 and C6are given by:

We can derive a general relationship between the coefficients, equilibrium separation and welldepth as follows. At the equilibrium separation, reqm, the potential energy is a minimum: in otherwords, V(reqm) = -. The derivative of the potential will be zero at the minimum:

1. van der Waals, J. H. (1908) Lehrbuch der Thermodynamik, Mass and Van Suchtelen, Leipzig, Part 1

V r( )Cn

rn

------Cm

rm

------ Cnrn

Cmrm=

XY XXYY=

reqm,XY1

2--- reqm,XX reqm,YY+( )=

reqm, XY 2

1

6---

=

V12-6 r( ) 4XY

r---

12 r---

6=

C12 XYreqm, XY12

=

C6 2XYreqm, XY6

=


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therefore:

so:

Substituting Cm into the original equation for V(r), at equilibrium, we obtain,

Rearranging:

Therefore, the coefficient Cn can be expressed in terms ofn, m, and reqm thus:

and, substituting into original equation for V(r),

In summary, then:

Some example reqm and parameters for various AMBER atom types of carbon are shown in Table

rd

dV nCn

rn 1+

-----------mCm

rm 1+

------------+ 0= =

nCn

rn 1+

-----------mCm

rm 1+

------------=

CmnCnr

m 1+

mrn 1+

---------------------n

m----Cnr

m n( )= =

Cn

reqmn--------

nCnreqmm n( )

mreqmm------------------------=

Cnmreqm

mnreqm

nreqm

m n( )

mreqmn

reqmm

----------------------------------------------

=

Cnm

n m--------------reqmn

=

Cmn

n m--------------reqm

m=

V r( )

m

n m--------------reqm

n

rn------------------------

n

n m--------------reqm

m

rm------------------------


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1.

Using the equations describing C12 and C6 above, the following new set of 12-6 parameters werecalculated (see Table 2). These parameters may be used with AutoDock version 2.4, or alterna-

tively, you may use or derive your own.

Table 1: AMBER parameters for carbon atom types.

AMBER atom typereqm

_--- /

/ kcal mol-1

C, C*, CA, CB, CC, CD, CE, CF, CG, CH, CI, CJ,CM, CN, CP

1.85 0.12

C2 1.925 0.12

C3 2.00 0.15

CH 1.85 0.09

CT 1.80 0.06

Table 2: Self-consistent Lennard-Jones 12-6 parameters.

Atoms i-j reqm,ij----------/

ij/ kcal mol-1

C12/ kcal mol-112

C6/ kcal mol-16

C-C 4.00 0.150 2516582.400 1228.800000

C-N 3.75 0.155 1198066.249 861.634784

C-O 3.60 0.173 820711.722 754.059521

C-S 4.00 0.173 2905899.052 1418.896022

C-H 3.00 0.055 29108.222 79.857949

N-C 3.75 0.155 1198066.249 861.634784

N-N 3.50 0.160 540675.281 588.245000

N-O 3.35 0.179 357365.541 505.677729

N-S 3.75 0.179 1383407.742 994.930149

N-H 2.75 0.057 10581.989 48.932922

O-C 3.60 0.173 820711.722 754.059521

O-N 3.35 0.179 357365.541 505.677729

O-O 3.20 0.200 230584.301 429.496730

O-S 3.60 0.200 947676.268 870.712934

O-H 2.60 0.063 6035.457 39.075098

S-C 4.00 0.173 2905899.052 1418.896022

S-N 3.75 0.179 1383407.742 994.930149


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The above parameters yield the following graphs, for C, N, O and H atom types; the curves in

order of increasing well-depth are: HH


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example, if the small molecule being docked is a hydrocarbon, then only carbon and hydrogengrid maps would be required. In practice, however, non-polar hydrogens would not be modelledexplicitly, so just the carbon grid map would be needed, for united atom carbons. This savesboth disk space and computational time.

2. Electrostatic Potential Grid Maps

In addition to the atomic affinity grid maps, AutoDock requires an electrostatic potential gridmap. Polar hydrogens must be added, if hydrogen-bonds are being modelled explicitly. Partialatomic charges must be assigned to the macromolecule. The electrostatic grid can be generated byAutoGrid, or by other programs such as MEAD2 or DELPHI3, which solve the linearized Pois-son-Boltzmann equation. AutoGrid calculates Coulombic interactions between the macromole-cule and a probe of charge e, +1.60219x10-19 C; there is no distance cuttoff used for electrostaticinteractions. A sigmoidal distance-dependent dielectric function is used to model solvent screen-

ing, based on the work of Mehler and Solmajer4

,

where: B = 0 - A; 0 = the dielectric constant of bulk water at 25C = 78.4; A = -8.5525, =0.003627 and k= 7.7839 are parameters.

Charges must be stored inPDBQformat in order for AutoGrid to read them. PDBQ is an aug-mented form of the standard PDB format, in which an extra column is used to store the partialatomic charges (hence the Q in PDBQ). Columns 71-76 of the PDB file hold the partialatomic charge (the older form of PDBQ contains charges in columns 55-61).

Charges can be assigned using a molecular modelling program. Unix shell scripts are provided toconvert from InsightII5 .car files (cartopdbq) and SYBYL6 .mol2 files (mol2topdbq).See also q.amber and q.kollua, in the appendices.

2. Bashford, D. and Gerwert, K. (1992) Electrostatic calculations of the pKa values of ionizable groups inbacteriorhodopsin,J. Mol. Biol., 224, 473-486; Bashford, D. and Karplus, M. (1990) pKas of ionizable

groups in proteins - atomic detail from a continuum electrostatic model.,Biochemistry, 29, 10219-10225;MEAD is available from Donald E. Bashford, Dept. Molecular Biology, Mail Drop MB1, The ScrippsResearch Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037.3. Gilson, M.K. and Honig, B. (1987)Nature, 330, 84-86; DELPHI is available from Biosym Technologies,9685 Scranton Road, San Diego, CA 92121-2777, USA.4. Mehler, E.L. and Solmajer, T. (1991) Electrostatic effects in proteins: comparison of dielectric andcharge models Protein Engineering, 4, 903-910.5. Biosym Technologies, 9685 Scranton Road, San Diego, CA 92121-2777, USA.6. Tripos Associates, Inc., 1699 South Hanley Road, Suite 303, St. Louis, Missouri 63144-2913, USA.

r( ) A B

1 keBr

+--------------------------+=


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3. Simulated Annealing in AutoDock

As already described in the Introduction, AutoDock uses Monte Carlo simulated annealing toexplore a wide range of conformational states. A job consists of a number of independent runs,each of which begins with the same initial conditions. A run is a sequence of constant temperatureannealing cycles. Each job can be seeded with a user-defined or a time-dependent random-numbergenerator seed.

When using the Monte Carlo approach, it is important to consider the algorithm for random num-ber generation. Ideally, the samples are uniform and uncorrelated in hyperdimensions; and theperiod is longer than the number of random values called for in a given simulation.

During each constant temperature cycle, random changes are made to the ligands current posi-tion, orientation, and conformation if flexibiliy was defined. The resulting new state is then com-

pared to its predecessor; if the new energy is lower than the last, this new state is accepted.However, if the new states energy is higher than the last, this state is accepted probabilistically.This probability depends upon the energy and cycle temperature (see the first equation on page 3).Generally speaking, at high temperatures, many states will be accepted, while at low tempera-tures, the majority of these probabilistic moves will be rejected. The user can select the minimumenergy state found during a cycle to be used as the initial state for the next cycle, or the last statecan be used. The best results tend to be achieved by selecting the minimum energy state from theprevious cycle.

Quaternion rotations7 have been implemented in handling the rigid body rotation of the smallmolecule. It was found that this gave finer control over the movement of the small molecule, and

gave better docked conformations than the alternative Eulerian rotations.It is advisable to do a short run to check the setup first. The initial annealing temperature shouldbe of the order of the average E found during the first cycle. This ensures that the ratio ofaccepted to rejected steps is high near the start. A typical automated docking or job may have aninitial annealing temperature of 500 and a temperature reduction factor of 0.85-0.95 /cycle. Grad-ual cooling is recommended, so as to avoid simulated quenching, which tends to trap systems inlocal minima. A relatively thorough search is given by 50Monte Carlo cycles, and a maximum of30,000 steps rejected or 30,000 steps accepted. 10 runs give a feel for the possible binding modes,and also an idea of their relative energies. When more than one run is carried out in a given job,cluster analysis or structure binning will be performed, based on structural rms difference, rank-

ing the resulting families of docked conformations in order of increasing energy. The defaultmethod for structure binning allows for symmetry rotations, as in a tertiary butyl which can berotated by +/-120, but in other cases it may be desirable to bypass this similar atom type checkingand calculate the rms on a one-for-one basis. A schedule of 100 runs, 50 cycles, 3,000 stepsaccepted, 3,000 steps rejected will provide more highly populated clusters, hinting at the densityof states for a given conformation. A short test job would be: 1 run, 50 cycles, 100 accepted, 100

7. Shoemake, K. (1985) Animating Rotation with Quaternion Curves SIGGRAPH 85, 19, 245-254.


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rejected steps.

The user must specify the maximum jump a state variable can make in oneMonte Carlo step. Thedefault values are: translation, 0.2 ; rigid-body rotation, 5; and torsion angle rotation, 5. Fur-thermore, these can be adjusted from cycle to cycle during each run, if the reduction factor fortranslations and rotations is given a (positive, non-zero) value less than one. At the start of eachcycle, this constant is multiplied onto the current jump-maxima to give the new maxima.

If desired, the states visited during a docking simulation can be sampled and output to a trajectoryfile. This file contains all the state variables required to define each sampled conformation, posi-tion and orientation of the small molecule. The user can specify the range of cycles to be sampled.This allows the selection of the last few cycles when the docking will be nearing the final dockedconformation, or the selection of the whole run.

4. Setting Up AutoGrid and AutoDock Jobs

Let us suppose that the user wishes to test AutoDock by trying to reproduce an x-ray crystallo-graphic structure of a small molecule-enzyme complex taken from the Brookhaven Protein DataBank. The first step is to create two PDB files, one containing all the heavy atoms of the enzyme,the second containing those of the small molecule. Both files should retain the extension .pdb.

Note: Care should be taken when the PDB file contains disordered residues, in which alternatelocation indicators (column 17) have been assigned; for each such residue, the user must selectonly one of the possible alternate locations (preferably that with the highest occupancy value).

We will discuss in the next sections, the steps needed to prepare the parameter files for AutoGridand AutoDock. If desired the user can specify rotatable bonds in the small molecule (receptorflexibility is not allowed). To help in the definition of rotatable bonds in the small molecule, thereis a tool called AutoTors, which prepares the small molecule input file for AutoDock, defining theroot, branches and torsions automatically.

5. Preparing the Small Molecule

Initially you must add hydrogens to all the heavy atoms in the small molecule, ensuring theirvalences are completed. This can be done using a molecular modelling package. Make sure thatthe atom types are correct before adding hydrogens.

Next, assign partial atomic charges to the molecule. AMPAC or MOPAC can be used to generatepartial atomic charges for the small molecule. Write these charges out in PDBQ format (seecartopdbq and mol2topdbq in Appendix 20).


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6. Modelling Hydrogen Bonds

Hydrogen bonds are often important in ligand binding. These interactions can be modelled explic-itly in AutoDock. Polar hydrogens can be allowed to rotate freely.

The partial atomic charges ofnon-polarhydrogens need to be unitedwith their heavy atoms. Thissaves having two types of hydrogen grids, thus conserving disk space and computational time. Tounite a non-polar hydrogens partial charge, the latter is added to that of the heavy atom to whichit is bonded. The hydrogen can then be deleted from the small molecule. This is repeated for allnon-polar hydrogens. AutoTors has a flag -h, which will unite non-polar hydrogens for you,automatically.

The user mustspecify the appropriate 12-10 parameters in the AutoGrid parameter file, and onthe correct lines. Pairwise atomic interaction energy parameters are always given in blocks of 7

lines, in the order: C, N, O, S, H, X, M. X and M are spare atom types: If there were phosphorusatoms in the receptor, X could be used as P. For example, to model donorhydrogens in the smallmolecule, 12-10 parameters would be needed in the hydrogen parameter block, but only for H-bond acceptors, N,O and S (second, third and fourth lines in the H-parameters). The other param-eters remain as 12-6 Lennard-Jones values (C,H,X and M). In order to keep the symmetry of pair-wise energetics (H-O is the same as O-H), the user must specify 12-10 parameters for H (fifthline) in the N, O and S-parameter blocks.

AutoGrid detects hydrogen bond parameters in the grid parameter file, if either n is not 12 or m isnot 6. If so, the pairwise interaction is modulated by a function of the cosine of the hydrogen bondangle. This takes into account the directionality of hydrogen-bonds.

7. Small Molecule Flexibility and Constraints

To allow flexibility in the small molecule, it is necessary to assign the rotatable bonds. It is a goodidea to have a plot of the small molecule, labelled by atom name, and a second labelled by atomserial number or ID. AutoDock can handle up to MAX_TORS rotatable bonds. This parameter isdefined in autodock.h, and is ordinarily set to 32.

Table 3: Self-consistent Hydrogen bonding 12-10 parameters.

Atoms i-j reqm,ij----------/

ij/ kcal mol-1

C12/ kcal mol-112

C10/ kcal mol-16

N-H 1.90 5.00 55332.873 18393.199

O-H 1.90 5.00 55332.873 18393.199

S-H 2.50 1.00 298023.224 57220.459


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Torsions are defined in the PDBQ file using a number ofkeywords. These keywords use the meta-phor of a tree. See the diagram below for an example. The root is defined as thefixedportion ofthe small molecule, off which rotatable branches sprout. Branches within branches are possible,and torsions are a special case of branches, where the two atoms at either end of the rotatable bondhave only two nearest neighbours (unlike branches which can have three or more). Rotatable frag-ments are moved in order from leaves to root.

The PDBQ keywords must be carefully placed, and the order of the ATOM or HETATM recordsmay need to be changed in order to fit into the correct branches. The following keywords are rec-ognized by the AutoDock PDBQ file parser:

ROOT, ENDROOT

BRANCH, ENDBRANCH

TORSION, ENDTORSION

CONSTRAIN

They can be abbreviated to no less than the first 4 letters. To assist the user in correctly placing

these keywords, and in re-ordering the ATOM or HETATM records in the small molecule PDBQfile, there is an interactive program called AutoTors to do this (see below).

Note: AutoTors, AutoGrid and AutoDock do not recognize PDB CONECT, neither do theywrite out CONECT records.

CONSTRAIN definesa single distance constraint, between two flexible parts of the small mole-cule. This retains only those conformations where this distance is within a certain range of values.In docking, a conformation which violates this constraint is instantly rejected; it does not incre-ment the rejections-counter, its energy is not evaluated, nor is the steps-counter incremented. Thecommand has the following syntax:

CONSTRAIN atom1 atom2 lower upper

The first two parameters are the atom serial numbers of the two atoms to be constrained, and thelast two are the lower and upper bounds for this distance, in Angstroms. This can be particularlyuseful when docking say two proteins: a loop from one protein can be cut out and the ends con-


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strained to have roughly the same value as in the original protein.

The next sections describe the input needed for AutoTors, and how to run it.

8. Input for AutoTors

This section describes input and output files used and generated by AutoTors. Input consists ofone or two files, depending on whether the small molecule is in our AutoDock-standard PDBQ-format, or in Sybyls mol2-format. PDBQ-format is the default; mol2-format is allowed with the-m flag (see below).

PDBQ-format:When using PDBQ format, AutoTors also needs a bond file. In this example, the bond file isoligo.bnd, and oligo.pdbq is the input PDBQ file; oligo.out.pdbq is created and contains allthe ROOT, BRANCH and TORS keywords needed to define the torsions selected by the user.

autotors oligo.bnd oligo.pdbq oligo.out.pdbq

The .bnd file, contains information about the covalent bonds in the small molecule. The bondsare described by the serial numbers of the atoms in the input PDBQ file, with one line per bond.For example, if C10 is the atom appearing on the first ATOM line in the PDBQ file, and it isbonded to N18 which appears on the 17th line in the PDBQ file, this information appears as a dis-crete line in the .bnd file as: 1 17. The output of the script pdbtoatm is an atm file; thiscan be converted to a bnd file, using atmtobnd. For example, to generate a bnd file, usesomething like this command:

+

O1

N2

N1

O3

O2H2

H21

H22

H12

H11

C1

C2 C3

C4

C5C6

C7

C8

C9

C10

H1

ROOT

BRAN

CH

TORS


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pdbtoatm vitc.pdbq | atmtobnd > vitc.bnd

Mol2-format:When using SYBYL-mol2 format, only one input file is required, in addition to -m flag. This isbecause the mol2 file contains both atom coordinates and bonding information. So, for eample the

following command would read in the lead.mol2 file and after interactively requesting whichtorsions to rotate, AutoTors would write out lead.out.pdbq:

autotors -m lead.mol2 lead.out.pdbq

AutoTors Output:The output filename is defined by the last AutoTors command-line argument. Output consists ofPDBQ-formatted lines, rearranged as required by AutoDock, according to the users specificationof the fixed ROOT portion of the molecule, and the allowed rotatable bonds in the rest of the mol-ecule. AutoTors inserts the ROOT, ENDROOT, BRANCH, ENDBRANCH, TORS, and END-TORS lines in the necessary places.

AutoTors Flags:-m

as described under Mol2-format above, this tells AutoTors to read in coordinates, partialcharges and bond information from a SYBYL-mol2 formatted file.

-h

merge or add the charges of non-polar hydrogen atoms to that of the carbon atom to which theyare bonded, and then delete these hydrogens from the molecule. In other words, the molecule is

converted from an all atom representation to a united atom representation. The net charge onthe molecule remains the same.

-o

read partial atomic charges from column 55 onwards (i.e. the olderPDBQ format). The default isthe new PDBQ format which has the charge data in the 71-st to 76-th columns.

-a

disallows torsions in amide bonds. This should normally be used, because amide bonds are par-tially conjugated and therefore cannot rotate freely.

-c

tells AutoTors to add a column at the end of each ATOM line, showing the number of bondednearest neighbours to each atom. If the -h flag is used as well, then the atoms with non-polarhydrogens are merged first, then the resulting merged structure is used to obtain this column ofatom connectivities.

The -m, -h and -a flag may appear in any order. The -o flag must be given after the -h flag.


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Placement of these flags should follow these two examples. Square brackets denote optional flags:

autotors [-h][-o][-a][-c] peptid.bnd peptid.pdbq peptid.out.pdbq

For SYBYL-mol2 input:

autotors -m [-h][-o][-a][-c] drug.mol2 drug.out.pdbq

9. Running AutoTors

There are two interactive phases in running AutoTors:

1) Root selection: After all the bonding data is read in and any cycles detected in the small mole-cule, the user designates which (adjacent) atoms are to be considered the ROOT. If cycles aredetected in the molecule, the first part of designating the ROOT is to select a cycle for the ROOT

by number orno cycle by entering 0. In either case the user next has the opportunity of enteringspecific atoms by number (after entering a on a menu provided) or quitting (selection q) whenall the desired root atoms are entered. (At this point it is not necessary to specify all of the rootatoms desired because the root will be expanded to include all atoms not in a BRANCH orTORS.)

2) Torsion selection: After the root has been specified, the program goes through the data andmakes a list of possible torsions. These are listed as possible torsions and the user next must editthis list as desired. For example, if all the possible torsions detected are to be used, the user simplyselects q and quits the torsions selection section. If a small number of the possible torsions are tobe eliminated, the user selects d and deletes the unwanted torsions. He stops deleting by enteringq. If only a few of the possible torsions are desired, it is possible to select only these few(instead of eliminating a large number of them) by selecting s from the menu. This selectionprocess is also ended by entering q.

Once either phase is ended by entering q, it is not possible to change what has been selected.Instead, the user should abort the program with -C and start again.

MAX_TORS: AutoDock is set up to allow a maximum number of torsions. IfAutoTors detectsmore torsions than are permitted, a warning to that effect is given and it is up to the user to reducethe number of torsions, either by deleting or selecting the appropriate number of torsions.MAX_TORS is defined in the file autodock.h; if this definition is changed, the autodock-executable must be re-made, using the appropriate Makefile.

10. Adding Polar Hydrogens to the Macromolecule

When modelling hydrogen bonds explicitly, it is necessary to add polar hydrogens to the macro-


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molecule also. Then the appropriate partial atomic charges can be assigned. This can be achievedby the users preferred method, e.g. using InsightII, Quanta, Sybyl, AMBER or CHARMm.Alternatively, one of the shell scripts described in the Appendix can be used. The charged macro-molecule must be converted to PDBQ format so that AutoGrid can read it.

Note that most modelling systems add polar hydrogens in a default orientation, typically assum-ing each new torsion angle is 0 or 180. Without some form of refinement, this can lead to spuri-ous locations for hydrogen-bonds. One option is to relax the hydrogens and perform a molecularmechanics minization on the structure. Another is to use a program like pol_h which takes asinput the default-added polar hydrogen structure, samples favourable locations for each movableproton, and selects the best position for each. This intelligent placement of movable polarhydrogens can be particularly important for tyrosines, serines and threonines.

11. Getting Started...

There are several Unix scripts available to help prepare default AutoGrid and AutoDock parame-ter files. They are described in more detail in the Appendix; see prepare, prepareII, prepare-gpf+dpf and prepare-dpf. The user must check these defaults, to ensure they look reasonable.The user can adjust the default parameters according to what is required. Each parameter isdescribed in the sections AutoGrid Parameter File Format and AutoDock Parameter File For-mat.

12. AutoGrid Parameter File Format

The input file is often referred to as a grid parameter file or GPF for short. The scriptsdescribed in the appendices give these files the extension .gpf. In the grid parameter file, theuser must specify the following spatial attributes of the grid maps:

1. the center of the grid map;2. the number of grid points in each of thex-,y- andz-directions; and3. the separation or spacing of each grid point.

In addition, the pairwise-atomic interaction energy parameters must be specified. The followinglines are required for each small molecule atom type, Y:

4. the grid map filename for atom type Y;5. seven lines containing the non-bonded parameters for each pairwise-atomic interaction, inthe following order: Y-C, Y-N, Y-O, Y-S, Y-H, Y-X, (Xis any other atom type) and Y-M(Mis a metal,say).


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Using coefficients Cn, Cm, n and m, the pairwise interaction energy, V(r) is given by:

Alternatively, the user can specify reqm, , n and m. By default, the Y-Xand Y-Mlines are copies ofthe Y-H line. But in some systems, such as receptors which consist of DNA/protein complexes,both sulphur andphosphorus can be present. In this scenario, the Y-Xline can be used for model-ling interactions with receptor-phosphorus atoms. A very rough approximation for phosphorusparameters is to borrow those of carbon.

The elecmap line in the grid parameter file is the filename of the electrostatic potential gridmap. The following parameter, dielectric, if negative, indicates that the distance-dependentdielectric function of Mehler and Solmajer3 will be used. If positive, however, the value of thatnumber will be used as a constant dielectric. For example, if the value were 40.0, then a constantdielectric of 40 would be used.

The AutoGrid parameter file format is described below. The type of each argument is describedusing C-style, %s = a character string; %d = a (decimal) integer; and, %f = a floating pointor real number.

AutoGrid Keywordsreceptor %s

Macromolecule filename, in PDBQ format.

gridfld %s

The grid field filename, which will be written in a format readable by AutoDock and AVS8. Thefilename extension mustbe .fld.

npts %d %d%d

Number ofx-,y- andz-grid points. Each mustbe an even integer number. When added to the cen-tral grid point, there will be an odd number of points in each dimension. The number ofx-,y- andz-grid points need not be equal.

spacing %f

The grid point spacing, in (see the diagram on page 8). Grid points must be uniformly spaced inAutoDock: this value is used in each dimension.

gridcenter %f %f %f

gridcenter auto

8. AVS stands for Application Visualization System; AVS is a trademark of Advanced Visual SystemsInc., 300 Fifth Avenue, Waltham, MA 02154.

V r( )Cn

rn

------Cm

rm

------


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The user can explicitly define the center of the grid maps, respectively thex,y andz coordinates ofthe center of the grid maps (units: , , .) Or the keyword auto can be given, in which caseAutoGrid will center the grid maps on the center of mass of the macromolecule.

types %s

1-letter names of the atom types present in the small molecule; e.g. if there are carbons, nitrogens,oxygens and hydrogens, then this line will be CNOH; there are no delimiters.

map %s

Filename of the grid map, for ligand atom type Y; the extension is usually .map.

nbp_coeffs %f %f %d %d

Either nbp_coeffs or nbp_r_eps keywords can be used to define Lennard-Jones or hydrogenbond interaction energy parameters. The keyword nbp_coeffs specifies coefficients and expo-nents, in the order CnCmnm, delimited by spaces; n and m are integer exponents. The units of

Cn and Cm must be kcal mol-1

n

and kcal mol-1

m

respectively; n and m have no units.nbp_r_eps %f %f %d %d

Alternatively, the user can employ nbp_r_eps to specify the equilibrium distance and welldepth, epsilon, for the atom pair. The equilibrium separation has units of and the well depth,epsilon, units of kcal mol-1. The integer exponents n and m must be specified too.

In either case, the order of the parameters must be: Y-C, Y-N, Y-O, Y-S, Y-H, Y-X, and Y-M. Repeat 1map line and the 7 nbp_coeffsor nbp_r_eps lines, for each atom type, Y, present in the smallmolecule being docked.

elecmap %s

Filename for the electrostatic potential energy grid map to be created; filename extension .map.

dielctric %f

Dielectric funtion flag: if negative, AutoGrid will use distance-dependent dielectric of Mehlerand Solmajer3; if the float is positive, AutoGrid will use this value as the dielectric constant.

fmap %s

(Optional.) Filename for the so-called floating grid map9; filename extension .map. In suchfloating grids, the scalar at each grid point is the distance to the nearest atom in the receptor. These

values could be used to guide the docking ligand towards the receptors surface, thus avoidingnon-interesting, empty regions.

9. This grid map is not used in AutoDock 2.4; its utility is under investigation, and may be included in a laterversion.


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Example AutoGrid Parameter FileAn example AutoGrid parameter file is given below:

______________________________________________________________________________

receptor 3ptb.pdbq #macromoleculegridfld 3ptb.maps.fld #grid.data.file

npts 60 60 60 #numxyzpoints

spacing .375 #spacing/Angstroms

gridcenter -1.930 14.070 16.224#center_of_grids or auto

types CNH #atom.type.names

map 3ptb.C.map #atomic.affinity.map

nbp_r_eps 4.00 0.1500 12 6#C-C non-bond Rij & epsilonij

nbp_r_eps 3.75 0.1549 12 6#C-N non-bond Rij & epsilonij

nbp_r_eps 3.60 0.1732 12 6#C-O non-bond Rij & epsilonij

nbp_r_eps 4.00 0.1732 12 6#C-S non-bond Rij & epsilonij

nbp_r_eps 3.00 0.0548 12 6#C-H non-bond Rij & epsilonij



map 3ptb.N.map #atomic.affinity.mapnbp_r_eps 3.75 0.1549 12 6#N-C non-bond Rij & epsilonij

nbp_r_eps 3.50 0.1600 12 6#N-N non-bond Rij & epsilonij

nbp_r_eps 3.35 0.1789 12 6#N-O non-bond Rij & epsilonij

nbp_r_eps 3.75 0.1789 12 6#N-S non-bond Rij & epsilonij

nbp_r_eps 1.90 5.0000 12 10#N-H non-bond Rij & epsilonij



map 3ptb.H.map #atomic.affinity.map

nbp_r_eps 3.00 0.0548 12 6#H-C non-bond Rij & epsilonij

nbp_r_eps 1.90 5.0000 12 10#H-N non-bond Rij & epsilonij

nbp_r_eps 1.90 5.0000 12 10#H-O non-bond Rij & epsilonij

nbp_r_eps 2.50 1.0000 12 10#H-S non-bond Rij & epsilonij

nbp_r_eps 2.00 0.0200 12 6#H-H non-bond Rij & epsilonij

nbp_r_eps 2.00 0.0200 12 6#H-H non-bond Rij & epsilonijnbp_r_eps 2.00 0.0200 12 6#H-H non-bond Rij & epsilonij

elecmap 3ptb.e.map #electrostatic.PE.map

dielectric -1. #distance-dep.diel=-1,constant>0

#fmap 3ptb.f.map #floating.grid

_________________________________________________________________

Note how hydrogen bonding is defined for oxygens. The ideal hydrogen bond would have anangle, , of 180 between the acceptor, the polar hydrogen and the donor, thus:

O H Ndonor

acceptor

probe

macromolecule


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As decreases, the strength of the hydrogen bond diminishes. There are no hydrogen bonds when is 90 or less.

If a line in the parameter file contains a 10 in the fourth column, AutoGrid will treat this atom-pair as hydrogen bonding. So in the example above, the last 3 lines in the mcp2_O.map blockwill be treated as hydrogen bonds. AutoGrid scans for any polar hydrogens in the macromole-cule. The vector from the hydrogen-donor, along with the vector from the probe-atom at the cur-rent grid point, are used to calculate the directional attenuation of the hydrogen bond. In thisexample, AutoGrid will calculate H-bonds between O-H, O-Xand O-M.

13. Running AutoGrid

AutoGrid requires an input grid parameter file, which usually has the extension .gpf. The com-mand is issued as follows:

% autogrid -p molecule.gpf -l molecule.glg &

where -p molecule.gpf specifies the grid parameter file, and -l molecule.glg the log file outputduring the grid calculation. The & ensures that the this job will be run in the background. Thiswhole line can be prefixed with the nice command to ensure other processes are not undulyaffected. The log file will inform the user of the maximum and minimum energies found duringthe grid calculations.AutoGrid writes out the grid maps in ASCII form, for readability and porta-bility; AutoDock expects ASCII format grid maps. The first six lines of each grid map hold headerinformation which describe the spatial features of the maps and the files used or created. Theseheaders are checked by AutoDock to ensure that they are appropriate for the requested docking.The remainder of the file contains grid point energies, written as floating point numbers, one perline. They are ordered according to the nested loops z( y( x ) ). A sample header is shown below:

______________________________________________________________________________

GRID_PARAMETER_FILE vac1.nbc.gpf

GRID_DATA_FILE 4phv.nbc_maps.fld

MACROMOLECULE 4phv.new.pdbq

SPACING 0.375

NELEMENTS 50 50 80

CENTER -0.026 4.353 -0.038

125.095596

123.634560

116.724602

108.233879

:

______________________________________________________________________________

As well as the grid maps, AutoGrid creates two AVS-readable files, with the extensions .fld, and.xyz. The former is a field file summarizing the grid maps, and the latter describes the spatialextent of the grids in Cartesian space. (To read the grid maps into AVS, use a read field module.)


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The -o flag can be used on the AutoGrid command line to signify that the .pdbq file specifiedin the grid parameter file is in old PDBQ format (charges are stored in columns 55-61).

14. AutoDock Parameter File Format

AutoDock 2.4 has a completely new and upgraded interface, based on keywords. This new inter-face is intended to make it easier for the user to set up and control a docking job, and for the pro-grammer to add new commands and functionality. The input file is often referred to as a dockingparameter file or DPF for short. The scripts described in the appendices give these files theextension .dpf.

Note: All delimiters where needed are white spaces. Default values, where applicable, are given insquare brackets [thus]. A comment must be prefixed by the # symbol, and can be placed at theend of a parameter line, or on a line of its own. Once again, the type of each keyword argument is

described using the C-standard, where:

%s = is an alphanumeric string, and in most cases, a valid filename;%d = is a decimal integer; and%f = is a floating point or real number.

Although in theory it should be possible to give these keywords in any order, not every possiblecombination has been tested, so it may be wise to stick to the following order.

AutoDock Keywordsseed %ld

seed time

Each job can be seeded with either a user-defined or a time-dependent random-number generatorseed. The first form explicitly defines the seed-value for the random number generation. The sec-ond uses a keyword, time, to obtain the number of seconds since the epoch. The epoch is refer-enced to 00:00:00 CUT (Coordinated Universal Time) 1 Jan 1970.

types %s

Atom names for all atom types present in small molecule. Each must be a single character, andonly one of: C, N, O, S, H, X, or M. The maximum number of characters allowed in this line isATOM_MAPS, which is defined in the autodock.h include file. Do not use any spaces to delimit

the types: they are not needed.

fld %s

Grid data field file created by AutoGrid and readable by AVS (must have the extension .fld).

map %s

Filename for the first AutoGrid affinity grid map of the 1st atom type. This keyword plus filename


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must be repeated for all atom types in the order specifed by the types command. In all map filesa 6-line header is required, and energies must be ordered according to the nested loops z( y( x ) ).

map %s

Filename for the electrostatics grid map. 6-line header required, and energies must be ordered

according to the nested loops z( y( x ) ).

move %s

Filename for the ligand to be docked. This contains most importantly, atom names, xyz-coordi-nates, anb partial atomic charges in PDBQ format. (Filename extension should be .pdbq).

about %f %f %f

Use this keyword to specify the center of the ligand, aboutwhich rotations will be made. (Thecoordinate frame of reference is that of the ligand PDBQ file.) Usually the rotation center of theligand is the meanx,y,z-coordinates of the molecule. Inside AutoDock, the about xyz-coordi-

nates are subtracted from each atoms coordinates in the input PDBQ file. So internally, theligands coordinates become centered at the origin. Units: , , .

tran0 %f %f %f

tran0 random

Initial coordinates for the center of the ligand, in the same frame of reference as the receptors gridmaps. The ligand, which has been internally centered using the about coordinates, has the xyz-coordinates of the initial translation tran0 x y z addedon. Every run starts the ligand from thislocation.

Alternatively, the user can just give the keyword random and AutoDock will pick random initial

coordinates instead.

If there are multiple runs defined in this file, using the keyword runs, then each new run willbegin at this same location.

The user mustspecify the absolute starting coordinates for the ligand, used to start each run. Theuser should ensure that the small molecule, when translated to these coordinates, still fits withinthe volume of the grid maps. If there are some atoms which lie outside the grid volume, thenAutoDock will automatically correct this, until the small molecule is pulled completely within thevolume of the grids. (This is necessary in order to obtain complete information about the energyof the initial state of the system.) The user will be notified of any such changes to the initial trans-

lation by AutoDock. (Units: , , .)

quat0 %f %f %f %f

quat0 random

Respectively: Qx, Qy, Qz, Qw. Initial quaternion (applied to small molecule) - Qx, Qy, Qz define theunit vector of the direction of rigid body rotation [1, 0, 0], and Qw defines the angle of rotation


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about this unit vector, in [0]. (Units: none,none,none, .)

Alternatively, the user can just give the keyword random and AutoDock will pick a random unitvector and a random rotation (between 0 and 360) about this unit vector. Each run will begin atthis same random rigid body rotation.

ndihe %d

Number of dihedrals or rotatable bonds in the small molecule. This may be specifed only if rotat-

able bonds have been defined using ROOT, BRANCH, TORS etc. keywords in the .pdbq filenamed on the move line. If this keyword is used, then the next keyword, dihe0, must also bespecified. Note that ifndihe and dihe0 are not specified and there are defined torsions in theligand PDBQ file, AutoDock assumes that the chi1, chi2, chi3, etc. are all zero, and does notchange the initial ligand torsion angles.

dihe0 %f %f %f ...

Initial relative dihedral angles; there must be ndihe floating point numbers specified on thisline. Each value specified here will be added to the corresponding torsion angle in the inputPDBQ file, at the start of each run. Torsion angles are only specified by two atoms, so the defini-tion of rotations is relative. Units: .

tstep %f

Maximum translation step [0.2 ]. Units: .

qstep %f

Maximum quaternion rotation step [5.]. Units: .

dstep %f

Maximum dihedral step [5.]. Units: .

trnrf %f

Per-cycle reduction factor for translations [1.].

quarf %f

Per-cycle reduction factor for quaternions [1.].

A quaternion.

Qx

Qy

Qz

Qw


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dihrf %f

Per-cycle reduction factor for dihedrals [1.].

barrier %f

(Optional) This defines the energy-barrier height applied to constrained torsions. When the tor-

sion is at a preferred angle, there is no torsion penalty: this torsions energy is zero. If the torsionangle falls within a disallowed zone, however, it can contribute up to the full barrier energy. Sincethe torsion-energy profiles are stored internally as arrays of type unsigned short, only positiveintegers between 0 and 65535 are allowed. [10000].

gausstorcon %d %f %f

(Optional) Adds a constraint to a torsion. The torsion number is identified by an integer. Thisidentifier comes from the list at the top of the AutoTors-generated input ligand PDBQ file (on theREMARK lines). An energy profile will be calculated for this torsion. An inverted Gaussian bellcurve is added for each new constraint. To completely specify each Gaussian, two floating point

numbers are needed: the preferred angle and the half-width respectively (both in degrees). Notethat the preferred angle should be specified in the range -180 to +180; numbers outside thisrange will be wrapped back into this range. This angle, , isrelative to the original torsion anglein the input structure. The half-width is the difference between the two angles at which the energyis half the barrier (B/2 in the diagram above). The smaller the half-width, the tighter the con-straint.

If you wish to constrain to absolute-valued torsion angles, it will be neccessary to zero the initialtorsion angles in the ligand, before input to AutoTors. The problem arises from the ambiguous 2-atom definition of the rotatable bond B-C. To identify a torsion angle unambiguously, 4 atoms

Energy/kcal

mol-1

0-180 180, preferred angle

B

0

B/2

Torsion angle /

B, barrier/ kcal mol-1

+ve -ve

0constraint

half-width

torsion


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must be specified:A-B-C-D:

The sign convention for torsion angles which we use is anti-clockwise (counter-clockwise) arepositive angles, clockwise negative. In the above diagram, looking down the bond B-C, the dihe-dral angleA-B-C-D would be positive.

There is no limit to the number of constraints that can be added to a given torsion. Each new tor-sion-constraint energy profile is combined with the pre-exisiting one by selecting the minimumenergy of either the new or the existing profiles.

showtorpen

(Optional) (Use only with gausstorcon) This switches on the storage and subsequent output oftorsion energies. During each energy evaluation, the penalty energy for each constrained torsion,as specified by the gausstorcon command, will be stored in an array. At the end of each run, thefinal docked conformations state variables are output, but with this command, the penalty energyfor each torsion will be printed alongside its torsion angle.

hardtorcon %d %f %f

(Optional) This command also adds a torsion constraint to the %d-th torsion, as numbered in theAutoTors-generated REMARKs. The first float defines the preferred relative angle, and the sec-ond specifies thefull width of the allowed range of torsion angles (both in degrees). This type oftorsion constraint is hard because the torsion is never allowed to take values beyond the rangedefined. For example, hardtorcon 3 60. 10. would constrain the third torsion to values between55 and 65.

intnbp_coeffs %f %f %d %d

Respectively: Cn; Cm; n; m. These are the internal pairwise non-bonded energy parameters for

flexible ligands, where:

These parameters are needed even if no rotatable bonds were defined in the ligand-PDBQ file.They are only used in the internal energy calculations for the ligand and must be consistent with

A

D

A

B

D

looking down the B-C bond

+veC

B

V r( )Cn

rn

------Cm

rm

------


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those used in calculating the grid maps. (Units: kcal mol -1 n; kcal mol-1 m; none; none, respec-tively)

intelec

(Optional) Internal electrostatic energies will be calculated; the products of the partial charges in

each non-bonded atom pair are pre-calculated, and output. Note that this is only relevant for flexi-ble ligands.

rt0 %f

Initial annealing temperature; this is actually the absolute temperature multiplied by the gasconstantR. [500. cal mol-1].R = 8.314 J mol-1 K-1 = 1.987 cal mol-1 K-1. (Units: cal mol-1.)

rtrf %f

Annealing temperature reduction factor, g [0.95 cycle-1]. See the equation at the bottom of page 5.At the end of each cycle, the annealing temperature is multiplied by this factor, to give that of the

next cycle. This must be positive but < 1 in order to cool the system. Gradual cooling is recom-mended, so as to avoid simulated quenching, which tends to trap systems into local minima.

linear_schedule

schedule_linear

linsched

schedlin

These keywords are all synonymous, and instruct AutoDock to use a linear or arithmetic temper-ature reduction schedule during simulated annealing. Unless this keyword is given, a geometricreduction schedule is used, according to the rtrf parameter just described. If the linear scheduleis requested, then any rtrf parameters will be ignored. The first simulated annealing cycle is

carried out at the annealing temperature rt0. At the end of each cycle, the temperature is reducedby (rt0/cycles). The advantage of the linear schedule is that the system samples evenly acrossthe temperature axis, which is vital in entropic calculations. Geometric temperature reductionschedules on the other hand, under-sample high temperatures and over-sample low temperatures.

runs %d

Number of automated docking runs [1].

cycles %d

Number of temperature reduction cycles [50].

accs %d

Maximum number of accepted steps per cycle [100].

rejs %d

Maximum number of rejected steps per cycle [100].


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select %d

State selection flag. This can be eithermfor the minimum state, or l for the laststate found duringeach cycle, to begin the following cycle [m].

outlev %d

Diagnostic output level. 0 = no output, 1 = minimal output, 2 = full state output at end of eachcycle; 3 = detailed output for each step. [1].

rmstol %f

RMS deviation tolerance for cluster analysis or structure binning [0.5], carried out after multi-ple docking runs. If two conformations have an RMS less than this tolerance, they will be placedin the same cluster. The structures are ranked by energy, as are the clusters. The lowest energy rep-resentative from each cluster is output in PDBQ format to the log file. To keep the original residuenumber of the input ligand PDBQ file, use the -k flag; otherwise the cluster-representatives arenumbered incrementally from 1. (Units: ).

rmsnosym

When more than one run is carried out in a given job, cluster analysis or structure binning willbe performed, based on structural rms difference, ranking the resulting families of docked confor-mations in order of increasing energy. The default method for structure binning allows for atomsimilarity, as in a tertiary-butyl which can be rotated by +/-120, but in other cases it may be desir-able to bypass this similar atom type checking and calculate the rms on a one-for-one basis. Thesymmetry checking algorithm scans all atoms in the reference structure, and selects the nearestatom of identical atom type to be added to the sum of squares of distances. This works well whenthe two conformations are very similar, but this assumption breaks down when the two conforma-tions are translated significantly. Symmetry checking can be turned off using the rmsnosym

command; omit this command if you still want symmetry checking.

trjfrq %d

Output frequency, n, for trajectory of small molecule, in steps [0]. If n = 0, then no trajectorystates will be output; otherwise, every nth state will be output. The state consists of 7 floatsdescribing the x,y,z translation, the x,y,z components of the quaternion unit vector, the angle ofrotation about the quaternion axis; and any remaining floats describing the torsions, in the sameorder as described in the input small molecule PDBQ file).

trjbeg %d

Begin sampling states for trajectory output at this cycle. [1]

trjend %d

End trajectory output at this cycle.[50].

trjout %s

Trajectory filename [.trj]. AutoDock will write out state variables to this file every


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trjfrq steps. Use the traj command in AutoDocks command mode to convert this trajectory ofstate-variables into a series of PDB frames. The traj command is described in Using theCommand Mode in AutoDock; see also Trajectory Files.

trjsel %s

Trajectory output flag, can be either A or E; the former outputs only acceptedsteps, while thelatter outputs eitheraccepted or rejected steps.

watch %s

Optional) Creates a watch file for real-time monitoring of an in-progress simulatedannealing job. This works only if the trjfrq parameter is greater than zero.

The watch file will be in PDB format, so give a .pdb extension. This file has an exclusive lockplaced on it, while AutoDock is writing to it. Once the file is closed, the file is unlocked. This cansignal to a watching visualization program that the file is complete and can now be read in, forupdating the displayed coordinates. This file is written at exactly the same time as the trajectory

file is updated

extnrg %f

External grid energy [1000.] assigned to any atoms that stray outside the volume of the grid dur-ing a docking. Units: kcal mol-1.

rmsref %s

The RMS deviation of any conformations generated during the docking will be calculated bycomparing the coordinates in the file specified by this command. This tends to be useful when theexperimentally determined complex structure is known. The order of the atoms in the PDB file

specified by this command must match that in the input PDBQ file given by themove command.These values of RMS will be output in the last column of the final PDBQ records, after the clus-tering has been performed.

cluster %s

(Clustering multi-job output only.)AutoDock will go into cluster mode. Use this commandonly to perform cluster analysis on the combined output, , of several jobs. Thiscommand can be very useful when many jobs have been distributed to several machines and runin parallel. The docking parameter file will need the following keywords: rmstol and types;and optionally write_all_cluster_members and/or rmsnosym. It is necessary to grep theREMARKS along with the ATOM records, since AutoDock parses the REMARKS to determinewhat the energy of that particular conformation was. See the second example .dpf below.

write_all_cluster_members

(Custering multi-job output only.) This command is used only with the cluster command, towrite out all members of each cluster instead of just the lowest energy from each cluster. Thisaffects the cluster analysis PDBQ output at the end of each job.


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Example AutoDock Parameter FileAn example of a commented AutoDock parameter file is given below:

______________________________________________________________________________

seed randomtypes CNOH # atom type names

fld 4phv.nbc_maps.fld# grid data file

map 4phv.nbc_C.map # C-atomic affinity map

map 4phv.nbc_N.map # N-atomic affinity map

map 4phv.nbc_O.map # O-atomic affinity map

map 4phv.nbc_H.map # H-atomic affinity map

map 4phv.nbc_e.map # electrostatics map

move xk263pm3.pdbq # small molecule

about -5.452 -8.626 -0.082 # small molecule center

tran0 -5.452 -8.626 -0.082 # initial coordinates/A

quat0 1. 0. 0. 0. # initial quaternion:unit-vector(qx,qy,qz);angle/deg(qw)

ndihe 10 # number of rotatable bonds

dihe0 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. # initial dihedrals/degtstep 0.2 # translation step/A

qstep 5. # quaternion step/deg

dstep 5. # torsion step/deg

trnrf 1. # trans reduction factor/per cycle

quarf 1. # quat reduction factor/per cycle

dihrf 1. # tors reduction factor/per cycle

intnbp 1272653.000 1127.684 12 6 # C-C internal energy non-bond parameters/Cn,Cm,n,m

intnbp 610155.100 783.345 12 6 # C-N internal energy non-bond parameters/Cn,Cm,n,m

intnbp 588883.800 633.754 12 6 # C-O internal energy non-bond parameters/Cn,Cm,n,m

intnbp 88604.240 226.910 12 6 # C-H internal energy non-bond parameters/Cn,Cm,n,m

intnbp 266862.200 546.765 12 6 # N-N internal energy non-bond parameters/Cn,Cm,n,m

intnbp 249961.400 445.918 12 6 # N-O internal energy non-bond parameters/Cn,Cm,n,m

intnbp 39093.660 155.983 12 6 # N-H internal energy non-bond parameters/Cn,Cm,n,mintnbp 230584.400 368.677 12 6 # O-O internal energy non-bond parameters/Cn,Cm,n,m

intnbp 38919.640 124.049 12 6 # O-H internal energy non-bond parameters/Cn,Cm,n,m

intnbp 1908.578 46.738 12 6 # H-H internal energy non-bond parameters/Cn,Cm,n,m

rt0 500. # initial RT

rtrf 0.95 # RT reduction factor/per cycle

runs 10 # number of runs

cycles 50 # cycles

accs 100 # steps accepted

rejs 100 # steps rejected

select m # minimum or last

outlev 1 # diagnostic output level

rmstol 0.5 # cluster tolerance/A

#rmsnosym # no symmetry checking in RMS calc.

trjfrq 7500 # trajectory frequency

trjbeg 45 # start trj output at cycle

trjend 50 # end trj output at cycle

trjout xk263pm3.trj # trajectory file

trjsel E # A=acc only;E=either acc or rej


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extnrg 0.0 # external grid energy

______________________________________________________________________________

In this case, the small molecule file xk263pm3.pdbq has been defined such that it contains 10rotatable bonds. The docking will be sampled every 7500 steps, from cycle 45 to cycle 50. Eitheraccepted or rejected states will be output. The trajectory file xk263pm3.trj will hold thestate information required to generate the coordinates later on. The external grid energy is set to0.0, which can allow greater freedom for ligand rotations during docking.

The next example .dpf shows how to use the cluster mode in AutoDock. The PDBQ files con-taining the final docked conformations have been extracted from the .dlgAutoDock log files(using the UNIX grep command), and stored together in vac1.new.nrg.pdb. The toler-ance for the RMS deviation is set to 1.5, so only conformations with this RMS or less will begrouped into the same cluster. All cluster members will be written out, instead of just the lowestenergy representative of each.

______________________________________________________________________________

types CNOH # atom_type_names

rmstol 1.5 # cluster_tolerance/A

write_all_cluster_members

cluster vac1.new.nrg.pdb # structure binning

______________________________________________________________________________

15. Running AutoDock

Once the grid maps have been prepared by AutoGrid, and the docking parameter file is ready, theuser is ready to run an AutoDock job. A docking is initiated using the following command:

autodock [-o][-k][-i][-u][-t] -p molecule.dpf [-l molecule.dlg] &

Input parameters are specified by -p molecule.dpf, and the log file containing results ofthe docking is defined by -l molecule.dlg. This is the normal usage ofAutoDock, andperforms a standard docking calculation.

-o

can be added to the command line, to signify that the input file specified in the docking parameterfile is in oldPDBQ format, with charges in columns 55-61.

-k

keep the original residue number of the input ligand PDBQ file. Normally AutoDock re-numbersthe starting position to residue-number 0, and any cluster-representatives are numbered incremen-tally from 1, according to their rank (rank 1 is the lowest energy cluster).


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-i

used to ignore any grid map header errors that may arise due to conflicting filenames. This over-rides the header checking that is normally performed to ensure compatible grid maps are beingused.

-u

returns a message describing the command line usage ofAutoDock.

-t

instructs AutoDock to parse the PDBQ file to check the torsion definitions, and then stop.

The Unix script job can be used to submit an AutoDock job, and then perform additional post-processing, such as profiling, extracting job-information and creating a field file for AVS displayof the docked results. See the Appendix for more details.

16. Using the Command Mode in AutoDock

AutoDock can be run in command mode, using the -c flag thus:

% autodock -p molecule.dpf -l molecule.clg -c &

When AutoDock has read in the grid maps specified in molecule.dpf, the program givesthe message COMMAND MODE and waits for the user to issue a command from the standardinput. These commands are described in more detail below. An alternative method of using thecommand mode is to edit a file containing the commands you wish AutoDock to execute (com-

mand.file) and channel the output to a file (command.output), thus:

% autodock -p molecule.dpf -l molecule.clg -c < command.file >

command.output &

AutoDock can be used in a UNIX pipe command. This is valuable when an alternative search pro-cedure is desired. Here, alt_search_proc issues commands to the standard output, andreads the results from the standard input. In this case, AutoDock is behaving as an energy serverfor alt_search_proc, the alternative search-procedure program.

There are eight recognized commands: AutoDocks command interpreter is not case sensitive.

eval Evaluate this states total energy.epdb Evaluate the energy of the named PDBQ file.outc Output the last states PDB-formatted Cartesian coordinates.oute Output (non-bond and electrostatic) energy breakdown, by atom.traj Convert trajectory file to PDB-formatted Cartesian coordinates.stop, exit, quit Stop the AutoDock command mode interpreter.


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EvalEvaluates the total energy of a state defined by the subsequent state variables. This command uti-lizes the trilinear interpolation routine in AutoDock along with the supplied grid maps defined inthe parameter file specified after the -p flag to return this energy. The internal energy of thesmall molecule is also taken into account, as dictated by the values of the torsion angles supplied

in the ntorlines following this eval command line; ntoris the number of torsion angles definedin the small molecule PDBQ file, as described in the section Defining Torsions in AutoDock.The usage of this command is:

eval %f %f %f %f %f %f %f} Tx, Ty, Tz, Qx, Qy, Qz, Qw (in )

%f } ith torsion angle, in .

:: ntorlines.

where: Tx, Ty, Tz are the coordinates of the center of rotation of the small molecule; Qx, Qy, Qz is theunit vector describing the direction of rigid body rotation, about which a rotation of angle Qw

degrees will be applied. The following ntorlines hold the torsion angles in degrees, given in thesame order as described in the AutoDock log file.

EpdbCalculates the energy of the molecule provided in the PDBQ file, thus:

epdb filename.pdbq

where: filename.pdbq is the PDBQ formatted coordinates of a molecule for which the interac-tion energy with the macromolecule will be returned. The -o flag supplied at the AutoDock exe-cution line specifies the old format of PDBQ, with charges in columns 55-61; otherwise it is

assumed that the charges are in columns 71-76.

This command is useful when the state variables for a given molecule are not known, e.g. the x-ray crystal conformation of the small molecule.

OutcReturns the coordinates of the small molecule at its current transformed position (in the form of aPDB REMARK). Thex,y,z coordinates will be determined by the state variables supplied to theeval command.

OuteReturns the total internal energy of the small molecule and the total energy of the complex, at thecurrent state variables. These two REMARK lines are written in PDB format, to the commandoutput channel and the log file.


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TrajConvert a .trj file into PDBQ format. Usage:

traj filename.trj

where filename.trj is the trajectory file written out by an earlier run ofAutoDock. This trajec-tory file contains the state variables for the states sampled during the docking simulation. The tor-sions are assumed to be in exactly the same order as the input ligand PDBQ file. The torsionangles in the trajectory file are relative to the the latters conformation.

See also the Appendix 20, script runtrj; and the next section, Trajectory Files.

Stop, Exit, QuitHalts the execution of AutoDock. A value of 0 is returned by the program, and the messageautodock: Successful Completion is written to the log file and standard error. Timing informa-tion is also written. Note: stop, exit and quit are synonymous.

17. Trajectory Files

A trajectory (of state variables) can be written out during a normal docking simulation, if the tra-jectory-frequency (set by the keyword trjfrq) in the docking parameter file is greater than zero.This value defines the output frequency, in steps, for states sampled during the run. The defaulttrajectory filename extension is .trj . These state variables are all that is needed to regenerate thecoordinates of the small molecule. The trajectory control parameter (either A or E) allows theuser to record only acceptedmoves (A); or, moves which are eitheraccepted or rejected (E). Justfor information, a sample .trj trajectory file is shown below; you will not need to create suchfiles (unless you feel like creating an animation!):

______________________________________________________________________________

ntorsions 2

run 1

cycle 1

temp 300.000000

state 1 A -3.745762 -1.432243 -9.518171 23.713793 23.076145 0.713534 -0.023818 0.700216

30.606248

-4.894825

2.661499:

:

state 6 R -12.679995 -1.452641 -9.259430 21.634645 23.135242 0.653369 -0.440832 0.615448

39.127316

-31.636299

10.261519

state 7 a -8.746072 -1.458231 -9.080998 21.356874 23.325665 0.648312 -0.448577 0.615200

41.075955

-37.935175


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11.918847

:

______________________________________________________________________________

There are several keywords: run and cycle are self-explanatory; ntorsions is the total num-ber of changing torsions in the ligand; temp is the annealing temperature for all subsequententries, unless otherwise stated. Each state record has the format:

state nstep acc_rej_code e_total e_internal x y z qx qy qz qw

where:nstep = the number of the step, within this cycle;acc_rej_code = A = an accepted move whose energy was lower than its preceedingstate;

= a = an acepted move whose energy was higher than its preceedingstate, which nevertheless passed theMonte Carlo probability test, at this temperature;

= R = a rejected move.

= e = an edge-hit, also a treated as a rejected move.e_total = total energy of the system, small molecule + macromolecule;e_internal = internal energy of small molecule only;x,y,z = translation of small molecule center;qx,qy,qz,qw = quaternion, which describes the small molecules orientation;

In order to get a coordinate-based trajectory file, for visualization, the command mode ofAutoDock must be used to regenerate the coordinates from the state variables. Use the trajcommand with the name of the pre-calculated trajectory file. For example, suppose there is a com-mand file called trj.conv.com that contains:

_____________________________________________________________________________

traj ligand.trj

stop

_____________________________________________________________________________

AutoDock would be executed a second time using the following command,

autodock -p ligand.dpf -l ligand.trj.conv.log -c < trj.conv.com >

trj.conv.out

18. Evaluating the Results of a Docking

At the end of an AutoDock execution, in which more than one run was performed, the programoutputs a list of clusters and their energies. The clustering or structure binning of docked confor-


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mations is determined by the tolerance specified in by the rmstol keyword. The best repre-sentative from each cluster (that with the lowest energy) is written out in PDBQ format at the endof the log file.

These structures can be read into any appropriate molecular modelling system and the resultscompared, where possible, with the experimental data. The table of ranked clusters shows the finaldocked energy for each conformation, and the RMS difference between the lowest energy mem-ber of the group and every other member. The RMS for the lowest member of the group is by def-inition zero. After this table, the structures are output in PDBQ format. Each conformation has aset ofREMARK records, one of which describes the RMS difference between itself and the coordi-nates specified in the original input PDBQ file.This can be useful for comparing how close eachdocked conformation is to the experimentally determined position.

19. Visualizing Grid Maps and Trajectories Using AVS

Grid maps can be visualised in AVS by using a read field module. The user must specify the.fld file that was created by AutoGrid, in order to read in the grid maps. An extract scalarmodule selects the grid map of interest, e.g. carbon affinity or electrostatics. The resulting gridmap data can be analyzed using arbitrary slicerand isosurface modules, in order to examine crosssections and iso-energy contours respectively. Negative energy contours are most informative forthe atomic affinity grid maps, since they reveal favorable regions of binding.

Trajectories can be read into AVS also using the read fieldmodule. The trajectory file is essen-

tially a set of stacked PDB frames, and must be read in as a two dimensional field (being thenumber of atoms in the small molecule, and the number of frames in the trajectory file). By pag-ing through this field, using the orthogonal slicer, continuous replay of the trajectory can beachieved using an animate integer module to control which PDBQ frame is selected by theorthogonal slicer. This animates the sequence of sampled states and allows the user to view in realtime the progress of the docking simulation.


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APPENDIX 20. Shell Scripts and Auxilliary Tools

cartopdbq

Usage: cartopdbq ligand.car > ligand.pdbq

Converts from Biosym InsightII .car format to PDBQ format

check-qsUsage: check-qs molecule

needs: molecule.pdbq

creates: molecule.err

Checks partial atomic charges in PDBQ file; any non-integral charges are reported.

clampUsage: clamp grid.map > grid.map.NEW

Clamps any AutoGrid map values that exceed ECLAMP (normally set to 1000.0)

deftorsUsage: deftors ligand

needs: ligand.mol2

creates: ligand.pdbq, ligand.err, ligand.bndand ligand.bnd.pdbq

Sets up rotatable bonds for AutoDock. This script launches AutoTors, with the -a, -h and -mflags; checks the charges in the output, with check-qs; creates a .bnd file with pdbqtobnd;

and creates an AVS .fld file using mkavsheader for trajectory viewing later on.

dpf-genUsage: dpf-gen ligand.pdbq > ligand.dpf

Generates a default AutoDock docking parameter file. You must edit the file before using it. Inparticular, you will need to edit the filename stem on the trjout line.

extjobinfoUsage: extjobinfo file.dlg > file.dlg.inf

Extracts information about a particular docking job. Columns contain: cycle, run, annealing tem-perature, minimum energy, change in energy, number of accepted moves, number of rejectedmoves, accepted/rejected ratio, total number of moves (accepted + rejected), time taken for thiscycle, average time per step.

genpdbqUsage: cat file.pdb file.tor | genpdbq > output.pdbq


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This is used to extract the order of the atoms and ROOT, BRANCH, TORS records from thefile.tor, and replace the atom lines with the new coordinates in file.pdb. The PDB ATOMrecords in file.tor must be converted into (non-standard) ATMNUM records, keeping just theatom serial number from the original PDBQ file.

get-coordsUsage: get-coords file1.vol > file1.txtThis is used as part ofprepare, prepare-gpf+dpf, prepare II and prepare III. Ittakes the .vol file created by pdb-volume and creates a line that can be used in the gridparameter file to specify the center of the maps.

gettrjdimUsage: tail -12 file.tlg | gettrjdim > file.tmp

Used in mktrjfld, to obtain the number of atoms and number of frames in the trajectory logfile, file.tlg.

gpf-genUsage: gpf-gen ligand.pdbq > ligand.gpf

This script is used to generate a grid parameter file. It takes as its input, a ligand.pdbq file,detects all atom types present, and creates the properly formatted parameter file for AutoGrid.This is used in prepare, prepare-gpf+dpf, prepareII and prepare III.

job, job2Usage: job dpfstem > dpfstem.joblog &

Launches a single AutoDock job. It assumes that dpfstem.dpf exists, and executesAutoDock using the arguments:

$bin/autodock -p dpfstem.dpf -l dpfstem.dlg

You must edit this script the first time you use it, so that the environment variables $root, $bin and$sh are correctly set equal to, respectively: the path to the root ofAutoDock tree, the architecture-dependent binary subdirectory and the Unix scripts subdirectory. The file dpfstem.joblogcontains the output from the job script.

The variant job2 takes two arguments, the first is as above, while the second is an AutoDockflag. For eaxmple:

Usage: job2 dpfstem -flag > dpfstem.joblog &

This script can be useful for passing one of the arguments -k (keep original ligand residue num-ber in output), -i (ignore grid map header mismatch errors) or -o (old format PDBQ file).

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