Insight II. Search Compare. March Scranton Road San Diego, CA / Fax: 858/

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1 Insight II Search Compare March Scranton Road San Diego, CA / Fax: 858/

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3 Copyright * This document is copyright 2000, Molecular Simulations Inc., a subsidiary of Pharmacopeia, Inc. All rights reserved. Except as permitted under the United States Copyright Act of 1976, no part of this publication may be reproduced or distributed in any form or by any means or stored in a database retrieval system without the prior written permission of Molecular Simulations Inc. The software described in this document is furnished under a license and may be used or copied only in accordance with the terms of such license. Restricted Rights Legend Use, duplication, or disclosure by the Government is subject to restrictions as in subparagraph (c)(1)(ii) of the Rights in Technical Data and Computer Software clause at DFAR or subparagraphs (c)(1) and (2) of the Commercial Computer Software Restricted Rights clause at FAR , as applicable, and any successor rules and regulations. Trademark Acknowledgments Catalyst, Cerius 2, Discover, Insight II, and QUANTA are registered trademarks of Molecular Simulations Inc. Biograf, Biosym, Cerius, CHARMm, Open Force Field, NMRgraf, Polygraf, QMW, Quantum Mechanics Workbench, WebLab, and the Biosym, MSI, and Molecular Simulations marks are trademarks of Molecular Simulations Inc. IRIS, IRIX, and Silicon Graphics are trademarks of Silicon Graphics, Inc. AIX, Risc System/ 6000, and IBM are registered trademarks of International Business Machines, Inc. UNIX is a registered trademark, licensed exclusively by X/Open Company, Ltd. PostScript is a trademark of Adobe Systems, Inc. The X-Window system is a trademark of the Massachusetts Institute of Technology. NSF is a trademark of Sun Microsystems, Inc. FLEXlm is a trademark of Highland Software, Inc. Permission to Reprint, Acknowledgments, and References Molecular Simulations usually grants permission to republish or reprint material copyrighted by Molecular Simulations, provided that requests are first received in writing and that the required copyright credit line is used. For information published in documentation, the format is Reprinted with permission from Document-name, Month Year, Molecular Simulations Inc., San Diego. For example: Reprinted with permission from Cerius 2 User Guide, Month 2000, Molecular Simulations Inc., San Diego. Requests should be submitted to MSI Scientific Support, either through electronic mail to support@msi.com or in writing to: * U.S. version of Copyright Page

4 MSI Scientific Support and Customer Service 9685 Scranton Road San Diego, CA To print photographs or files of computational results (figures and/or data) obtained using Molecular Simulations software, acknowledge the source in the format: Computational results obtained using software programs from Molecular Simulations Inc. dynamics calculations were done with the Discover program, using the CFF91 forcefield, ab initio calculations were done with the DMol program, and graphical displays were printed out from the Cerius 2 molecular modeling system. To reference a Molecular Simulations publication in another publication, no author should be specified and Molecular Simulations Inc. should be considered the publisher. For example: Cerius 2 Modeling Environment, Month San Diego: Molecular Simulations Inc., 1999.

5 Contents 1. Introduction 1-1 What Does Search_Compare Do? Starting Search_Compare Using This Guide Additional Information Note on Command Names Theory 2-1 Superimposing Molecules RMS Fitting Electrostatic Potential Similarity Steric Shape Similarity Field_Fit (Electrostatic & Steric Similarity) Steric Clash-checking Systematic Conformational Searching Standard Searches Use of Distance Constraints Distance Maps Distance Maps used in Series Screening by Energy Criteria Vector Maps Screening Duplicate Conformations Implementation 3-1 Volumes Overlapping Molecules Systematic Conformational Searching on Ring Systems Use of Energy Criteria in Systematic Conformational Searching Vector Mapping Background Jobs Search Compare i

6 4. Command Summary 4-1 Volume Pulldown Create Boolean Grid_Setup Align Color Label Display List Overlap Pulldown Define Field_Fit Display SC_Search Pulldown Set_Ring_Closure Set_Rot_Bond Set_Distance Set_Dist_Map Scale_Radii Set_Energy_Params Set_Filter_Params SCS_Run Load Vector_Map Pulldown Create Color Label Display_Vector List Conformer Pulldown Display Distance_Map Pulldown Boolean Construct_Graph Spreadsheet Pulldown Graph Pulldown Background_Job Pulldown Methodology 5-1 Using the Volume Pulldown Aligning Molecules and Volumes Specifying How Volumes will be Calculated Creating Volumes Preparing the System ii Search Compare

7 Specifying the Volume s Size Setting Whether and How a Volume Is Displayed 5-4 Calculating the Volume Displaying Information About the Volume Boolean Operations on Volumes Determining How Many Volumes Are Displayed after a Boolean Operation Changing How Existing Volumes Are Displayed Cancelling a Volume Calculation Using the Overlap Pulldown Using Highlights Defining Atom Pairs Setting Up Torsions Checking the Specifications Correcting Mistaken Entries Executing the RMS Fitting Calculation Executing the Field Fitting Calculation Using the SC_Search Pulldown Standard Searches Setting up Parameters for the Standard Search and Background Job Defining the Bonds to be Rotated Scaling Atomic Radii Setting Distance Constraints Setting Up the Background Job Running the Background Job for Standard Searches5-19 Initiating the Search Monitoring the Job Killing a Background Job Loading Search Results into Insight II Advanced Searches Setting up an Advanced Search and Background Job 5-21 Setting up Distance Map Calculations Setting up Energy Calculations Initiating the Background Job for Advanced Searches 5-26 Using the Distance_Map Pulldown Boolean Operations on Distance Maps Setting up a Boolean Distance Map Calculation Visualizing a Distance Map as a Graph Constructing a Distance Map Graph of an N-Dimensional Distance Map Constructing a Distance Map Graph From a Trajectory Using the Vector_Map Pulldown Creating and Editing a Vector Map Search Compare iii

8 Controlling the Display of a Vector Map Using the Conformer Pulldown Displaying Desired Conformers Saving and Using Lists of Conformers Manipulating the List of Conformers Tutorial 6-1 Pilot Online Tutorials Creating and Comparing Molecular Volumes: Superimposing Molecules with the Overlap Pulldown: Conformational Searching: Creating and Using Vector Maps: Running Background Jobs: A. References A-1 B. Glossary B-1 C. File Formats C-1 Input Files C-1 Search_Compare Parameter File (.scs_prm) C-1 Format of Search_Compare Parameter File C-2 Example Search_Compare Parameter File C-6 Output Files C-7 Search_Compare Log File (.scs_log) C-8 Files Used and Produced by Advanced Searches C-8 D. Utilities D-1 scs_xdrtor D-1 Purpose D-1 Syntax D-1 Errors and Warnings D-2 iv Search Compare

9 1 Introduction What Does Search_Compare Do? The Search_Compare module contains the pulldowns Volume, Overlap, SC_Search, Vector_Map, Conformer, Distance_Map, Spreadsheet, Graph, and Background_Job. The commands in Search_Compare enable you to calculate and operate on molecular volumes, to superimpose two or more molecules, to search systematically for the sterically allowed conformations of a molecule, and to quickly find and examine conformations of interest after they have been generated. The Volume pulldown contains the commands for calculating and operating on molecular volumes the space enclosed by a molecule s van der Waals surface. You can also change how a volume is displayed. The Overlap pulldown enables you to simultaneously superimpose multiple flexible molecules using two approaches: Generating an overall rms best fit of all atoms that have been selected for superimposition. Optimizing the molecular field similarity, based on electrostatic potentials and steric shape. You may specify certain bonds in the molecules as rotatable. During superimposition, these torsions will be rigidly rotated in the process of finding the best fit. No limit is imposed on the number of molecules, atom pairs, or torsions specified. The SC_Search and Background_Job pulldowns are used to set up and run a systematic conformational search and to load the search results into Insight II for viewing and analysis. The search is a systematic exploration of the conformational space accessible Search_Compare 1

10 1. Introduction to a molecule and is determined by continuously rotating one or more bonds through desired ranges. You can now use both distance constraints and a more powerful distance mapping facility in searches, for example, to find common conformations among a series of molecules having similar activity or to see whether a drug candidate molecule can adopt some desired conformation. In addition, the energy evaluators and minimizers of Discover are directly accessible from Search_Compare. You can perform searches with energy calculation or minimization, thus screening out unstable conformers (and saving disk space). You can also subject the conformers resulting from previous searches to energy calculations without having to leave the Insight II environment. The Vector_Map pulldown allows you to examine atomic movements that occurred during a conformational search so as to quickly find conformations in which pairs of atoms are located at specific points in space. The Conformer pulldown is used to display conformations in the currently loaded trajectory associated with vector maps and graphs. The Distance_Map pulldown contains commands that allow you to perform boolean operations on distance maps, and to construct distance map graphs either interactively or from a systematic search run with a distance map. For more information on the Spreadsheet, Graph, and Background_Job pulldowns refer to the Insight II User Guide. Starting Search_Compare Search_Compare is invoked by selecting Search_Compare from the Module pulldown in Insight II. 2 Search_Compare

11 Using This Guide Using This Guide Chapter 2, Theory, in this User Guide presents the theories upon which the functionalities of Search_Compare are based. It is written mainly for the typical scientist-user of Search_Compare. Chapter 3, Implementation, presents some information on how the functionalities are implemented. Chapter 4, Command Summary, briefly summarizes the main functions of each command. Chapter 5, Methodology, explains how to use Search_Compare, while Chapter 6, Tutorial, shows how to use it in a series of tutorial lessons. Appendices A, References, and B, Glossary, contain the scientific references cited in this guide and a glossary of terms and symbols. The file formats and a utility are documented in Appendices C, File Formats, and D, Utilities. Additional Information In addition to this printed Search_Compare documentation, on-line help is available and activated by clicking the? icon located on the icon bar in the Insight II window. Technical information that is mainly of use to programmers and system administrators is contained in the Biosym Products System Guide. Note on Command Names In referring to commands that are used when running Search_ Compare through the Insight II menus, this guide uses the format Pulldown/Command, since you use the mouse to select the pulldown first, before the command name appears. Note, however, that if you enter commands on the command line near the bottom of the Insight II window, the names must be entered in the format Command Pulldown or simply Command (whichever appears at the top of the equivalent parameter block). Search_Compare 3

12 1. Introduction 4 Search_Compare

13 2 Theory Superimposing Molecules A common use of molecular modeling is to find the conformational resemblance and the molecular field similarity of a group of molecules to a template molecule. The template molecule may be a lead compound, or a desired structure that is complementary to a receptor molecule. Two of the most powerful techniques in conformational comparison are provided in Search_Compare. The first method is to superimpose the molecules by minimizing the distances between specified atomic nuclei. The second method is to align the molecules using their electrostatic potentials and steric shapes. These processes are accomplished by rotating and translating the molecules. Very often, you may also want to allow some bonds in these molecules to be rotated during the fitting process. This allows you to answer questions such as: If these bonds can be rotated, do these molecules have conformations similar to the template molecule? What are the closest conformations to the template molecule that these molecules can achieve? RMS Fitting In the Overlap pulldown, the function to be minimized is the sum of squares of the distances between all atoms to be superimposed, as in Eq. 1: Search_Compare 1

14 2. Theory = M 1 M p = 1 q = p + 1 N( p, q) i = 1 ( r ip r iq ) 2 Eq. 1 where: M = the number of molecules; N(p,q) = the number of atoms between the p th and the q th molecules to be aligned; and r = the transformed atomic coordinate of the i th ip atom in the p th molecule. This is described as: r = f r 0, θ, t, φ Eq. 2 where: r 0 = the original atomic coordinate; θ = the rotation angles of the molecule; t = the translation vector of the molecule; and φ = the angles of all torsions in the molecule to be optimized. Eq. 1 can be minimized by conventional nonlinear least-squares methods. The RMS value reported by the Overlap/Define calculation is: RMS = F N pair Eq. 3 where F is calculated using Eq. 1, and N pair = M 1 p = 1 M q = p + 1 N( p, q) Eq. 4 2 Search_Compare

15 Superimposing Molecules Electrostatic Potential Similarity A number of different techniques have been proposed and applied to electrostatic potential similarity calculation, which is becoming a well-established modeling technique (Good, 1992). Different formulas for similarity determination have been proposed. Here, we use the Hodgkin index as discussed by Good for electrostatic potential similarity calculation SF ( a, b) = 2 P a P b dv P adv + P bdv Eq. 5 In Eq. 5, P a and P b are the electrostatic potentials for molecules a and b, which are dependent on the atomic charges and distance according to Eq. 6: P r = n Q i r R i i = 1 Eq. 6 where: n = the number of atoms in the molecule; r = the coordinate where electrostatic potential is to be evaluated; R i = the coordinate position of atom i; and Q i = the charge assigned to atom i. The value of the function, SF, ranges from -1, maximum dissimilarity, to 1, indicating identical potentials. A value of 0 corresponds to two molecules with zero electrostatic potential overlap, either because the molecules are far apart or because the value of the positive overlap equals the value of the negative overlap. For multiple molecules, Eq. 7 is used for the similarity calculation and optimization: Search_Compare 3

16 2. Theory SF = M 1 M 2 M ( M 1) SF ( a, b ) a = 1 b = a + 1 Eq. 7 where M = the number of molecules. Here, the SF function again ranges from -1 (inverse) to 1 (identical). Steric Shape Similarity The molecular steric similarity of two molecules is calculated with Eq. 5, where P a and P b are the steric functions for molecules a and b, which are Lennard-Jones potentials as in Eq. 8 ( 9-6 potential) or Eq. 9 ( 12-6 potential), depending on forcefield choice P r = i=1 e - k n s - r 9 s - r 6 Eq. 8 P r = i=1 e - k n s - r 12 s - r 6 Eq. 9 where: n = the number of atoms in the molecule; r = the coordinate where steric potential is to be evaluated; k = a constant; e = epsilon for the atom type; s = sigma for the atom type. The function SF ranges from 0, meaning zero steric overlap (molecules are too far apart), to 1 indicating identity. Field_Fit (Electrostatic & Steric Similarity) The combined similarity is calculated using Eq Search_Compare

17 Superimposing Molecules SF = w SF ( steric) + ( 1 w) SF ( electrostatic) Eq. 10 w = user-specified weighting factor, ranging from 0 to 1. The combined similarity function SF ranges from -1, meaning maximum dissimilarity, to 1, indicating identity. Steric Clash-checking To avoid van der Waals clashes in flexible fitting, a penalty function is added to the similarity function or the RMS function during the optimization process, as in Eq. 8. F ( Optimize) = SF F ( penalty) Eq. 11 where: SF = the similarity function or RMS function. The following penalty function is used: F ( penalty) = i= r o r n 2 Eq. 12 F ( penalty) 0 when r 2 2 = r o Eq. 13 where: n = the number of atom pairs between rotatable segments in the molecule; r 2 o = the sum of the VDW radii; r 2 = the distance between the two atoms. To provide a degree of softness in the penalty function, scaling factors are applied to the VDW radii of atoms. The scaling factors are: 1-4 interactions0.85 H-bond candidates0.65 Others0.95 Search_Compare 5

18 2. Theory Systematic Conformational Searching Systematic conformational searches can provide relatively quick answers to several types of problems. For example, determining which of a series of molecules that appear to be related is able to assume the correct conformation for binding to a particular receptor restricts the number of compounds that must be tested in more expensive or time-consuming ways. Standard Searches Systematic conformational searching explores the torsional space sterically available to a molecule. A dihedral angle is stepped through a range of values, and each resulting conformation is accepted if its interatomic distances do not indicate steric clashes between van der Waals radii of the constituent atoms and if optional user-defined distance constraints are not violated. Steric clashes are not checked for atoms whose interatomic distances do not change during the search. However, if you do not minimize your molecule with respect to the current forcefield the search may return no conformations, since it may detect steric atom clashes in the initial molecule. Consequently, it is generally a good idea to optimize a molecule s geometry with Discover before performing systematic conformational searching. Note that, in a standard search, only the steric interactions are examined the energy is not calculated unless you specifically request it. Use of Distance Constraints Since the search process varies selected torsions through all combinations of angles (within desired ranges), systematic searches can generate undesirably large numbers of conformations. The number of generated conformations can be greatly reduced, thus increasing computational efficiency, by using pair-wise interatomic distances derived from experimental data or from previous searches on related molecules as constraints. In this way, only 6 Search_Compare

19 Systematic Conformational Searching those conformations whose interatomic distances lie within desired ranges are generated. Distance Maps Distance Maps used in Series One way of rejecting molecules having interatomic distances that lie outside the desired ranges is to set up specific interatomic distance constraints, as mentioned under Standard Searches. Another, more efficient way of limiting accepted conformations to those that are similar to several possible conformations, is based on a distance map. This is a map of the pair-wise interatomic distances being monitored, in N-dimensional space where N = the number of atom pairs. Each interatomic distance is divided into equal increments (referred to as the resolution), thus forming a set of lattice points in the distance space. Each conformation is then mapped to a specific lattice point, taking into account all the monitored interatomic distances in that conformation. More than one valid conformation can map to a single lattice point. Naturally, a map with smaller resolution (more increments) has more points than one with a larger resolution, but then each point represents fewer conformations. Figure 1. shows an example of a two-dimensional distance map, where the distances between two pairs of atoms are mapped for a series of conformers. If, in addition, we wanted to monitor or constrain the distance between a third pair of atoms in each conformer (say, atoms 1 and 4), the associated distance map could be illustrated as a three-dimensional block, where the z-axis would be the distance between atoms 1 and 4. There is no limit to the number of distances we can monitor in this fashion. When conformational searches are being performed on a series of molecules with the idea of finding a geometry common to all of them, computational efficiency can be increased further by using the results obtained for each molecule in the series to restrict searches on subsequent molecules. In effect, distance maps are used as active filters to restrict the searches to regions of conformational space that contain sterically allowed conformations that are common to the set of compounds. This type of analysis might actually lead to a null set, indicating the absence of a common geometry. If so, the hypothesis that the functional groups (or biophores) Search_Compare 7

20 2. Theory conformers: A B C D distance between atoms 3 and A C B 2.0 D distance between atoms 1 and 2 Figure 1. Construction of a Two-Dimensional Distance Map The solid circles represent the map points of conformations A D, and the open circles represent the map points of many molecules (not shown) whose conformations are relatively close to conformation C. For these molecules, differences in the distances between atoms 1 and 2 are not resolved, while the distances between atoms 3 and 4 are resolved into four groups. Note that the resolutions for the two axes (i.e., for the two interatomic distances being monitored) do not have to be identical. 8 Search_Compare

21 Vector Maps correspond may be incorrect. Alternatively, a search may lead to a set of several possible geometries. In this case, their relative likelihoods must be tested and analyzed further by other means. Screening by Energy Criteria The number of conformers that satisfy van der Waals clash-checking and distance constraints can still be very large. Many of these conformers may be energetically unfavorable. By evaluating the total energy of each conformer and screening out those conformers whose energy values are a specified amount above that of the minimum-energy conformation, a much smaller set of stable conformers can be obtained for further examination. Very often, instead of simply calculating the conformers' energies, it may be preferable to perform energy minimization for each conformer before screening out unstable conformers. The resulting conformers are the most stable, minimized conformations of the molecule under the specified search conditions. Performing a systematic conformational search using this option enables multiple stable conformers to be found. This has a great advantage over optimization of a single structure. In the latter case, only one conformer is obtained, which may correspond to a local rather than a global minimum in the available conformational space. Discover functionality is used to calculate and minimize the energy. Several minimizers are used in sequence: steepest descents, followed by conjugate gradients, followed by a quasi-newton Raphson method known as BFGS (formerly VA09A). Please refer to the Implementation section and to the Discover documentation for additional details. Vector Maps Even with the application of various screening methods during a search, large amounts of data may still remain to be analyzed. Vector maps are a useful analytical tool for examining these results. They provide a means of graphically displaying regions of space swept out by a pair of atoms during a systematic conformational search. Search_Compare 9

22 2. Theory A vector map consists of a set of line segments representing the coordinates of a pair of atoms for every conformation within a trajectory resulting from a systematic search. The connected atom pair is the same for each frame or conformer of the search, but since the atoms locations change from one frame to the next, a series of lines (vectors) is generated that shows the position of the atom pair for each trajectory frame or search conformation. A vector map refers to both the atom pair and the Insight II trajectory that has been loaded with the Trajectory/Get command in the Analysis module or the SC_Search/Load command in the Search_ Compare module. Vector maps are a useful tool for finding which conformations of a search or trajectory have atoms located at specific points in space. There is no requirement that the atom pair used to define the vector map be a bonded atom pair. Defining a vector map for a nonbonded pair of atoms is a useful means of visualizing relative orientations and distances between a pair of atoms for each trajectory frame. Screening Duplicate Conformations Very often a systematic torsional search will produce duplicate conformations. This is especially true when the molecule you are searching has local symmetry and/or you are running with energy minimization. In the former case this is because rotating a symmetrical side-group (e.g., a phenyl group by 180 degrees) produces an identical looking conformation the difference only being in the labeling of the topologically equivalent atoms. In the latter case, several torsional conformations may minimize to the same conformer. Systematic search offers controls for governing how duplicates are removed. These are essentially based on the conformational energy difference (when energy searches are performed) and/or the RMS difference between the atomic coordinates of superimposed conformations. By default, the parameters for duplicate removal are set with low tolerances so only duplicate conformations are removed. However, by setting higher tolerances more 10 Search_Compare

23 Screening Duplicate Conformations conformations will be screened out as duplicates, resulting in a more diverse set of conformations from a search. Duplicate removal based on RMS can be customized for use of topological symmetry and for the subset of atoms it is applied to. By specifying the set of atoms used for RMS calculations, duplicate removal can be made to focus on just the regions of interest within the molecule s conformation. For example, you may not care about the geometry of a particular side chain of your molecule and so atoms in this region could be excluded from the list of RMS atoms. Search_Compare 11

24 2. Theory 12 Search_Compare

25 3 Implementation Volumes In the Insight II environment, a volume is composed of a Boolean grid and, optionally, its contour. When a volume is created, the algorithm constructs a grid that spans the operand molecule(s) or volumes and turns on grid points that lie within the van der Waals surface of the molecule(s) or that satisfy the requested volumetric Boolean operation. The size and extent of this grid can now be controlled by the user (see Chapter 5, Methodology). An additive amount and/or a scale factor can be applied to each van der Waals radius before the molecular volume is created. (These are applied only during volume creation and do not change the van der Waals radii referred to by other functions in Insight II.) Overlapping Molecules Eq. 1 is minimized using the Levenburg Marquardt method (Marquardt, 1963), which is very efficient for nonlinear least-squares problems. Minimization by this method varies smoothly between the extremes of the steepest-descent and the Newton methods. The former is used far from the minimum, with the algorithm switching continuously to the latter as the minimum is approached. The Levenburg Marquardt method requires the first derivatives of the minimizing function to be known. Here, the first derivatives with respect to all variables are calculated using the analytical solutions derived from Eq. 1. You cannot superimpose pseudoatoms, since this is not supported by the current version of the Overlap functionality. Search_Compare 1

26 3. Implementation In the electrostatic calculation, P r in Eq. 6 is replaced by a Gaussian function approximation of two terms, as in Eq. 14. The integrals in Eq. 5 have a simple form based on exponent values and the distance between atom centers (Good et al., 1992) e r2 r e r2 Eq. 14 The Lennard-Jones potential in Eq. 8 or Eq. 9 is approximated by the following Gaussians over a range of r covering just inside the repulsive part of the potential, as follows: LJ 9 6 ( ε,σ,r) ε exp 6.81 ( r σ) 2 LJ 12 6 ( ε,σ,r) ε exp ( r σ) 2 Eq. 15 Before the overall optimization is performed (by molecule rotations, molecule translations, and torsion rotations), the molecules may be aligned using the dipole, quadrupole moments, and moments of inertia to find the best starting position. Systematic Conformational Searching on Ring Systems Conformations of rings can also be examined in torsion space by including ring closure bonds in the search parameters. These bonds are broken during search calculations, allowing the torsions of the now pseudo acyclic structure to be freely adjusted. In order to properly close the ring, closure constraints are applied. These constraints maintain the distance between the two atoms forming the ring closure bond and the bond valence angles, within reasonable tolerances. During search calculations, when the ring closure bond is temporarily cleaved, the chirality of the closure bond atoms can be lost if these atoms are stereo centers. To account for this, and also to properly reorient in space the side chains off the 2 closure bond atoms, a torsional rotation is applied to realign the original closure 2 Search_Compare

27 Systematic Conformational Searching on Ring Systems C1 C2 C6 IT IT C3 RB RB RB Figure 2. Ring Closure Constraints distancec1-c2 2 anglesc6-c1-c2 C1-C2-C3 torsion1-4 interactions across bond C1-C2 Closure bond Implicit torsions (IT) Rotatable bonds (RB) bond axis with the new closure bond axis position. This torsion is called an implicit torsion, and is automatically handled by the search algorithm (Lipton and Still, 1988). Since there can be large geometry variations localized at the two closure bond atoms, it is strongly recommended that you optimize the geometries of the conformers so that these deviations are distributed over the entire structure. When searching conformations of ring systems, you must specify which ring closure bonds will be broken during search calculations. This is done using the SC_Search/ Set_Ring_Closure command. As explained on page 2, two implicit torsions are associated with each closure bond. These torsions are automatically assigned by the search algorithm.therefore, they are not allowed as rotatable bonds. Note that a ring closure bond must be in a unique ring, and if the bond is broken, its two adjacent ring bonds (implicit torsions) can Search_Compare 3

28 3. Implementation rotate freely without breaking other rings. A fusion bond (a bond whose atoms are in two or more rings that share two or more atoms) cannot be defined as a ring closure bond. In addition, bonds adjacent to fusion bonds or connecting spiro atoms in spiro rings are not allowed as ring closure bonds. Use of Energy Criteria in Systematic Conformational Searching Energy calculation or minimization is performed on a conformer using Discover functionality, after the conformer has passed checking for van der Waals clashes and satisfied any distance constraints. If energy evaluation is requested for a search, the final number of conformers found is restricted by the user-defined maximum number of conformers and energy threshold. The energy threshold is used first, to screen out the conformers whose energy values are a specified amount above the minimum energy the energy value of the most stable conformer among all the conformers. If the number of conformers passing this phase of screening is too large, only the most stable conformers are selected, with the number of conformers being limited to the user-specified maximum value. When energy minimization is performed, the resulting conformers are sorted according to their energy values from lowest to highest, and the atomic coordinates are stored in an archive (.arc) file. If desired, duplicate conformers, whose energy and coordinates are nearly identical to those of an already-stored conformer, can be screened out. You can control this screening process by specifying the thresholds for the energy and rms values. Note that there is no constraint on the torsion angles and distances during the minimization process. Therefore, the minimized conformers may not satisfy the original distance constraints, if any were present. Discover functionality is used to calculate and minimize the energy. Several minimizers are used in sequence: steepest descents, followed by conjugate gradients, followed by a 4 Search_Compare

29 Vector Mapping quasi-newton Raphson method known as BFGS (formerly called VA09A). Maximum derivative criteria are used to control when to switch to the next algorithm in the cycle: conjugate gradients is started when the maximum derivative falls below 10, and BFGS is started when the maximum derivative falls below 1.0. The total number of iterations for the complete minimization cycle and the final convergence criterion the maximum derivative are specified by the user. Note that optimization terminates when the total number of iterations is exceeded even if the convergence criterion has not been satisfied. It is also possible to specify whether or not to include charges or cross terms in the Discover energy calculation. Please refer to the Discover documentation for additional details. Vector Mapping Due to the nature of systematic searches, it is likely that atoms will occupy the same position in space in more than one valid conformation resulting from the search. Consequently, the same line segment in a vector map may occur in multiple trajectory frames loaded from a search. When this occurs, a duplicate vector is not generated, but instead a list of associated frames is generated for each vector of the vector map. You may use the Conformer/Display command to view any or all of the associated frames of a given vector. Background Jobs Much of the computational work within Biosym products is performed by background jobs that are run using the Insight II program as the user interface. Background jobs run concurrently with the interactive Insight II program; this is possible because, once started, they do not require user interaction. If you have access to more than one computer (mainframe or workstation), you may want to run some of the background jobs on a different computer (the remote host) than the one that is running the Insight II software (the local host). Search_Compare 5

30 3. Implementation Running the background job on a remote host involves several general requirements: The actual background job program must exist as an executable image for that host. Files transferred between local and remote hosts must be readable and writable on both hosts. You must have an account on the remote host, and sufficient disk space to contain all the input, temporary, and output files produced by that background job. The local host must be able to communicate statuses, submit jobs, and copy files to the remote host. Making an executable image compatible with a remote host involves recompiling and relinking the program; this is done by Biosym for host types that the company supports. The size and complexity of the background job s program greatly affects the difficulty of this step. The issue of file compatibility is primarily problematic when the data are not represented as text (ASCII). These files are called binary files and typically store data in a machine-dependent way, which varies from one host type to another (e.g., IRIS and Convex use different formats for some types of data). This problem is most often addressed either by using an additional program that filters the data or by converting the actual file formats to use machineindependent representations especially those specified by Sun called XDR (external data representation). 6 Search_Compare

31 4 Command Summary The Search_Compare module contains several pulldowns in addition to the core pulldowns on the top menu bar. They are: Volume, Overlap, SC_Search, Vector_Map, Conformer, Distance_Map, Spreadsheet, Graph, and Background_Job. What the commands in these pulldowns do is summarized briefly below. For more detailed explanations of individual commands, refer to the on-line help facility (accessed by clicking the? icon). For information on how to use these commands, see Chapter 5, Methodology. Volume Pulldown The Volume pulldown contains commands that allow you to calculate molecular volumes, perform Boolean operations on molecules and volumes, create graphical representations of molecular volumes, and modify the appearance of the graphical representations. Create The Volume/Create command is used to calculate volumes for molecules, assemblies of molecules, and sets of conformations in trajectories. It also has the ability to create several types of graphical representations of the volumes. Boolean The Volume/Boolean command is used to perform Boolean volumetric operations involving both molecules and volumes. Search_Compare 1

32 4. Command Summary Grid_Setup The Volume/Grid_Setup command is used to specify the size, location, and resolution of the volume s grid. Align The Volume/Align command is used to align a volume to the coordinate system of a reference molecule. Color The Volume/Color command is used to change the color of a volume s point cloud, contour, or label. Label The Volume/Label command is used to add, move, and remove volume labels. The text of the label is the volume name. Display The Volume/Display command is used to independently specify which components of the volume to display the label, contour, and point cloud. A contour can be displayed as a line contour or a solid contour. List The Volume/List command is used to report the volume (in cubic angstroms) occupied by an existing volume and the resolution of the volume s grid. 2 Search_Compare

33 Overlap Pulldown Overlap Pulldown The Overlap pulldown contains commands allowing you to superimpose a group of molecules using either rms alignment or molecular field similarity optimization. You can perform either rigid or flexible fitting. In the latter case, you can select bonds in the molecules that are rotated in the optimization process. After the optimization process, the molecules are aligned on the screen automatically. You can form an assembly object in order to move and rotate all molecules together. You can also display each molecule with a distinctive color. In addition, you may create a table detailing rms or similarity values for each pair of molecules. Define The Overlap/Define command is used to define atoms to be superimposed and rotatable bonds to be optimized during the superimposition process. This command is also used to execute the rms overlap calculation. You use this command to add, delete, and list atom pairs and rotatable bonds. Field_Fit The Overlap/Field_Fit command is used to compare molecular fields combinations of electrostatic potentials and steric shape among two or more molecules, and/or to superimpose these molecules by optimizing the similarity as a function of position. In the fitting process, bonds among the molecules can be rotated to find the conformations that have the highest similarity. Display The Overlap/Display command specifies whether or not to display selected atom pairs or rotatable bonds with special highlights and whether to show labels of selected atoms. Search_Compare 3

34 4. Command Summary SC_Search Pulldown The SC_Search pulldown contains commands that allow you to set up a systematic conformational search, run it, and load the results into Insight II. A systematic conformational search is set up by specifying the parameters for the search with the commands Set_Ring_Closure, Set_Rot_Bond, Set_Distance, Set_Dist_Map, Scale_Radii, and Set_Energy_Params. The commands in the Background_Job pulldown are used to prepare for running the systematic conformational search as a background job. The search calculation is initiated with the SCS_Run command. The results of a search are loaded into the Insight II program with the Load command. Set_Ring_Closure The SC_Search/Set_Ring_Closure command is used to create and edit a list of ring closure bonds and the corresponding ring closure constraints. This command is used for systematic searching on cyclic systems. You must first define the closure bonds that are broken during search calculations, then you can proceed with definition of rotatable bonds through the SC_Search/Set_Rot_Bond command. Set_Rot_Bond The SC_Search/Set_Rot_Bond command is used to create and edit a list of rotatable bonds for the systematic conformational search. These bonds constitute the torsion angles about which the systematic conformational search is performed. Set_Distance The SC_Search/Set_Distance command is used to create a list of atom atom distance constraints that constitute a set of acceptance criteria for conformations generated in a search. It allows you to create a new specification, read a list of specifications from a pre- 4 Search_Compare

35 SC_Search Pulldown vious systematic search parameter file, or extract distance constraints from the output file of a previous search. Set_Dist_Map The SC_Search/Set_Dist_Map command is used to set up a new distance map or a distance map that maps the results of a prior search. Scale_Radii The SC_Search/Scale_Radii command is used to apply scaling factors to the van der Waals radii of all atoms, of hydrogen bond donor/acceptor pairs, or of vicinal (1 4) atoms. Set_Energy_Params The SC_Search/Set_Energy_Params command specifies how energy calculation and screening will be performed when the SCS_Run command is invoked. This command specifies the maximum number of conformers to keep, the energy threshold for screening out unstable conformers, whether or not to perform energy minimization for each conformer, and several parameters used by the energy calculation routines. Set_Filter_Params The SC_Search/Set_Filter_Params command is used to control and customize the duplicate removal process applied to the conformations produced by a systematic search. For example, if local symmetry in the search molecule is to be considered, you specify the RMS and energy tolerances and the set of atoms considered when comparing conformations. You can also specify which atoms are used for the RMS duplicate matching by specifying one of a list of atom types, e.g., Heavy Atoms (all atoms other than hydrogens) or a specific list of atoms that you create and edit yourself. Search_Compare 5

36 4. Command Summary SCS_Run The SC_Search/SCS_Run command is used to initiate a systematic conformational search; to estimate the number of conformations that will be found, prior to actually running the search; or to perform post-processing on the results of a previous search (such as energy filtering or distance map calculation). The progress of a search can be monitored by using the Background_Job/Completion_Status command. You can interrupt a searching job and keep the partial results by executing the Background_Job/Kill_Bkgd_Job command with the Save_Output parameter toggled on. Load The SC_Search/Load command is used to load the results of a systematic conformational search into the Insight II program. The results are loaded in the form of a trajectory. Trajectories can be loaded from both XDR_TOR and ARC files and it is possible to load only part of a trajectory. The commands in the Vector_Map, Distance_Map, and Conformer pulldowns, and the commands in the Analysis or DeCipher modules can then be used to analyze the results. You can monitor the angles of defined torsions and distances between constrained atoms by toggling the Dihedral_Monitor and Distance_Monitor parameters on. These monitors can later be removed using the Dihedral/Measure and the Distance/Measure commands. If you stop a loading process by pressing the <Esc> key, the conformers loaded before the time of the interruption are kept. Note that, to optimize performance, the torsions are not necessarily searched in the order in which they were input. The order of conformations in the results reflects the search order rather than the input order. 6 Search_Compare

37 Vector_Map Pulldown Vector_Map Pulldown The Vector_Map pulldown contains commands that allow you to create vector maps. Create The Vector_Map/Create command allows you to create a vector map by specifying a name and a pair of atoms. A trajectory must be loaded before a vector map can be created. Color The Vector_Map/Color command is used to change the color of a vector map s vectors and label. Label The Vector_Map/Label command is used to add, move, and remove vector map labels. The label is the name of the vector map. Display_Vector The Vector_Map/Display_Vector command is used to blank or unblank individual vectors in the vector map. List The Vector_Map/List command is used to report information about the vector map. Various levels of detail are possible. Search_Compare 7

38 4. Command Summary Conformer Pulldown The Conformer pulldown contains one command, Display, that allows you to derive lists of conformations, perform boolean operations on the list of conformations, and display the conformations in the lists. Display The Conformer/Display command is used to display conformations in the currently loaded trajectory associated with vector maps and graphs. Distance_Map Pulldown The Distance_Map pulldown contains commands that allow you to perform boolean operations on distance maps, and to construct distance map graphs either interactively or from a systematic search run with distance map. Boolean The Distance_Map/Boolean command allows you to perform a number of different boolean operations on distance maps. The distance maps used must have the same number of dimensions, although the dimensions can be mapped in any order. Corresponding distances in the distance maps must also have the same resolution and bin boundaries. Construct_Graph The Distance_Map/Construct_Graph command allows you to visualize distance map data. Up to 3 dimensions of an N-dimensional distance map can be visualized at a time. This is done by 8 Search_Compare

39 Spreadsheet Pulldown projecting any dimension of an N-dimensional distance onto the X, Y, or Z axes of a graph. Spreadsheet Pulldown Please see the Insight II User Guide for detailed information on this pulldown. Graph Pulldown Please see the Insight II User Guide for detailed information on this pulldown. Background_Job Pulldown Please see the Insight II User Guide for detailed information on this pulldown. Search_Compare 9

40 4. Command Summary 10 Search_Compare

41 5 Methodology This section is a general description of how to use the Search_ Compare module. The on-line help (accessed by clicking the? icon), contains further details about what individual commands and parameters do, and Chapter 6, Tutorial, shows examples of their use. Using the Volume Pulldown Aligning Molecules and Volumes The results of a volumetric operation depend on the relative spatial orientation of the operands, so before creating a volume or performing a volumetric Boolean operation, you may need to align the operand molecules or volumes. Objects can be aligned by using the Overlap pulldown in the Search_Compare module, using the Transform/Move or Transform/Superimpose commands from the upper menu bar, or connecting to one of the objects in order to orient it with the dials or mouse. The Volume/Align command can be used to realign a volume with its reference molecule. The volume that the command is applied to is specified by the Volume Name parameter, and the Reference Molecule parameter indicates the molecule to which the coordinate system of the volume will be aligned. Specifying How Volumes will be Calculated The Volume/Grid_Setup command is used to set specifications for volumes to be created. It does not affect already-existing volumes. Search_Compare 1

42 5. Methodology As mentioned in Chapter 3, Implementation, the algorithm for creating a volume works by constructing a grid that spans the operand object and then turns on the grid points that lie within the scaled van der Waals surface of the object (or that satisfy a volumetric Boolean operation). You can control the extent of this grid, its resolution, and the thickness of the border around the grid. The Grid Style parameter determines the extent of the grid, that is, the amount of space in the Insight II world occupied by the grid. Setting Grid Style to Enclosure instructs Insight II to automatically determine the extent of the grid needed to enclose the operands. When Grid Style is set to Extents, you can define the Lower Bounds and Upper Bounds (i.e., the two diagonally opposite corners) of the cubic space occupied by the grid, by filling in the X, Y, and Z coordinates of those points. Setting Object_Coords to off means that these coordinates are specified according to the Insight II program s world axes. When Object_Coords is on, these bounds are interpreted in the coordinate space of the object specified by Reference Object. Please refer to the Insight II documentation for a discussion of world and object space coordinates. The Border Space parameter specifies the border (in angstroms) that is added around the grid, and the grid resolution (also in angstroms) is specified by the Grid Step parameter. The border should generally be twice the grid resolution, to allow the algorithm to function properly. Creating Volumes Preparing the System Volumes can be created for molecules, assemblies, and sets of conformations in trajectories, with the Volume/Create command. The Volume Level parameter specifies whether to create a volume from a Molecule, an Assembly, or a Trajectory. The name of the object for which the volume will be calculated is supplied to the Operand Name parameter. You may pick a molecule from the Insight II screen, choose a desired operand from the Operand Name value-aid, or enter its name by typing in the Operand Name parameter box. If you type in a molecule name, it may contain wild card characters (*). If it does, the molecules specified 2 Search_Compare

43 Using the Volume Pulldown Specifying the Volume s Size are placed in an assembly, and the first molecule name to resolve from the wild card becomes the reference molecule. Then the command executes as if the Volume Level had been set to Assembly. If a volume is to be created for a molecule, no other parameters must be set before going on to calculate the volume. However, if you want to change the default volume size or specify how to display it (see page 3), you should do so before filling in the Operand Name by picking a molecule or its name, since this is a trigger parameter, meaning that the command executes automatically after it is filled in. If a volume is to be created from an Assembly, you must specify both an Operand Name (i.e., the name of the assembly) and a Reference Molecule. The coordinate system of the reference molecule is used for the new volume. For a volume to be created from a Trajectory, the trajectory must first be loaded by using the SC_Search/Load command in the Search_Compare module or the Trajectory/Get command in the Analysis or DeCipher modules. The coordinates of the molecule or assembly to which the trajectory belongs will be updated to match the coordinates of the first frame of the trajectory if the Update_Mol_Coords parameter is on. This can be useful for preventing misalignment between the trajectory and the molecule or assembly. The Atom Move Tol value specifies a minimum distance that an atom must move before its new position is factored into the volume calculation. If the Frame_Range parameter is on, the trajectory frames to be included in the volume calculation are specified with the Start, Last, and Step parameters. If the Frame_ Range parameter is off, the trajectory frames are specified in the Frame Spec parameter as a single number (n) or a range of numbers (n-m, >n, <n, >=n, or <=n, where n and m are integers). You can set the relative size of a volume before you create it, by specifying a scaling factor and/or an additive amount. The VDW Scale parameter is a number that each atom s van der Waals radius is multiplied by, and the VDW Increment is a number in angstroms that is added to each atom s van der Waals radius. These adjustments are applied before the volume is created. If both Search_Compare 3

44 5. Methodology Setting Whether and How a Volume Is Displayed Calculating the Volume are specified, the scaling factor is applied first. These adjustments apply only within the context of volume creation. The Make_Contour parameter controls whether or not a contour is calculated for the volume. If Make_Contour is on, the contour is calculated and displayed as soon as the volume calculation is finished. If Make_Contour is off, the contour does not appear, but can be calculated and displayed later (see Changing How Existing Volumes Are Displayed, page 6). The Create_Solids parameter controls whether the contour is displayed as a solid contour (on), or as the default line contour (off). The volume s style can also be changed later. The name of the new volume is specified by New Volume Name, and the name of the new assembly that will contain the volume and the molecules from which it is derived is specified by New Assembly Name. Selecting Execute starts the volume calculation. When the calculation finishes, the dials are automatically connected to the assembly to which the volume belongs. Applying Volume/Create to a molecule results in calculation of the molecular volume and, optionally, display of the volume contour around the molecule. The molecule and volume are automatically associated into an assembly, and a linkage is created between the molecule and its volume. This means that if you connect to and move either the molecule or its volume, the other member of the linkage also moves. The linkage is imposed in order to maintain alignment between the molecule and volume. You can remove an object from the linkage with the Assembly/Remove command. Applying the Volume/Create command to an assembly results in calculation of the union volume of the molecules in the assembly and, optionally, display of the volume contour around the assembly. The new volume is added to the operand assembly, and a linkage, as described above, is created between the molecules and the volume. Applying the Volume/Create command to a trajectory results in calculation of the union volume for the conformations in the trajectory and, optionally, display of the volume contour around the 4 Search_Compare

45 Using the Volume Pulldown molecule to which the trajectory belongs. The molecule and its volume are automatically associated into an assembly, and a linkage, as described above, is created between the molecule and the volume. Displaying Information About the Volume After a volume is created, its size in cubic angstroms appears in the information area of the Insight II screen. The Volume/List command can also be used to report the volume (in cubic angstroms) occupied by an existing volume and the resolution of its grid. If the Output_File parameter is on, this information is written to the file specified by the File Name. If Output_ File is off, the volume is displayed in the textport. After you are finished examining the information, you can put the textport away by selecting Textport off at the bottom of the Insight II window. Boolean Operations on Volumes The Volume/Boolean command performs Boolean operations on molecules and existing volumes. The coordinate system of the first operand for a volumetric Boolean operation, specified by the Reference Object parameter, is used for the new volume. The Reference Object must be a single object, so wild-carding is not allowed. Both objects involved in Boolean operations may be a molecule or a volume. The second object is specified by the Operand 2 parameter. For union or intersection operations, the second object may be a single object or an assembly of objects, and its specification may contain wild card characters. For difference and exclusive-or operations, Operand 2 must be a single object. To choose which Boolean operation to perform, you need to set the Boolean Operation parameter to Union, Intersection, Difference, or XOR (exclusive- or ). The results of volumetric operations are also volumes. The union volume is the sum of the operand volumes. The intersection volume is the part of the operand volumes that is common to both operands. The difference volume is the part of a given operand volume that is not shared by a second operand volume. The XOR volume is the part of the operand volumes that is contained in one operand or the other, but not in both. Search_Compare 5

46 5. Methodology Before executing the command, you may want to set several other parameters: the Make_Contour, Create_Solids, VDW Scale, and VDW Increment parameters function exactly as in the Volume/ Create command (see page page 3). You can also control how many volumes will remain in the display area after the Boolean operation is executed (see below). The name for a new volume created by a Boolean operation is specified by New Volume Name. This new volume can be added to any of the assemblies to which the operands belong, or a new assembly can be created, as desired, by appropriately setting the Assembly Name parameter. To maintain alignment, a linkage, as described above, is created between the new volume and the operand volumes. Once the Boolean operation is complete, the size of the new volume in cubic angstroms appears in the information area, and the dials are connected to the assembly to which the volume belongs. Determining How Many Volumes Are Displayed after a Boolean Operation As part of the Volume/Boolean command, you have the choice of preserving the current status of all operand volumes (by choosing Preserve for the Old Volumes Status), blanking operand volumes (by choosing Blank for the Old Volumes Status), deleting all operand volumes (by choosing Delete for the Old Volumes Status), or overwriting an operand volume (by entering its name as the New Volume Name). If either the reference object or the second operand is a molecule rather than a volume, then the Old Volumes Status parameter has no effect on that operand. A volume can also be removed after it is created, with the Object/ Delete command on the upper menu bar. Changing How Existing Volumes Are Displayed A volume is composed of a Boolean grid that indicates which points are inside ( on points) and outside ( off points) the volume and, optionally, a contour of the grid. The Volume/Display command is used to set whether a given volume, specified by the Volume Name, is displayed as: 6 Search_Compare

47 Using the Volume Pulldown A line contour (toggle the Contour parameter on and set Solid_ Contour to off). A point cloud representation of all on grid points (toggle the Point_Cloud parameter on). A solid contour (toggle both the Contour parameter and the Solid_Contour parameter on the Flip_Normals option changes which side of the solid contour is lit). The line contour and point cloud can be displayed simultaneously. If you try to display a volume contour that does not exist, the contour is created and then displayed. Note that displaying a point cloud can significantly slow the graphical response of Insight II. You can use the Volume/Label command to: Label a volume (set Remove to off and set the desired Position Option and Volume Name). Change the position of a label (set Remove to off and set the desired Position Option and Volume Name). Remove a label (set Remove to on and input the Volume Name). The Volume Name becomes the text of the label. The Position Option allows you to place the label Above, Below, to the Left, or to the Right of the volume or to specify absolute coordinates by filling in the X, Y, and Z Label Coordinates. You can also use the Volume/Display command to show or hide labels by toggling the Label parameter on. You can use the Volume/Color command to change the color of a volume s Contour, Point_Cloud, or Label. The Color parameter is easiest to fill in by picking a color from the right side of the Palette value-aid or by mixing a custom color with the sliders on the left side and then accepting it by choosing the block above the sliders. The commands in the Object pulldown can also be applied to volumes. Cancelling a Volume Calculation You can cancel a volume calculation that was started with the Volume/Create or Search_Compare 7

48 5. Methodology Volume/Boolean command before it finishes. To do this, place the cursor in the Insight II window and press the <Esc> key. This aborts the calculation (if the Resultant Volume message appears, processing has gone too far for the command to be cancelled). Using the Overlap Pulldown To superimpose multiple molecules via the Overlap pulldown, you first must define the atoms to be superimposed. Then, if desired, you can define rotatable bonds. After that, you can execute the overlap calculation and study the results. To perform molecular similarity calculation/optimization, specify the molecule to use by defining one atom pair between two molecules. Then start the calculation using the Field_Fit command. Using Highlights Before defining any overlap specifications, you can specify some highlight options, to show what atoms are to be superimposed and which bonds are rotatable. Atom labels can be used to highlight selected atoms. Link highlights are dashed lines connecting atoms to be superimposed. Torsion highlights draw dashed colored lines along the specified rotatable bonds. To specify these highlight options, you select the Overlap/Display command. You then turn atomic labels, link highlights, and torsion highlights on or off by toggling the Display_Label, Display_ Link, and Display_Torsion parameters. These highlights can be turned on or off at any time. If the highlights are turned on, they remain on either until they are turned off explicitly or until the overlap specifications are deleted (they may also be deleted indirectly through deletion of the molecules). Defining Atom Pairs For an rms overlap calculation, you must specify one-to-one correspondences for atoms in the molecules to be superimposed. For an 8 Search_Compare

49 Using the Overlap Pulldown electrostatic potential calculation, however, you need only specify one atom pair between the corresponding molecules. You may choose any atoms, since the atom pair is only used to indicate which molecules to use in the electrostatic potential calculation. You specify these atom pairs with the Overlap/Define command, by setting Definition Action to Add, choosing Atoms as the Definition Type, setting the Define Pick Level to Atom, and supplying the names of the two atoms to be superimposed. This is done by picking atoms in the molecules in the display area, to fill in the Overlap Spec 1 and Overlap Spec 2 parameters. Each atom must belong to a different molecule. You may also specify two groups of atoms to be superimposed simultaneously. The group of atoms may be atoms in a molecule, a subset, or a monomer/residue. In this case, the numbers of atoms in the two groups must be the same, and the atoms are paired automatically by Insight II according to the internal sequence. To do this, you again set the Definition Action to Add and the Definition Type to Atoms. But now you set Define Pick Level to Molecule, Subset, or Monomer/Residue. You then pick atoms in the molecule as before, to fill in the Overlap Spec 1 and Overlap Spec 2 parameters. You should always verify that the automatic specifications are what you expected (see Checking the Specifications). Note that you can define atom pairs among several molecules, if you want to superimpose more than two molecules. For example, if you want to superimpose atom p in molecule A, atom q in molecule B, and atom r in molecule C on one another, you only need to define the p q and q r atom pairs. Wildcards are allowed for Overlap Spec 1 and Overlap Spec 2, but the atoms specified by Overlap Spec 1 must be in the same molecule. Overlap Spec 2 may specify atoms from a different molecule, as long as the numbers of specified atoms in each molecule are the same. These atom pair specifications remain in the memory throughout the Insight II session. You can modify them by adding or deleting at any time. However, they cannot be saved or restored using a file. Search_Compare 9

50 5. Methodology Setting Up Torsions You can specify that some of the bonds in the molecules will be able to rotate during the fitting process. Only single or triple bonds that are not part of a ring and do not contain a terminal atom (an atom bonded to only one other atom) are allowed. To specify rotatable bonds, you need to set the Definition Action to Add and the Definition Type to Torsion. You may specify a) one bond or b) a group of bonds at a time, by setting the Define Pick Level parameter to a) Atom or b) Molecule, Subset, or Monomer/ Residue, as desired. In specifying one bond, you need to supply the names of the two atoms forming the rotatable bond, by picking atoms in the molecule in the display area. This fills in the Overlap Spec 1 and Overlap Spec 2 parameters. In specifying a group of bonds at once, you only need to specify the name of the molecule, subset, or monomer/residue as Overlap Spec 1 (again by picking), and all rotatable bonds are automatically added to the specification. You may use wildcards in Overlap Spec 1 and Overlap Spec 2 to specify a group of bonds. These torsion specifications remain in the memory throughout the Insight II session. You can modify them by adding or deleting at any time. Checking the Specifications To list all defined atom pairs or rotatable bonds, set the Definition Action to List and the Definition Type to Atoms or Torsion. After you execute the command, the textport appears, displaying a list of atom pairs or rotatable bonds. Put the textport away by selecting the Textport off at the bottom of the Insight II screen. Correcting Mistaken Entries To remove the specification of an atom pair, you must set the Definition Action to Delete, the Definition Type to Atoms, the Deletion Mode to Links, and the Define Pick Level to Atom. Pick the two atoms in the molecule in the display area, which fills in the Overlap Spec 1 and Overlap Spec 2 parameters. You can remove 10 Search_Compare

51 Using the Overlap Pulldown a group of specifications in the same way, except that you need to set the Define Pick Level to Molecule, Subset, or Monomer/Residue. Specifications of individual rotatable bonds are removed in the same way, except that the Definition Type is set to Torsion and the Deletion Mode is not used. To remove all rotatable bond specifications pertaining to a group of atoms, set the Define Pick Level to Molecule, Subset, or Monomer/Residue and pick one atom in the molecule, subset, or monomer/residue. To remove all atom pair specifications that relate directly to a given atom (links), set the Deletion Mode to Atoms and fill in only Overlap Spec 1, by picking that atom on the screen. To remove specifications for all atoms that are linked (directly or indirectly) to a given atom, set the Deletion Mode to Chains before picking the atom. To remove all specifications for atom pairs or rotatable bonds, set the Definition Action to Clear and set the Definition Type to Atoms or Torsion. You then change the Definition Action back to Add to input any specifications that are still needed, as explained above. Executing the RMS Fitting Calculation After all atoms and rotatable bonds have been specified, you can accept these definitions by setting the Definition Action to End_ Definition. Before you execute the command to start the calculation, you may want to set three optional features first, to cause the superimposed molecules to be displayed in different colors and (or) to all be associated into an assembly. Toggling the Color_By_Molecule parameter on helps you to distinguish the molecules after they are superimposed. Toggling the Form_Assembly parameter on and entering a name for the Assembly Name parameter allows you to rotate or move them all together. Toggling the Create_Table parameter to on enables you to create a table listing RMS values between each pair of molecules. Executing the command then starts the overlap calculation. Search_Compare 11

52 5. Methodology After the calculation finishes, all molecules are automatically aligned on the screen, and the final value of the rms difference between their atomic coordinates is displayed in the information area. Executing the Field Fitting Calculation All molecules containing at least one linked atom (defined by the Overlap/Define command) will be used in the field similarity calculations. Note that the atom pairs are only used to indicate which molecules to use. Therefore, you only need to define one atom pair (any atoms) for each pair of molecules. There are two types of calculations you can perform. The first type calculates the field similarity using the molecules current position. You start this calculation by toggling the Optimize_Fit parameter off and selecting Execute. You may also move the molecules using the mouse and then execute the command to monitor the similarity. In addition, you can toggle the Create_Table parameter on and fill in the Table Name parameter to create a table listing the similarities between each pair of molecules. The second type of calculation optimizes the similarity by moving the molecules (molecular translation, molecular rotation, and torsion rotation). You start the calculation by toggling the Optimize_ Fit parameter on and selecting Execute. There are several optional features you may select. The Pre_Align parameter specifies whether to align molecules using their dipole moments and quadrupole moments to the best starting position, or to simply use the current position as the starting position for the optimization. Toggling the Color_By_Molecule parameter on helps you to distinguish the molecules after they are superimposed. Toggling the Form_Assembly parameter on and entering a name for the Assembly Name parameter allows you to rotate or move them all together. Toggling the Create_Table parameter on and entering a name for the Table Name parameter creates a table detailing the similarities between each pair of molecules. The overall similarity is also listed (in the upper left corner cell). 12 Search_Compare

53 Using the SC_Search Pulldown Using the SC_Search Pulldown The SC_Search pulldown is used to perform systematic conformational searching. This search method generates a set of molecular conformations that both are sterically reasonable and satisfy an optional set of user-defined distance constraints. The usual purpose is to apply information about one set of active analogs to delimit the interesting conformational space for another, more flexible analog. Systematic conformational searching runs as a background job. Insight II is used to set up and initiate the background job and, when the job completes, to load the search results for viewing and analysis. Two pulldowns are used in conformational searching: the SC_Search pulldown is used to set up search parameters, initiate the background job, and load the search results; and the Background_Job pulldown is used to set up background job parameters and monitor the job as it runs. The systematic conformational search procedure consists of four steps: 1. Setting up parameters for the search and the background job. 2. Running the background job. 3. Preparing the results for analysis. 4. Analyzing the search results. The first step is accomplished by specifying a list of rotatable bonds with the SC_Search/Set_Rot_Bond command. In addition, you may define a list of distance constraints with the SC_Search/Set_Distance command, define a distance map with the SC_Search/Set_ Dist_Map command, and specify energy criteria with the SC_ Search/Set_Energy_Params command. You can also specify scaling factors for distances between nonbonded atoms, between vicinal atoms, and between hydrogen bond donor/acceptor pairs, with the SC_Search/Scale_Radii command. Background job parameters are set with the Background_Job/Setup_Bkgd_Job command. Search_Compare 13

54 5. Methodology The second step is accomplished by executing the SC_Search/ SCS_Run command, which creates a command file containing specified information that was set up in Step 1 and submits it to the searching program. You can check on the status of the background job with the Background_Job/Completion_Status command. The searching program runs in the background and notifies you when it is finished. The third step is accomplished in several ways. Executing the SC_ Search/Load command loads the search results into Insight II in the form of a trajectory. You may set up runs that use or refine the results of an earlier search, which involves reiterating steps one and two. The fourth step depends on your particular purpose for generating conformations. The commands in the Vector_Map pulldown, Distance_Map pulldown, Conformer pulldown, and the Analysis or DeCipher modules are useful for analyzing the results of systematic conformational searches. The first three steps are presented in detail below. Each of the SC_ Search and Background_Job commands and parameters is explained. Standard search procedures are presented in the first section, and searches involving the creation or use of distance maps and energy criteria follow in a separate section. Standard Searches Setting up Parameters for the Standard Search and Background Job Three kinds of search parameters can be defined in standard searches: rotatable bonds, optional interatomic distance constraints, and optional radius-scaling parameters. Defining the Bonds to be Rotated The SC_Search/Set_Rot_ Bond command is used to create and edit a list of rotatable bonds. You can add rotatable bonds to the list by setting Rot Bond Operation to Add. Rotatable bonds are added to the list in two ways: by reading in a list of rotatable bonds from the command file of a previous search or by defining each rotatable bond interactively. 14 Search_Compare

55 Using the SC_Search Pulldown To read rotatable bonds from a prior input file, you need to set Rot Bond Mode to Prior_Input and specify the File Name (search parameter files have the extension.scs_prm). Then fill in the Molecule Name by picking the molecule in the graphics area of the screen, choosing its name from the Molecule Name value-aid, or typing in the Insight II name of the molecule containing the rotatable bonds. To specify rotatable bonds interactively, set Rot Bond Mode to User_Defined. Several other pieces of information are needed: the two atoms defining the rotatable bond (Atom 1 and Atom 2); the Range (in degrees relative to the current dihedral angle of the bond you want to rotate Relative_Range must be on); and the Increment for stepping through the range (also in degrees). Alternatively, you can define a particular dihedral by toggling Relative_Range parameter off and specifying the four atoms that define the dihedral (the parameters Atom 1, Atom 2, Atom 3, and Atom 4). In this case, you specify, following the IUPAC convention, the Range of absolute dihedral angle values (in degrees) through which to search and the Increment. Continue adding definitions in the same manner until all desired rotatable bonds are specified. Wildcards can be used to define a group of torsions in one command execution. To do this, toggle the Relative_Range parameter to on, and specify the torsions using the Atom 1 and Atom 2 parameters. This adds all valid single bonds (including ring bonds) specified by the two parameters. For example, if you type > MOL:*:CA and > MOL:*:* for Atom 1 and Atom 2, respectively, all valid single bonds connecting to all CA atoms in MOL are specified. You can remove rotatable bonds from the list by setting the Rot Bond Operation parameter to Delete, specifying the two atoms of the rotatable bond (the Atom 1 and Atom 2 parameters), and executing the command. You can remove all rotatable bonds from the list by setting the Rot Bond Operation parameter to Clear and executing the command. Search_Compare 15

56 5. Methodology You can review the current list of rotatable bonds by setting the Rot Bond Operation parameter to List and executing. After examining the information in the textport that appears, you can put away the textport by selecting the Textport off at the bottom of the Insight II window. Scaling Atomic Radii Each conformation generated during a search is checked for steric clashes before it is accepted as a valid conformer. Steric clashes occur when two atoms try to approach closer than the sum of their radii. The SC_Search/Scale_Radii command is used to set the radii at which steric clashes are considered to occur. You can apply different scaling factors to the van der Waals radii of all atoms that are neither vicinal nor a member of a hydrogen-bonding pair (VdW), to the radii of vicinal (1 4) atoms (Vicinal), and to the radii of hydrogen bond donor acceptor pairs (H_Bond). Scaling factors less than 1.0 allow atoms to approach each other more closely than their unscaled van der Waals radii. Setting Distance Constraints The SC_Search/Set_Distance command is used to create and edit a list of upper and lower bounds for particular interatomic distance constraints. These distances constitute additional acceptance criteria for conformations produced by the search. You can add distances to the list by setting the Distance Operation to Add. Distances are added to the list in three ways: By setting the Distance Mode parameter to Prior_Input and reading in a set of distance constraints from the parameter file of a previous search. By setting the Distance Mode parameter to Prior_Output and deriving distance constraints from the results of a previous search. By setting the Distance Mode parameter to User_Defined and specifying the atoms and bounds for each distance constraint. To read distance constraints from a Prior_Input file, you specify the File Name (of an.scs_prm file) and the Molecule Name for the molecule to which the distance constraints should be applied. To read distance constraints from a Prior_Output file, you specify the File Name (of an.scs_tor or.xdr_tor file), the two atoms whose interatomic distance you want to constrain (the Atom 1 and Atom 16 Search_Compare

57 Using the SC_Search Pulldown 2 parameters), and the two atoms of the previous search (these need not be from the same molecule) whose longest and shortest interatomic distances should become the upper and lower bounds of the new distance constraint (the Prior Search Atom 1 and Prior Search Atom 2 parameters). The minimum value of their distance during the previous search becomes the lower bound, and the maximum value of their distance becomes the upper bound. If the Interactive_Calc parameter is on, the bounds are calculated immediately and printed in the information area. If Interactive_Calc is off, the bounds calculation is deferred until the search job is initiated. To enter distance constraints in the User_Defined mode, you specify the two atoms whose interatomic distance you want to constrain (Atom 1 and Atom 2) and the Lower and Upper bounds of acceptable values for the distance (in angstroms). You can remove a distance from the list by setting the Distance Operation parameter to Delete and specifying the two atoms (Atom 1 and Atom 2) to remove from the list. Setting the Distance Operation parameter to Clear removes all distance constraints from the list. You can review the current list of distance constraints by setting the Distance Operation parameter to List. After examining the information in the textport that appears, you can put away the textport by selecting Textport off at the bottom of the Insight II window. Setting Up the Background Job Use of the Background_Job pulldown is optional. If it is not used, the default is to run all jobs on the local host in Cont_Insight mode. If you prefer the latter mode, you do not need to read the remainder of this subsection. When the Setup_Bkgd_Job command is used, the background job list shows only those background jobs that are both run from the current module and can be run on a remote host. If the module contains only one job, the parameter is automatically filled in. The list of hosts shows only those hosts that are associated with that background job in the background_job_hosts file at your site. It is possible for you to specify a remote host that is unavailable (off line, for instance) or for which you have no login account. Search_Compare 17

58 5. Methodology Every background job submitted via the generic background utility is assigned a job number. This number is displayed in the information area when the job is submitted (e.g., Starting systematic search job on iris5 as job 1). You should note the job number when the job is submitted, since it can be used later to check on the job s completion status or to kill the job. The Setup_Bkgd_Job command does not actually run the command; it simply records your host and Execution_Mode preference. The default host is Local. Your selected host and Execution_ Mode are used for any subsequent background jobs of the specified program for the duration of the Insight II session. When you start up a new session, all background job parameters are reset to their default values. The Execution_Mode parameter allows you to choose to run a given background job concurrently (Cont_Insight), run the job interactively (Wait_For_Job), or simply create the necessary command files to submit the job, but not actually execute them (Cmd_ File_Only). The Send_Mail parameter instructs the system to send you an electronic mail message upon completion of the background job. This parameter is not active if Execution_Mode is set to Wait_For_ Job. You may find Send_Mail useful when running long jobs where you exit Insight II before the job completes. The Save_Cmd_Files parameter allows you to save the command file used to submit the background job (bkgd_job_run_name#.csh). Otherwise this file is deleted when the job completes. This parameter is not active when Execution_Mode is set to Cmd_File_Only. All background jobs return a completion status. The completion status is an integer code that indicates success, failure, and/or reason for failure of the job. The status code is always displayed when you are notified that the job has completed. If you consistently want to send background jobs to another host, you can modify your personal Insight II start-up file to invoke Setup_Bkgd_Job for each module/background job(s) that you want to automatically assign. Note that you must first change to the module in which the background job s interface is found before using Setup_Bkgd_Job to set a preference for that background job. 18 Search_Compare

59 Using the SC_Search Pulldown The Completion_Window parameter can be used to prevent the notification window from appearing when the background job completes. The default setting is on. Running the Background Job for Standard Searches Initiating the Search The SC_Search/SCS_Run command is used to estimate the number of conformations that will be generated with the current set of parameters, or to initiate a systematic conformational search. Only those SCS_Run parameters that are relevant to standard searches are explained here (the parameters used in advanced searches are explained under Advanced Searches, page 21). To estimate the number of conformations that will be obtained with a particular set of parameters before actually performing the search, set Estim_Num_Confs to on. The estimate takes into account all the currently defined parameters applicable to the molecule specified in Molecule Name. However, no search is initiated. To initiate a standard search, turn Estim_Num_Confs to off and set Search Type to Standard. You need to specify the molecule on which you want to perform a search (the Molecule Name parameter), and rotatable bonds must have been defined for that molecule before a search can be performed. Existing distance constraints and scaling factors are taken into account in the search calculations. In addition to Search Type, you can set the Search Method parameter, which may be set to one of two values. Setting this to Torsion_ Search indicates the search molecule will be used to generate a new set of conformations from a torsion search that uses the torsions and step values specified by the Set_Rot_Bond command. If the Search Method parameter is set to Prior_Search, the set of initial conformations is taken from an existing trajectory file (XDR_ TOR or ARC) for the search molecule. With this setting some of the SCS_Run command parameters are renamed or not available, which reflects the fact that you are running the search to filter or process an existing set of conformations. By default, a fixed atom (that is, one whose coordinates remain fixed during the calculations) is chosen by the algorithm. However, you may want to choose a specific atom to remain fixed, by Search_Compare 19

60 5. Methodology Killing a Background Job specifying it with the Anchor Atom parameter. You also need to supply a Run Name. This name serves as the prefix for all the files generated by the search. Exactly what files are generated depends on the options that are set. Monitoring the Job The Completion_Status command has three modes of operation. The One_Job option displays a brief message in the information area of the screen, indicating whether a specific job has completed. Certain background jobs generate a status file containing additional information while they are running. If this additional status information is available, it is displayed in the textport. If All_Jobs is chosen, the job number, job name, run name, status code, and job status are displayed in the textport for every job submitted during the current Insight II session. The Look_Up_Status option is used to find the meaning of a return status code. The Report_Mode parameter is used to indicate what information you would like the command to return: status of one job, status of all jobs, or the meaning of a return status code from a particular job. The Job_Number parameter becomes active when One_Job is selected. It is used to specify a specific background job that you want to monitor. The Background_Job and Status parameters become active when Look_Up_Status is chosen. They are used to specify a status code that you would like to look up. The Kill_Bkgd_Job command is used to stop execution of a background job by killing the process in which it is running and, optionally, deleting its output files. The Job_Number parameter is used to specify which background job to kill. A value-aid containing a list of all currently running background jobs is provided. If the Save_Output parameter is toggled on, then all output files generated by the background job are saved when the job is killed. 20 Search_Compare

61 Using the SC_Search Pulldown Loading Search Results into Insight II The default setting for this parameter is off, in which case all output files are deleted. Please note that, if you kill a background job and keep the output files, the files contain only the partial results of the searching process. The SC_Search/Load command is used to load the results of a search back into Insight II for further analysis. Search results are usually contained in an.xdr_tor or.arc file. Ordinarily, the entire trajectory (i.e., all the conformers found by the search) is loaded, but you can choose to load only selected parts of large sets of results. You select which file to load with the Torsion File parameter and tell Insight II which molecule the file applies to with the Molecule Name parameter. To load a complete set of search results, set the Selection Mode to Range. The default values for the Trajectory Spec parameters (Start, Last, and Step) should be accepted (1, end, and 1, respectively). To limit the amount of data loaded, you may do one of three things: Leave the Selection Mode as Range, but change the values of Start, Last, and/or Step. Choose Specified as the Selection Mode, and specify the desired trajectory frames in Frame Spec as a single number (n) or a range of numbers (n-m, >n, <n, >=n, or <=n, where n and m are integers). Choose From_File as the Selection Mode, to indicate that the desired frame numbers are specified in an ASCII file (specified as the File Name) that you have created with any editor or by using the Output_list option of the Conformer/Display command (see page 34). Advanced Searches Setting up an Advanced Search and Background Job Several kinds of search parameters can be defined for searches involving distance maps or energy calculations: rotatable bonds, Search_Compare 21

62 5. Methodology interatomic distance constraints, a distance map, radius-scaling parameters, and criteria for energy filtering.all of these parameters are optional, with the exception of rotatable bonds. Table 1 summarizes the process of setting parameters for various types of searches using the distance map and energy features. Note that some of these search types cannot be combined, such as distance map creation and energy calculation. Rotatable bonds, interatomic distance constraints, and radius scaling, as well as background job parameters, are set up exactly as described for the standard searches. You may refer to Setting up Parameters for the Standard Search and Background Job on page 14 for further information on using these commands. After an advanced search, results are loaded in the same way as they are in standard searches (see above). Setting up Distance Map Calculations The SC_Search/Set_ Dist_Map command is used to set up a distance map (defined in Chapter 2, Theory). Each pair-wise interatomic distance in the map is defined by its lower and upper bounds and is broken into increments (the increment size is the resolution). When a distance map is created during a search, each sterically allowed conformation is mapped to a specific point of the map, which is then recorded as a valid point in the distance map. (A new distance map is set up with the Set_Dist_Map command, but is not actually created until a distance-type search is run with the SCS_Run command.) You set up a new distance map by setting Dist_Map Operation to Add and Prior_Search_Dmap to off, then specifying the atom pair (the Atom 1 and Atom 2 parameters) that defines one distance, the Lower and Upper bounds for that distance, and the Resolution. Continue defining atom pairs in the same way, until you have as many atom pairs as you want to monitor. Each distance in a distance map may have a different resolution. All distances are in angstroms. A distance map can also be used as a constraint on a subsequent search, which means that only conformations that map to a valid point in the constraining distance map are accepted. This is particularly useful for identifying common geometries among a series of 22 Search_Compare

63 Using the SC_Search Pulldown Table 1 active compounds. To constrain your distance map to the results of a prior search, set the Prior_Search_Dmap to on. The resolution and bounds for your new map are then derived from the distance Search_Compare 23