PyRy3D Manual
Contents
1. As a result when o sort options were used the user receives a text file with model names sorted according to PyRy3D score NOTES e all input files required for this tool should follow the same format rules as PyRy3D itself e much more options of ranking models are available in PyRy3D GUI launched with UCSF Chimera Extension see chapter 6 6 for details Also GUI provides graphical interface to run all these options 6 5 Input Generator Preparing input files for PyRy3D can be sometimes hard and time consuming For this reason we implemented a tool that automatically generates files ready for PyRy3D run called InputGenerator py distributed together with PyRy3D source The simplest way to use it is by running regular command line program without delivering MultiFASTA sequences for components In this case InputGenerator py is called straightaway and PyRy3D generates this file for the user based on coordinates in PDB files However this tool offers a lot more options such as e renaming residues and chains of 3D coordinates of components e defining disordered flexible regions e coming back to original names and numbering of components operation reverse to generating input for PyRy3D e renaming chains and renumbering residues in files with restraints according to changes applied to structures and sequences e and many more NOTES e much more options of automatic generation of input files for PyRy3D program are available in Py2. include disorder flexibility while modeling IDENTIFY DISORDERS True SIMUL DD FREQ 0 5 0 5 4 PyRy3D Output As a result of the simulation PyRy3D returns a set of PDB files comprising the best scored conformations generated during the modeling Files are saved on disc with frequency defined by the user parameter WRITE_N_ITER in configuration file and they are named according to the following formula Outname score iteration _temperature pdb Where Outname name of output assigned by the user in o option by default pyryresult Score score assigned by PyRy3D the closer to zero the more model fulfills the restraints Iteration number of iteration in which the particular model was created Temperature value of temperature parameter indicating the annealing progress in iteration when this model was saved 5 Examples In this chapter we present how to run a command line tool to perform some specific modeling tasks The same operations can be done with server and GUI We would like to recommend e Acommand line tool for extensive runs of PyRy3D multiple runs with large number of steps e GUI for learning how PyRy3D works but also to select the most accurate values of settings parameters e Server for running single runs of PyRy3D they might consist of large numer of steps Multiple runs of server might lead to increase a queue 1 Docking structures with restraints gt gt python pyry3d py fast d c
3. applied to all complex components All these files can be used or reused in various post processing tasks such as models clustering Additional tools that accompany this distribution are explained in the Section 6 titled Additional Tools 2 Command line options To get help with PyRy3D command line just type python pyry3d py help and a list of all available options will appear with short descriptions s input file sequence The sequences of complex components are written in a single text file in MultiF ASTA format where each sequence is called as the corresponding chain ID in PDB file the defline of each sequence starts with gt and chain ID c simulation config file The config_file contains a variety of important program s settings see section 2 0 for details 0o output files _ basename If this is not specified then the program will create a pyryresults directory and copy output files there r restraints _ file This is the file specifying restraints The details on the format of the file for restraints Filtrest3D are discussed below The format is somewhat strict and should be followed or an error will be issued d archive with structures This is tar or tgz archive containing 3D coordinates of complex components Required format is PDB and only one chain can be stored in a single PDB file m density map file This is the shape of the complex defined by the electron density map Negative st
4. as number of clashes violation of restraints or leaving empty spaces in density map default available rae parameter name description value values Weight of a penalty for atoms residues outside simulation area OUTBOX 0 to 10 Weight of a penalty for free spaces inside density MAP_FREESPACE map 0 to 10 Weight of a penalty for collisions only CA and C4 CLASHES atoms are considered 1 to 10 CLASHES_ALLATOMS Weight of a penalty for collisions all atoms are 1 to 10 considered Weight of a penalty for violation of distance RESTRAINTS i restraints 0 to 10 Weight of a penalty for occupation of map points with low density values DENSITY 0 to 10 Weight of a penalty for disagreement with SAXS curve CHI2 0 to 10 Weight of a penalty for disagreement with user RGE defined radius of gyration RG_VAL 0 to 10 Weight of a penalty for violation of symmetry SYMMETRY restraints 0 to 10 Movements frequencies Describe how often particular types of movements are applied to components default available er parameter name description value values ROTATION_FREQ frequency of rotations ROTATION_COV_FREQ frequency of rotations around covalent bonds TRANSLATION_FREQ frequency of translations EXCHANGE_FREQ frequency of components exchange SIMUL_DD_FREQ 0 25 fom ROTATION_ALL_ FREQ 0 25 pe ROTATION_WHOLE_FRE 0 25 oor Q e SLATION_ALL_FR 0 25 Otol frequency
5. files folder the folder that contains a set of models generated by a single PyRy3D simulation These models will serve as frames for the animation A fraction of models that will serve as frames in the Record I n of the files the user can define the fraction of models that will constitute the animation s frames For example if the user has generated 1000 files but wants the animation to be faster and only show 100 of them the n number should equal 10 Record 1 10 of the files After defining the fraction the Number of frames label should inform the user about the resulting total number of frames that will be recorded The output movie s file path using the Save movie file as field The output movie s format using the Movie format menu The folder that will store frame images before generating a movie file Chimera must first take screenshots of each of the frames and store all of them in one location The user can define this location using the Save images in field Movie size the width and height of the output movie Create ranking of multiple complexes The PyRy3D Chimera Extension allows for a quick evaluation of multiple complexes in order to create a ranking based on criteria chosen by the user e g distance restraints Thanks to this tool a user can evaluate complexes based on different set of PyRy3D configuration parameters level of clashes quality
6. identification of most probable solutions The input for clustering tool delivered with PyRy3D is just a folder containing a set of models saved in PDB files Typically those PDB files originate from multi instance runs of PyRy3D The output of clustering is a set of PDB files corresponding to subsequent clusters Usage gt gt cluster complexes py lt options gt o output i input t threshold n struct number Example usage gt gt cluster complexes py i alternativemodels t 10 n 1000 clustering_result txt where alternativemodels is a name of folder containing alternative models to be clustered 10 is the RMSD threshold in the set for clustering 1000 is the number of best scored models to be considered while clustering clustering result txt isa name of text file with clustering results there are also some additional options s for selecting best N structures criterion from selection of pyry3d for score assigned to models by PyRy3D cc for cross correlation coefficiency m for selecting clustering criterion from selection of RMSD GDT_TS and TMSCORE r for choosing representation of models here clustering can be performed for full atom models r fa only Calfa or C4 atoms r ca or for centers of mass r sphere sort this option generates new folders for five largest clusters containing PDB files assigned to those groups As a result when o sort options were used to run the t
7. of simulation of disordered fragments frequency of rotations where all components are moved simultaneously frequency of rotations where all components are moved simultaneously around common centre of mass frequency of rotations where all components are moved simultaneously Information about complex Set of different parameters describing modeling complex such as density threshold of density map values measured with Eo g or definitions of mobile and immobile components Z foe THRESHOLD oo SAXSRADIUS foo Value set must occur in a density map positive float value positive float value e g D movable 5 5 5 0 10 1 0 1 10 10 100 1 0 1 1 530 To fix a molecule use fixed parameter no default values paramet er is optional MOVE_STATE ChainName ChainBound 1 gt ChainBound2 no default AtomBound Number1 AtomBound Namel AtomBound Number2 AtomBound Name2 e g values paramet er is optional COVALENT_BONDS Eed oe parameter name name description Ee oe how many complex volumes will describe density map e g KVOL 2 indicated that map volume will be twice as big as complex volume calculated from its sequence float value describing density map threshold float value describing dammy atom radius float value describing radius of gyration for a complex CRYSOL_PATH path to CRYSOL binaries Indicates limited values of moves rotations translat
8. or 5 10 A between the Ca residue X in chain A and any residue of the fragment Y Z in chain B or sets of residues e g 10 A between B sheet A171 C182 of chain A and an a helix A57 D62 of chain B e If C alpha atoms of V10 and D34 should be in at least 4 5 Angstroms distance dist V10 A D34 X gt 4 5 e To restraint SG atom of the residue C140 to be within 4 angstroms from OE1 atom of residue E33 dist C140_near_E33 C140 A E33 E SG OE1 lt 4 e To define distance between two regions here any residue in range 163 175 in chain Z should be within 4 8 Angstroms from any residue 306 310 in chain A dist E163 F175 Z 306 310 A lt 4 8 Surface accessibility Surface accessibility allows for defining entities atoms residues components fragments that are positioned on the surface of the whole complex such as exposed enzyme active sites surface_access 163 165 Z lt 1 0 Solvent accessibility The residue burial exposition to the solvent may be given as a range of the relative accessible solvent area ASA expressed in percentages e g residue X is exposed in 40 50 to define residues exposed to solvent in a particular component e To define aminoacid 45th in chain A as having between 40 to 90 accessibility Glycine 56th as having 30 to 60 accessibility and alanine 20th as having between 80 to 90 accessibility use the following formu
9. Glu45 40 90 Restraint weights Default restraint weight is 1 0 but it can be changed DEFAULT_WEIGHT lt 2 all penalties defined in the restraint file will be multiplied by 2 dist V10 A 34 36 B CA lt 4 5 weight lt 2 C alpha penalty will be multiplied by 2 0 Comments e the residue numbers specified in the restraints file must correspond to ones in input PDB file e chain id is required in all restraint types to indicate which component the user refers to 3 4 Format of the configuration file c simulation config file The argument is a file containing simulation configuration parameters With this option the user can configure the simulation in a more advanced way The configuration file is a text file with one parameter specified in each line There can be blank lines each line must contain a command or must be empty Comments starts with Also if no configuration file is provided by the user PyRy3D is run with default parameters see the tables below in this paragraph Config file options These are some of the options that can be included in a typical configuration file STEPS N This specifies the number of unified iterations in a simulated annealing simulation WRITE N ITER N This specifies how many iterations should occur before the conformation is saved on disk or appended to the trajectory file In general there probably should be at least 10 000 iterations of PyRy3D bef
10. PyRy3D version 4 6 User Manual 1 PyRy3D Functionality PyRy3D is a method for building multi resolution models of large macromolecular complexes using restraints derived from biochemical data The components proteins nucleic acids and any other type of physical objects including e g solid surfaces can be represented as rigid bodies e g based on atomic coordinates of structures determined experimentally or modeled computationally or as flexible shapes e g for parts whose structure is dynamic or unknown PyRy3D run can be initiated with input files that include sequences of all complex components in MultiFASTA format and structures of some components ar tar archive containing files in PDB format Additionally information about restraints can be provided in Filtrest3D format as well as a complex density map in CCP4 format or SAXS curve as dat file The output from PyRy3D consists of a set of pdb files with generated models and a file containing information about PyRy3D parameters used and PyRy3D scores for models saved on disk All models generated by PyRy3D are saved in PDB format and the filenames contain information about a score assigned by the program number of simulation step and current temperature a parameter characteristic for simulated annealing algorithm For the user s request a trajectory of complex simulation trace file with extension trafl can be produced and a text file with a history of all movements
11. Ry3D GUI launched with UCSF Chimera Extension see chapter 6 6 for details 6 6 GUI We have created a tool that associates PyRy3D with UCSF Chimera a popular program for interactive visualization and analysis of molecular structures PyRy3D Chimera Extension is a plugin that provides a user friendly graphical interface letting the user to generate a set of PyRy3D input files interactively or to calculate a score for a set of different components arrangements based on default or user defined parameters directly from the extension s interface It also offers many features designed to make the understanding and interpretation of PyRy3D results much easier PyRy3D Chimera Extension installation The graphical user interface that we developed for PyRy3D is an extension to a popular molecular viewer called UCSF Chimera It is not a standalone program so in order to use it you must first install the viewer STEP 1 Download and install UCSF Chimera version 1 9 or higher STEP 2 Download BioPython 1 63 here and unpack it Do not install it yet UCSF Chimera comes with its own Python copy In order to use BioPython within Chimera on Linux you need to install the library using Chimera s Python copy for Windows Install BioPython 1 63 using the regular installer for Linux Install BioPython 1 63 using Chimera s Python copy Yes install BioPython even if you have BioPython on your system already 1 Open the terminal 2 Change y
12. ain map can also be provided here PyRy3D accepts files in ccp4 and mre format y SAXS curve file This is the shape of the complex measured with SAXS and saved as a curve in dat format f full atom representation This command allows to save output models in full atom representation even if reduced representation was used during the simulation v history of movements This option allows to save a history of movements applied to components during the simulation The file is also required to reconstruct full atom representation of the model e energy plot This option creates PyRy3D score plot t eran file This option allows to save a simulation trajectory 2 1 Format of sequence input file s input file sequence The sequences of complex components are written in a single text file in MultiFASTA format where each sequence s header contains its corresponding chain ID in PDB file the defline of each sequence starts with gt and chain ID For example gt A MMMET gt B AAUUCCGG gt X_protein WERTY gt D_DNA AAATCG A sequence corresponds to protein structure with chain id A B to RNA structure with chain id B X protein to protein with unknown structure and D_ DNA refers to DNA sequence with no 3D data no PDB provided by the user for X and D sequences 2 2 Format of structure input files d archive with structures This is a single tar or tgz archive containing 3D coordina
13. built the full atom model from the history of movements used during simulation Where history txt isa name of file where history of modeling movements is saved in format recognizable by PyRy3D Fullatom pdb is a name for PDB file where the fullatom model will be saved NOTES e Fullatom representation is reproduced only for the best scored model generated during a single run of PyRy3D e Fullatom pdb and history txt files are also used in PyRy3D GUI to record a movie of simulation 6 Starting modeling procedure from predefined orientations of some components gt gt python pyry3d py fast d coordinates tar c config file txt If the user wants to start a simulation from predefined model it is possible to achieve it with PyRy3D in two ways e Ifall components should have predefined starting positions set the START_ORIENTATION parameter in configuration file as True by default is False and modeling starts with random positions of components START_ORIENTATION True e If only some components should have predefined starting positions use MOVE_STATE parameter and define how much these components can change their positions during simulation MOVE STATE movable A 5 5 5 10 10 10 360 360 360 1000 1000 1000 5 30 7 Limiting movements of some components gt gt python pyry3d py fast d coordinates tar c config txt Movements of components can be either disabled e MOVE STATE B fixed in configuration file MOVE STATE parameter is s
14. epared file Map file an electron density map Sorting methods using the Sort results by menu on the bottom of the tab the user can choose if the ranking should be based on the overall PyRy3D score or on one of its components After defining all the necessary parameters the user can click the Create ranking button to start evaluating multiple complexes Clustering complexes An option to cluster models generated from multiple PyRy3D simulations is also available from the PyRy3D Chimera Extension The last tab of the Extension titled Clustering contains a set of parameters necessary to perform a clustering of multiple complexes Input directory the directory that contains the files that will be clustered Map file electron density map file Score type the score that will be used to arrange the files in the output file Measure type the measure that will be used to compare the structures Representation the models can be compared based on their full structures fa on their backbones only ca or on their approximate shapes sphere Number of structures the number of highest scored structures from the indicated directory that will be shown in the clustering output Clustering threshold the threshold that will be used to group similar models into clusters Output directory the directory that will contain the
15. er and more understandable In order to achieve this goal we ve created a set of features that allow for visualizing how PyRy3D scores particular structures with given set of parameters The user can show every element of PyRy3D scoring function such as regions outside the simulation box or regions inside the density map The Results display window pops up after finishing the automatic evaluation of visualized complex and after finishing a PyRy3D simulation It contains a PyRy3D score and its individual components and a set of buttons allowing the user to Display the simulation box Display the simulation grid Highlight the complexes disordered regions Highlight collisions between components Highlight regions outside the simulation box Highlight regions inside the density map Highlight regions of the map that are empty Highlight regions outside of the density map Display user defined restraints if provided Each feature can be visualized in user s color of choice After a user picks a color from the palette the square button will displayed the user chosen color Generating input files PyRy3D Chimera Extension offers a possibility to quickly generate files that can be used to perform simulations using the PyRy3D server or standalone version Even though you can run PyRy3D simulations from the Chimera Extension s interface it s much more efficient to perform the simulations using the original command
16. et as fixed for chain B Or only limited e MOVE STATE movable A 5 5 5 0 1 0 1 0 1 10 10 10 0 1 0 1 1 5 30 chain A can move but not more than defined ranges 8 Modeling disordered flexible fragments of some components gt gt python pyry3d py fast d coordinates tar s sequences fasta c config file txt In this particular case the user must provide several files coordinates tar an archive with PDB coordinates of components sequences fasta MultiFASTA file with full length sequences of all components config_file txt a text file with chosen values of PyRy3D parameters When modeling disorder flexibility the user must set two parameters IDENTIFY DISORDERS True Which turns on the procedures to find missing fragments in PDB files that will be treated as disordered flexible during the simulation SIMUL DD FREQ 0 5 0 1 Which defines how often a conformation of disordered flexible parts will be changed at the beginning and at the end of simulation 0 5 and 0 1 respectively NOTES Be very careful when preparing input data for modeling with flexible disordered regions Follow the guidelines below Sequences in MultiFASTA file must contain full length sequences with sequence of disordered fragments included Structures in PDB files must not contain any coordinates for disordered flexible regions remove any lines corresponding to those atoms Numbering of residues in PDB files must be in agreement with the number
17. et of clusters 6 4 Ranking Using rank_models py tool attached to PyRy3D distribution the user can create a ranking of alternative models according to PyRy3D scoring function For instance let s consider that a user generated a large set of alternative complex models using docking tool that does not allow to define distance restraints or other restraints such as symmetry solvent accessibility etc With help of rank_models py tool one can evaluate these structures according to their agreement with distance restraints from chemical crosslinking FRET or some other sources Also other types of restraints can be considered for this evaluation Additionally a ranking can be created with different PyRy3D configuration parameters values such as weights for clashes penalty or for quality of fit into the map e g to check which model from the set of alternatives fits most accurately to the map Usage gt gt rank _models py lt options gt m map d structures s sequences c config r restraints Example usage gt gt rank Models py m map ccp4 d structures s Sequences asta c config txt r restraints txt where m map ccp4 electron density map of the complex optional d structures name of a folder containing models to be ranked s sequences fasta name of file with sequences of components c config txt file with PyRy3D configuration parameters r restraints txt file with restraints
18. ing in MultiFASTA file Here are some examples o if residues 1 5 in chain A are disordered their sequence is provided in FASTA file but PDB file containing chain A starts from residue number 6 o if residues 50 55 are disordered their sequence is provided in FASTA file but PDB file contains residues 1 49 and then from 56 till the end of the chain o if a structure of entire component is unknown then provide the full length sequence in MultiFASTA file but call this sequence by chain name followed by and a molecule type e g A_protein B_RNA C_DNA to indicate that this component will be treated as a simulated volume Also no PDB file is provided for such a component General remarks The number of structures you will obtain from a single run is always STEPS WRITE_N_ITER 1 PyRy3D can be run without providing a configuration file by the user In such a case the program is run with default parameters with values as mentioned in 3 4 Chapter of this Manual For each run PyRy3D automatically generates pyry log text file with values of parameters used and detailed scores assigned for models saved on disk 6 Additional Tools Additional tools are also included with the distribution 6 3 Clustering Clustering is a tool for processing a set of alternative models by finding and grouping similar structures together into groups called clusters etc Benchmarks demonstrated that clustering provides is very useful in
19. ions for particular component is used to indicate which components are linked by a covalent bond A ZSD 10 CA 11 CA True if user sets start conformation False if True False not True if user wants PyRy3D to add missing or IDENTIFY_DISORDERS False True False disordered fragments into the structures False if not Simulation parameters default available ele parameter name description value values Genetic or Simulated Annealing for Simulated simulated Annealing annealing default or ReplicaEx change for replica exchange roulette roulette cutoff is available only for genetic simulation mode tournament each X steps replicas will be exchanged by default every 10 of simulation steps SIMMETHOD REDUCTMETHOD REPLICAEXCHANGE_FR EQ simulation algorithm integers 400 375 300275 stot REPLICATEMPERATURE integer S 230229 values of 200 175 150 125 any size 100 ANNTEMP from range annealing temperature in simulated annealing 1 to 100 procedure min 1 max 3 STEPS ne een Corresponds to number of simulation steps to ee perform not limited CA only calfas c4 default cacb Type of structure representation list of temperatures for all replicas COMPONENT_REPRESE NTATION coarse grain 3p 3points fa es a C GRIDRADIUS Radius of single grid cell do limits Parameters indicated how many ti
20. la access 45 A 40 90 Gly56 A 30 60 A20 80 90 A weight lt 1 5 Relation Relation restraint is specific to PyRy3D it is not available in Filtrest3D itself and is used to compare two distances e g data from FRET like a distance between residues X Y of chain A and residues U Z of chain B is larger smaller equal to a distance between the Ca residue X in chain C and any residue of the fragment Y Z in chain D e Relation restraint is applied to define relation between two distances e g distance between residue 3 in chain A and residue 5 in chain B should be smaller than distance between residue 30 in chain A and residue 500 in chain B relation 3 A 5 B lt 30 A 500 B Distance from point The distance can be defined between a residue or a set of residues and a point in 3D space PointDistance pointdist 100 101 D 20 0 20 0 20 0 lt 5 5 Symmetry Symmetry allows to provide distances that should be of equal length in the complex This particular restraint can be applied to introduce symmetry between identical copies of oligomeric assemblies symmetry 1 AM 1 B 0 1 B 1 GC CA CA 1 A 1 HN Conjunction and alternative To declare composite restraint having score of minimum or or maximum and of two other restraints the following restraint blocks can be used and access Gly56 30 60 access
21. line based PyRy3D software The Extension s third tab Input Gen contains a set of checkboxes By checking and un checking those checkboxes one can choose the types of input files that will be saved in the older of choice selected with Generate files in option and clicking the Generate defined input files The types of input files are Structures generated using the PDB files loaded into Chimera using the first tab Density map simple copy of map loaded into Chimera using the first tab Sequences automatically generated based on the structures loaded into Chimera Restraints file is copied from a localization indicated in Restraints file field Configuration file generated using the parameters defined by the user in the Parameters window Generating movies After the simulation is finished a user can investigate the whole process thanks to the animation generator The tool lets to generate animations that can be saved in one of many popular video file formats A user can find the parameters necessary to generate an animation on the fourth tab called Animations The camera s position does not change during the entire animation s recording For this reason the user has to first define the camera s angle by opening and positioning his density map in the Chimera window using standard manipulation tools To generate a move a user is supposed to define PyRy3D output
22. mes SIMBOX simulation box diameters is bigger than the longest distance inside a density map mor mor 1 to Maximal rotation angle for single component move MAXTRANS Maximal translation vector for single set component move Specific parameters to control simulation progress default available bugis parameter name description value values WRITE_N_ITER how often PyRy3D save a model on disk PARAMSCALINGRANGES psa a eee should parameter na foa ere ranon fea O SS aanas fe OO ee Some examples use density map volume equal to 10 complex volumes KVOL 10 use density map with density values gt threshold equal to 1 5 THRESHOLD 1 5 perform 50 000 simulation steps STEPS 50 000 assign default value for a particular component THRESHOLD X do not move component B during simulation MOVE STATE B fixed limit movements of component B MOVE STATE B 555 1 1 1 20 20 20 100 100 100 0 5 5 5 refers to rotation around X Y Z axis in single move X up and down Y right left Z back forward 1 1 1 refers to translation in single move 20 20 20 refers to maximal change in X Y Z coordinates during simulation due to rotations 100 100 100 refers to maximal change in X Y Z coordinates during simulation due to translations 0 0 are specific to rotation around covalents bonds start simulation from components orientations as provided in PDB files START ORIENTATION True
23. ms sets of atoms or even X Y Z points in 3D space Restraint options A restraint can be thought of as a flexible tether that drives the selected atoms towards points located within a certain distance by applying a penalty for distances that deviate from that range It can also provide a reward when a desired distance is achieved The penalty and reward are positive and negative contributions to the total energy of the simulated system respectively Restraint command formats The restraints format implemented in PyRy3D was based on Filtrest3D format The description of the latter can be found under the following website http filtrest3d genesilico pl readme html In PyRy3D some new types of restraints occurred e g surface accessibility distance from point in 3D space relation or symmetry General restraint file syntax is Comment DEFAULT WEIGHT lt 1 0 RESTRAINT TYPE NAME RESTRAINT DECLARATION RESTRAINT DECLARATION There are following restraint types restraint type name in parentheses e distance dist e surface accessibility surface_access e solvent accessibility access e distance from point in space pointdist e symmetry symmetry e relation between distances relation e logical operators and or Distance restraints Distance restraints allow to define the permitted range of distances between the residues e g 5 A between any atom of residue X and residue Y
24. of fit into map or verify structures built with other tools imagine having 1000 models generated with docking tool without any prior data about their possible interactions and then creating a ranking with PyRy3D that sorts models according to their agreement with external data such as distances from chemical crosslinking or FRET The Ranking tab is the fifth tab of the PyRy3D Chimera Extension Within this tab a user can define The ranked models source using the radio buttons in the first field of the tab a user can choose to load the input models from Chimera s window or from a previously prepared folder In the latter case the files should be already tared using for example the Extension s input generator The configuration file s source using the tools available in the second field the user can define if the configuration file is supposed to be generated automatically based on the Parameters window or if the user s previously prepared file should be used If experimental restraints will be used if so the user can check the Use restraints file from hard drive radio button and point to the file s location using the Restraints file field The sequences file s source using the tools available in the fourth field the user can define whether the sequences are supposed to be generated automatically based on the structures loaded into Chimera or if they should be loaded from a previously pr
25. ool a user will obtain new folders for five largest clusters containing PDB files assigned to those groups text file containing cluster members Clustered conformers number 3 poz2dna43_ 238 979_225000_4 46819e 06 pdb score 238 979 poz2dna46_ 240 066_295000_1 44237e 08 pdb score 240 066 poz2dna4_ 240 49_30000_0 0154917 pdb score 240 49 Clustered conformers number 3 poz2dna24_ 244 255_40000_0 00280232 pdb score 244 255 poz2dna39_ 245 184_285000_0 000100337 pdb score 245 184 poz2dna26_ 248 681_125000_0 000107724 pdb score 248 681 Clustered conformers number 2 poz2dna34_ 234 2963_300000_4 13923698943e 05 pdb score 234 2963 poz2dna54_ 235 7814_300000_6 45055979476e 06 pdb score 235 7814 Clustered conformers number 2 poz2dna92_ 239 959_80000_0 00641644 pdb score 239 959 poz2dna45_ 244 967_160000_8 1394e 07 pdb score 244 967 where for each cluster a number of members is given 3 3 2 and 2 in the above example and names of PDB files belonging to these groups with corresponding scores assigned by PyRy3D NOTE the current implementation of the clustering tool doesn t provide any cut off for a number of clusters Actually it identifies subsequent clusters until it exhausts the input data In such cases only the first few clusters are meaningful the remaining clusters are usually insignificant The user should inspect the clustering output to identify the percentage of jst md 3rd structures that are contained in the and so on s
26. oordinates tar m densitymap ccp4 r restraints txt NOTES e In this case be sure that residues used in restraint file are provided in corresponding PDB file with structures 4 Modeling with usage of SAXS data PyRy3D can work either with ab initio reconstruction or with raw data SAXS curve In first case the input is regular PDB file in the second DAT file gt gt python pyry3d py fast d coordinates tar x abinitio pdb OR gt gt python pyry3d py fast d coordinates tar y curve dat Where Abinitio pdb is the ab initio reconstruction calculated from SAXS data Curve dat is SAXS curve NOTES e When ab initio model is used for modeling PyRy3D performs docking in similar way as in case of a density map the algorithm of scoring agreement with complex shape is similar e When SAXS curve is used PyRy3D runs CRYSOL and scores agreement of theoretical curve calculated from obtained model with the curve delivered by the user e For y option CRYSOL must be available on disk and CRYSOL_PATH should be given in configuration file to folder with crysol bin 5 Reproduce full atom model from a reduced representation gt gt python pyry3d py fast d coordinates tar f fullatom pdb v history txt To reduce computational time of simulation the user can use reduced representation of complex structure In order to reproduce full atom representation of final model generated by PyRy3D f and v options can be used PyRy3D will re
27. oordinates tar r restraints txt Where Coordinates tar contains PDB files of complex components Restraints txt contains information about docking restraints NOTES e In this case MultiFASTA file with sequences of components is generated automatically Sequences are derived from PDB files found in coordinates tar archive e Bear in mind that structures in coordinates tar archive must be prepared before running PyRy3D each file should contain different chain name and the atoms residues defined in restraints txt file should occur in PDB files the same names and numbers in PDB file and restraints file e If configuration file is not delivered by the user PyRy3D is run with default parameters see 3 4 section of this Manual to check their values e fname of output is not provided PyRy3D puts all models into pyryresults folder 2 Docking into electron density map gt gt python pyry3d py fast d coordinates tar m densitymap ccp4 Where Coordinates tar contains PDB files of complex components Densitymap ccp4 is the map with electron density map or one from negative stain in CCP4 format NOTES e In this case FASTA file with sequences of components is generated automatically from PDB files found in coordinates tar archive e Docking of components into the map will start from random locations of components inside the map 3 Docking into density map with use of restraints gt gt python pyry3d py fast d c
28. ore a write is done to generate a meaningful structural change However if the user desires to have a chain of quite similar structures this value can be low ANNTEMP floatl float2 This specifies the temperature parameter of the simulation and is applied only to Simulated Annealing algorithm Floatl refers to the temperature at the beginning of the simulation float2 to the temperature at the end SIMMETHOD name of algorithm Algorithm to perform sampling of conformational space Available options Genetic for genetic algorithm or SimulatedAnnealing for simulated annealing default or ReplicaExchange for replica exchange Comments e The final temperature can be lower or higher that initial temperature In the second case the system is being gradually heated e The final temperature can be also equal the initial temperature In such a case the program maintains same temperature during entire simulation e The order of parameters in configuration file is not important The format of the file up to this point should be as follows STEPS 1000 WRITE_N_ITER 20 ANNTEMP 135 O90 SIMMETHOD SimulatedAnnealing There are some additional terms that can be set up the use in the configuration file These options include the following Scoring function parameters This is a group of parameters that control a scoring function and correspond to weights assigned for particular features of generated model such
29. our working directory to the previously unpacked BioPython folder it must contain the setup py file 3 Type the following command CHIMERA here is the location where you have your Chimera installed CHIMERA bin chimera nogui silent script setup py install 4 To check if the library was installed correctly open Chimera go to Tools gt General Controls gt IDLE and type gt gt import Bio If it doesn t return any exception it means BioPython is installed correctly STEP 3 Place the PyRy3D_Extension folder in share directory located in the main UCSF Chimera installation directory Loading input files The Extension s first tab called Models allows you to load your input files for further analysis Use Add density map and Add structures buttons to list your shape descriptor and components of the complex After you re done with preparing the list of your files which can be seen in Maps ready to open and Structures ready to open fields click the Load data button to load them into Chimera Defining simulation evaluation parameters Before running an automatic complex evaluation or a classic PyRy3D simulation a user can modify the default set of parameters In order to do so go to the Extension s second tab Sim Score There a user can use the parameters window to manually modify different parameters or load his her own configuration file using the Get pa
30. output files After defining all the necessary parameters press the Start clustering button to start the clustering process More materials about PyRy3D Chimera Extension can be found on the following website http genesilico pl pyry3d tutorials
31. rameters from configuration file option Automatic complex scoring Aside from performing PyRy3D modeling one can use PyRy3D Chimera Extension to evaluate particular arrangement of components that is set interactively in UCSF Chimera This feature lets to use knowledge and or intuition to manually arrange the components in the space and then check how it would be scored by PyRy3D After loading your input files using the Models tab go to the Extension s second tab Sim Score choose your output folder using the Browse button next to the Output directory field and click Calculate PyRy3D score for displayed complex Performing PyRy3D simulation The main purpose of PyRy3D software is conducting Monte Carlo simulations in order to find the best arrangement of components in a macromolecular complex PyRy3D Chimera Extension also allows for using this feature directly from the Extension s interface After the simulation is finished a diagram is being displayed showing the gradual changes of the complex score with the simulation progress After loading input files using the Models tab go to the Extension s second tab Sim Score choose your output folder using the Browse button next to the Output directory field and click Perform PyRy3D Simulation Results Analysis One of the most important goals of PyRy3D Chimera Extension is to make the PyRy3D s results analysis easi
32. tes of all complex components Required format is PDB and only one chain can be stored in a single PDB file Comment e The format of the PDB file has some very strict requirements i e it should be a simple structure Chain IDs for all components must be unique you cannot provide two structures with A chain ID For each PDB file a sequence must be provided in MultiFASTA file Do not use heteroatoms HETATOM use ATOM lt instead Program supports non standard base notations the readable residues are A C G U T but also most modified residues and some ligands are supported Be sure that there are no duplicated atoms or residues in provided structures program will complain about it Ina single PDB file only one chain should be provided if more chains are given only the first one will be considered by PyRy3D If one want to treat e g protein and DNA as a single component during simulation the easiest way is to prepare two separate PDB files and define COVALENT_BONDS explained further in this manual for these two molecules in the configuration file provided for PyRy3D In such a case PyRy3D will treat those two molecules as one e g if protein A is rotated DNA is rotated the same way at the same time 2 3 Format of long range restraint files r restraints F116 The input restraint file contains information on the type and weight importance of the restraints between entities defined as ato
Download Pdf Manuals
Related Search