INSTALLATION AND IMPLEMENTATION OF C-I-TASSER SUITE (Copyright 2020 by Zhang Lab, University of Michigan, All rights reserved) (Version 1.0, 2020/10/10) 1. What is C-I-TASSER Suite? The C-I-TASSER Suite is a composite package of programs for protein structure prediction and function annotations. The Suite includes the following programs: a) C-I-TASSER: A hierarchical program for protein structure prediction b) DeepMSA: A program for multiple sequence alignmnet generation c) MUSTER: A threading program for protein template identification d) CEthreader: A contact-based threading program for protein template identification e) LOMETS2: A meta-server approach consisting of multiple threading programs f) ResTriplet,TripletRes,ResPre,ResPLM and DeepPLM: Deep-learning-based programs for residue-residue contact prediction g) SPICKER: A clustering program for structure decoy selection h) HAAD: Quickly adding hydrogen atoms to protein heavy atom structure i) EDTSurf: Construct triangulated surfaces of protein molecules j) ModRefiner: Construct and refine atomic model from C-alpha traces k) NWalign: Protein sequence alignments by Needleman-Wunsch algorithm l) PSSpred: A program for Protein Secondary Structure PREDiction m) ResQ: An algorithm to estimate B-factor and residue-level error of models n) COACH: A function annotation program based on COFACTOR, TM-SITE and S-SITE o) COFACTOR: A program for ligand-binding site, EC number & GO term prediction p) TM-SITE: A structure-based approach for ligand-binding site prediction q) S-SITE: A sequence-based approach for ligand-binding site prediction 2. How to install the C-I-TASSER Suite? a) download the C-I-TASSER Suite 'C-I-TASSER-1.0.tar.bz2' from http://zhanglab.dcmb.med.umich.edu/C-I-TASSER/download and unpack 'C-I-TASSER-1.0.tar.bz2 by > tar -xvf C-I-TASSER-1.0.tar.bz2 The root path of this package is called $pkgdir, e.g. /home/yourname/C-I-TASSER-1.0. You should have all the programs under this directory. You can install the package at any location on your computer. b) Download C-I-TASSER and COACH library files from https://zhanglab.dcmb.med.umich.edu/C-I-TASSER/download http://zhanglab.dcmb.med.umich.edu/BioLiP/ A script 'download_lib.pl' is provided in the package for automated library download and update of the libraries. We recommend putting the library files under the path /home/yourname/ITLIB. c) Third-party software installation: While the majority of programs in the package 'C-I-TASSER-1.0.tar.bz2' are developed in the Zhang Lab herein the permission of use is released, there are some programs and databases (including blast, nr, GOparser, uniclust30,uniref90 and metaclust) which were developed by third-party groups. A default version of blast and nr are included in the package. It is user's obligation to obtain license permission from the developers for all the third-party software before using them. In addition, your system needs to have Java, python2, python3 (which supports pytorch 0.3.0) installed. To use DeepMSA, you need download uniclust30, uniref90 and metaclust from http://gwdu111.gwdg.de/~compbiol/uniclust/2017_04/uniclust30_2017_04_hhsuite.tar.gz , ftp://ftp.uniprot.org/pub/databases/uniprot/uniref/uniref90/uniref90.fasta.gz , and https://metaclust.mmseqs.org/2017_05/metaclust_2017_05.fasta.gz. after you unpack them, put the entire folder to the C-I-TASSER library folder, (i.e. where the folder you put your PDB, MTX, DEP folders). Then rename the folder uniclust30_xxx_xxx to uniclust30, uniref90_xxx to uniref90, metaclust_xxx to metaclust. Then use $pkgdir/contact/DeepMSA/bin/esl-sfetch to create .ssi index for uniref90 and metaclust, here $pkgdir means the path where you put the C-I-TASSER suite package. For example, if the uniref90 database in uniref90 folder is named as uniref90.fasta, then go to uniref90 folder, run $pkgdir/contact/DeepMSA/bin/esl-sfetch --index uniref90.fasta, you will find a new file named as uniref90.fasta.ssi after the command done. Then do the same thing to metaclust database. If you use different version of uniclust30, uniref90 or metaclust, please go to $pkgdir/I-TASSERmod/runI-TASSER.pl, change the variables $hhbdbdir = "$libdir/uniclust30"; $jacdbdir = "$libdir/uniref90"; $hmsdbdir = "$libdir/metaclust"; $hhbdb = "$libdir/uniclust30/uniclust30_2017_04"; $jacdb = "$libdir/uniref90/uniref90.fasta"; $hmsdb = "$libdir/metaclust/metaclust.fasta"; 3. Bug report: Please report and post bugs and suggestions at C-I-TASSER message board: http://zhanglab.dcmb.med.umich.edu/forum ####################################################### # # # 4. Installation and implementation of C-I-TASSER # # # ####################################################### 4.1. Introduction of C-I-TASSER C-I-TASSER (Contact-guided Iterative Threading ASSEmbly Refinement) is a new method extended from I-TASSER for high-accuracy protein structure and function predictions. Starting from a query sequence, C-I-TASSER first generates inter-residue contact maps using multiple deep neural-network predictors, including NeBcon, ResPRE, and TripletRes. It then identifies structural templates from the PDB by multiple threading approach LOMETS2, with full-length atomic models assembled by contact-map guided replica-exchange Monte Carlo simulations. The large-scale benchmark tests showed that C-I-TASSER generates significantly more accurate models than I-TASSER, especially for the sequences that do not have homologous templates in the PDB. For function annotation, the C-I-TASSER structure model is matched through the function library (BioLiP) to identify functional template. The biological insights (including ligand-binding, enzyme classification, and gene ontology) are inferred from the functional templates by COACH based on the consensus of predictions from COFACTOR, TM-SITE and S-SITE. 4.2. How to run C-I-TASSER? a) Main script for running C-I-TASSER is $pkgdir/I-TASSERmod/runI-TASSER.pl. Run it directly without arguments will output the help information. b) The following arguments must be set (mandatory arguments). One example is: "$pkgdir/I-TASSERmod/runI-TASSER.pl -libdir /home/yourname/ITLIB -seqname example -datadir /home/yourname/C-I-TASSER-1.0/example" -libdir means the path of the template libraries -seqname means the unique name of your query sequence -datadir means the directory which contains your sequence c) Other arguments are optional whose default values have been set. User can reset one or more of them. One example of command line is: "$pkgdir/I-TASSERmod/runI-TASSER.pl -pkgdir /home/yourname/C-I-TASSER-1.0 -libdir /home/yourname/ITLIB -seqname example -datadir /home/yourname/C-I-TASSER-1.0/example -runstyle parallel -homoflag benchmark -idcut 0.3 -LBS true -EC true -GO true -java_home /usr -python2 /usr/bin/python2 -python3 /usr/bin/python3 -cit true" -pkgdir means the path of the I-TASSER package. default is to guess by the location of runI-TASSER.pl script -java_home means the path contains the java executable "bin/java" (your system needs to have Java installed) -runstyle default value is "serial" which means running I-TASSER simulation sequentially. "parallel" means running parallel simulation jobs in the cluster using PBS/torque job scheduling system. "gnuparallel" means running parallel simulation jobs on one computer with multiple cores using GNU parallel -homoflag [real, benchmark],"real" will use all templates, "benchmark" will exclude homologous templates -idcut sequence identity cutoff for "benchmark" runs, default value is 0.3, range is in [0,1] -ntemp number of top templates output for each threading program, default is 20, range is in [1,50] -nmodel number of final models output by I-TASSER, default value is 5, range is in [1,10] -LBS [false or true], whether to predict ligand-binding site, default is false -EC [false or true], whether to predict EC number, default is false -GO [false or true], whether to predict GO terms, default is false -restraint1 specify distance/contact restraints (read more at http://zhanglab.dcmb.med.umich.edu/I-TASSER/option1.html ) -restraint2 specify template with alignment (read more at http://zhanglab.dcmb.med.umich.edu/I-TASSER/option4.html ) -restraint3 specify template name without alignment (read more at http://zhanglab.dcmb.med.umich.edu/I-TASSER/option2.html ) -restraint4 specify template file without alignment (read more at http://zhanglab.dcmb.med.umich.edu/I-TASSER/option3.html ) -temp_excl exclude specific templates from template library (read more at http://zhanglab.dcmb.med.umich.edu/I-TASSER/option6.html ) -traj this option means to deposit the trajectory files -light this option means to run I-TASSER in fast mode (each simulation runs by default 5 hours maximum) -hours specify maximum hours of simulations (default=5 when -light=true) -outdir where the final results should be saved (default value is set to data_dir) -python2 path to python 2, for example /bin/python -python3 path to python 3 for contact prediction, need to support pytorch 0.3.0, for example /bin/python3 -cit whether use predicted contacts guiding the C-I-TASSER simulation or not (default=true) NOTE: a) Outline of steps for running C-I-TASSER by 'runI-TASSER.pl': a1) standardize 'seq.fasta' to 'seq.txt' and get the sequence length a2) run 'deepmsa' to generate deep multiple sequence alignment run 'psiblast' to generate 'chk', 'out', 'pssm', 'mtx' files run 'PSSpred' to get 'seq.dat', 'seq.dat.ss' run 'solve' to get 'exp.dat' run 'pairmod' to get 'pair1.dat' and 'pair3.dat' a3) run 'restriplet','tripletres','respre','resplm' and 'deepplm' to predicted contact maps a4) run 'LOMETS2' threading programs sequentially run 'mkinit.pl' to generate restraints a5) run C-I-TASSER simulation a6) run SPICKER clustering program run 'get_cscore.pl' to get confidence score run 'EMrefinement.pl' to get full-atomic models run 'get_rsq_bfp.pl' to get local accuracy and B-factor estimations a7) run 'runCOACH.pl' to generate ligand-binding sites, EC number and GO terms predictions. b) 'seq.fasta' is the query sequence file in FASTA format, which is the only needed input file for running C-I-TASSER. This file should be put in $datadir before running this job. c) C-I-TASSER structure assembly simulations contains multiple independent runs by decided by protein type. This number can be modified if the user wants to run more simulations, especially for big protein without good templates. d) If working on a cluster with multiple nodes, it is recommended to set $runstyle="parallel". You need have PBS server installed in your system. Parallel jobs will run faster since jobs are distributed among different nodes. The default setting $runstyle="serial" will run all the jobs on a single computer. e) If the job has been executed partially and encounter some error, you can rerun the main script without modification. It will check the existing files and start from the correct position. 4.3 System requirement: a) x86_64 machine, Linux kernel OS, Free disk space of more than 60G. b) Perl and java interpreters should be installed. GO:Parser should be installed if you want to predict GO terms c) Basic compress and decompress package should be installed to support: tar and bunzip2. d) If you are using computer clusters, job management software PBS server should support 'qsub' and 'qstat'. If using other job management software, such as SGE and Slurm, some changes should be made following the instructions at: http://zhanglab.dcmb.med.umich.edu/bbs/?q=node/3561 4.4. How to cite C-I-TASSER and C-I-TASSER Suite? 1. Wei Zheng, Chengxin Zhang, Yang Li, Robin Pearce, Eric W. Bell, Yang Zhang. Folding non-homology proteins by coupling deep-learning contact maps with I-TASSER assembly simulations. In preparation, 2020. 2. Wei Zheng, Yang Li, Chengxin Zhang, Robin Pearce, S. M. Mortuza, Yang Zhang. Deep-learning contact-map guided protein structure prediction in CASP13. Proteins: Structure, Function, and Bioinformatics, 87: 1149-1164 (2019). 3. Y Zhang. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9: 40 (2008). 4. A Roy, A Kucukural, Y Zhang. I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols, 5: 725-738 (2010). 5. J Yang, R Yan, A Roy, D Xu, J Poisson, Y Zhang. The I-TASSER Suite: Protein structure and function prediction. Nature Methods, 12: 7-8 (2015) ################################################################### # # # 5. Installation and implementation of contact predictors # # # ################################################################### 5.1. Introduction of ResTriplet, TripletRes, ResPRE, ResPLM and DeepPLM TripletRes and ResTriplet are deep-learning based contact predictors using three co-evolutionary features: the covariance matrix (COV) proposed by DeepCov; the precision matrix (PRE) formulated by ResPRE; and the coupling parameters of the inverse Potts model obtained through pseudolikelihood maximization (PLM). ResPRE is our in-house contact-map predictor, which consists of two consecutive steps of precision matrix-based feature generation and deep residual neural network-based contact inference. ResPLM is also an in-house contact-map predictor similar to ResPRE. The only difference is that ResPLM was trained using the PLM feature. DeepPLM is our in-house contact-map prediction approach that has the same deep-learning architecture as ResPRE, except it uses different features that are generated by CCMpred. 5.2. How to install contact programs? When you unpack the C-I-TASSER Suite, ResTriplet, TripletRes, ResPRE, ResPLM and DeepPLM programs are already installed. 5.3. How to run contact programs? The contact main script is $pkgdir/I-TASSERmod/runCONTACTS.pl. The running option of this program is similar to that in runI-TASSER.pl. By running the program without argument, you can print all the running options. 5.4. How to cite contact? If you are using the TripletRes program, you can cite: Yang Li, Chengxin Zhang, Eric W Bell, Wei Zheng, Dongjun Yu, Yang Zhang. Deducing high-accuracy protein contact-maps from a triplet of coevolutionary matrices through deep residual convolutional networks. submitted, 2020. If you are using the ResTriplet program, you can cite: Yang Li, Chengxin Zhang, Eric W. Bell, Dongjun Yu, Yang Zhang. Ensembling multiple raw coevolutionary features with deep residual neural networks for contact-map prediction in CASP13. Proteins: Structure, Function, and Bioinformatics, 87: 1082-1091 (2019). If you are using the ResPre program, you can cite: Yang Li, Jun Hu, Chengxin Zhang, Dong-Jun Yu, and Yang Zhang. ResPRE: high-accuracy protein contact prediction by coupling precision matrix with deep residual neural networks. Bioinformatics, 35: 4647-4655 (2019). If you are using the ResPLM and DeepPLM programs, you can cite: Wei Zheng, Yang Li, Chengxin Zhang, Robin Pearce, S. M. Mortuza, Yang Zhang. Deep-learning contact-map guided protein structure prediction in CASP13. Proteins: Structure, Function, and Bioinformatics, 87: 1149-1164 (2019). ####################################################### # # # 6. Installation and implementation of MUSTER # # # ####################################################### 6.1. Introduction of MUSTER MUSTER (MUlti-Sources ThreadER) is a protein threading algorithm to identify the template structures from the PDB library. It generates sequence-template alignments by combining sequence profile-profile alignment with multiple structural information. 6.2. How to install MUSTER program? When you unpack the C-I-TASSER Suite, MUSTER program is already installed. 6.3. How to run MUSTER program? The MUSTER main script is $pkgdir/I-TASSERmod/runMUSTER.pl. The running option of this program is similar to that in runI-TASSER.pl. By running the program without argument, you can print all the running options. 6.4. How to cite MUSTER? If you are using the MUSTER program, you can cite: S Wu, Y Zhang. MUSTER: Improving protein sequence profile-profile alignments by using multiple sources of structure information. Proteins, 72: 547-556 (2008). ####################################################### # # # 7. Installation and implementation of CEthreader # # # ####################################################### 7.1. Introduction of CEthreader CEthreader is a novel threading algorithm, which first predicts residue-residue contacts by coupling evolutionary precision matrices with deep residual convolutional neural-networks. The predicted contact maps are then integrated with sequence profile alignments to recognize structural templates from the PDB. 7.2. How to install CEthreader program? When you unpack the C-I-TASSER Suite, CEthreader program is already installed. 7.3. How to run CEthreader program? The CEthreader main script is $pkgdir/I-TASSERmod/runCEthreader.pl. The running option of this program is similar to that in runI-TASSER.pl. By running the program without argument, you can print all the running options. 7.4. How to cite CEthreader? If you are using the CEthreader program, you can cite: W Zheng, Q Wuyun, Y Li, SM Mortuza, C Zhang, R Pearce, J Ruan, Y Zhang. Detecting distant-homology protein structures by aligning deep neural-network based contact maps. PLOS Computational Biology, 15: e1007411 (2019). ####################################################### # # # 8. Installation and implementation of LOMETS2 # # # ####################################################### 8.1. Introduction of LOMETS2 LOMETS2 (Local Meta-Threading-Server) is meta-server approach to protein fold-recognition. It consists of 11 individual threading programs: CEthreader, mCEthreader, eCEthreader, MUSTER, PPA, dPPA, dPPA2, sPPA, wPPA, wdPPA, wMUSTER. The mCEthreader and eCEthreader are variances of CEthreader which includes different scoring functions. The last 7 programs are variances of MUSTER which includes different optimized energy terms. 8.2. How to install LOMETS2 program? When you unpack the C-I-TASSER Suite, LOMETS2 programs are already installed. 8.3. How to run LOMETS2 program? The LOMETS2 main script is $pkgdir/I-TASSERmod/runLOMETS.pl. The running option of this program is similar to that in 'runI-TASSER.pl'. By running the program without argument, you can print all the running options. 8.4. How to cite LOMETS2? If you are using the LOMETS2 program, you can cite: Wei Zheng, Chengxin Zhang, Qiqige Wuyun, Robin Pearce, Yang Li, Yang Zhang. LOMETS2: improved meta-threading server for fold-recognition and structure-based function annotation for distant-homology proteins. Nucleic Acids Research, 47: W429-W436 (2019) S Wu, Y Zhang. LOMETS: A local meta-threading-server for protein structure prediction. Nucleic Acids Research, 35: 3375-3382 (2007). ####################################################### # # # 9. Installation and implementation of DeepMSA # # # ####################################################### 9.1. Introduction of DeepMSA DeepMSA is a new open-source method for sensitive MSA construction, which has homolo- gous sequences and alignments created from multi-sources of whole-genome and metagenome databases through complementary hidden Markov model algorithms. 9.2. How to install DeepMSA program? When you unpack the C-I-TASSER Suite, DeepMSA program is already installed. 9.3. How to run DeepMSA program? The DeepMSA main script is $pkgdir/contact/DeepMSA/scripts/build_MSA.py. The running option of this program is similar to that in runI-TASSER.pl. By running the program without argument, you can print all the running options. 9.4. How to cite DeepMSA? If you are using the DeepMSA program, you can cite: C Zhang, W Zheng, S M Mortuza, Y Li, Y Zhang. DeepMSA: constructing deep multiple sequence alignment to improve contact prediction and fold-recognition for distant-homology proteins. Bioinformatics 36: 2105-2112 (2020). ####################################################### # # # 10. Installation and implementation of SPICKER # # # ####################################################### 10.1. Introduction of SPICKER SPICKER is a clustering algorithm to identify the near-native models from a pool of protein structure decoys. 10.2. How to install SPICKER program? When you unpack the C-I-TASSER Suite, SPICKER program is already installed at $pkgdir/I-TASSERmod/spicker45d 10.3. How to run SPICKER program? To run SPICKER, you need to prepare following input files: 'rmsinp'---Mandatory, length of protein & piece for RMSD calculation; 'seq.dat'--Mandatory, sequence file, for output of PDB models. 'tra.in'---Mandatory, list of trajectory names used for clustering. In the first line of 'tra.in', there are 3 parameters: par1: number of decoy files par2: 1, default cutoff, best for decoys from template-based modeling; -1, cutoff based on variation, best for decoys from ab initio modeling. par3: 1, closc from all decoys; -1, closc clustered decoys From second lines are file names which contain coordinates of 3D structure decoys. All these files are mandatory. See attached 'rep1.tra1' for the format of decoys. 'CA'-------Optional, native structure, for comparison to native. Output files of SPICKER include: 'str.txt'-----list of structure in cluster; 'combo*.pdb'--PDB format of cluster centroids; 'closc*.pdb'--PDB format of structures closest to centroids; 'rst.dat'-----summary of clustering results; A detailed readme file can be found at http://zhanglab.dcmb.med.umich.edu/SPICKER/readme 10.4. How to cite SPICKER? If you are using the SPICKER program, you can cite: Y Zhang, J Skolnick, SPICKER: Approach to clustering protein structures for near-native model selection, Journal of Computational Chemistry, 25: 865-871 (2004). ####################################################### # # # 11. Installation and implementation of HAAD # # # ####################################################### 11.1. Introduction of HAAD HAAD is a computer algorithm for constructing hydrogen atoms from protein heavy-atom structures. The hydrogen is added by minimizing atomic overlap and encouraging hydrogen bonding. 11.2. How to install HAAD program? When you unpack the C-I-TASSER Suite, HAAD program is already installed at $pkgdir/abs/mybin/HAAD 11.3. How to run HAAD program? Hydrogen atoms in a PDB file(xx.pdb) can be added by running "./HAAD xx.pdb", the output is "xx.pdb.h". In "xx.pdb.h", the label in column 57 presents the label for the atoms that have been added by HAAD. When the value of the label is less than 2, the position of the added atom has higher confidence. 11.4. How to cite HAAD? If you are using the HAAD program, you can cite: Y Li, A Roy, Y Zhang, HAAD: A Quick Algorithm for Accurate Prediction of Hydrogen Atoms in Protein Structures, PLoS One, 4: e6701 (2009). ####################################################### # # # 12. Installation and implementation of EDTSurf # # # ####################################################### 12.1. Introduction of EDTSurf EDTSurf is a program to construct triangulated surfaces for macromolecules. It generates three major macromolecular surfaces: van der Waals surface, solvent-accessible surface and molecular surface (solvent-excluded surface). EDTsurf also identifies cavities which are inside of macromolecules. 12.2. How to install EDTSurf program? When you unpack the C-I-TASSER Suite, EDTSurf program is already installed at $pkgdir/bin/EDTSurf 12.3. How to use EDTSurf program? EDTSurf -i inputfile ... Specific options: -o prefix of output files (default is the prefix of inputfile) -t triangulation type, 1-MC 2-VCMC (default is 2) -s surface type, 1-VWS 2-SAS 3-MS (default is 3) -c color mode, 1-pure 2-atom 3-chain (default is 2) -p probe radius, float point in [0,2.0] (default is 1.4) -h inner or outer surface for output, 1-inner and outer 2-outer 3-inner (default is 1) -f scale factor, float point in (0,20.0] (default is 4.0) Molecule is scaled by this factor to fit in a bounding box. Scale factor is the larger the better, but will increase the memory use. Our strategy is first enlarging the molecule to check if it exceeds the maximum bounding box. If yes, then reset a proper scale factor to fit the molecule in the maximum bounding box. By running EDTSurf itself, it will print out a brief description on how to use the program. A detail description of EDTSurf is available at http://zhanglab.dcmb.med.umich.edu/EDTSurf/ 12.4. How to cite EDTSurf? If you are using the EDTSurf program, you can cite: D Xu, Y Zhang, Generating Triangulated Macromolecular Surfaces by Euclidean Distance Transform. PLoS ONE 4: e8140 (2009). ####################################################### # # # 13. Installation and implementation of ModRefiner # # # ####################################################### 13.1. Introduction of ModRefiner ModRefiner is a standalone program for atomic-level protein structure construction and refinement. It includes two steps: (1) construct main-chain models from C-alpha trace; (2) build side-chain models and atomic-level structure refinement. 13.2. How to install ModRefiner program? When you unpack the C-I-TASSER Suite, ModRefiner program is already installed at $pkgdir/I-TASSERmod/ModRefiner.pl 13.3. How to use ModRefiner program? ModRefiner supports following four options: a) add side-chain heavy atoms to main-chain model without refinement > ModRefiner.pl 1 ID MD IM ON b) build main-chain model from C-alpha trace model > ModRefiner.pl 2 ID MD IM RM ON c) build full-atomic model from main-chain model > ModRefiner.pl 3 ID MD IM RM ON d) build full-atomic model from C-alpha trace model > ModRefiner.pl 4 ID MD IM RM ON ID: the path of the C-I-TASSER package, e.g. '/home/yourname/C-I-TASSER-1.0' MD: directory which contains the initial model, e.g. '/home/yourname/C-I-TASSER/5.0/example' IM: the initial model to be refined, e.g. 'mode1.pdb' RM: reference model that refined model is driven to, e.g. 'combo1.pdb'. Only CA trace is needed and the length can be not full which will make the refinement of the missing region flexible. If you don't have the reference model, use the name of IM instead. ON: the output name of the refined model, e.g. 'model1_ref.pdb' By running the program without argument, you can print a brief description of how to use the program. 13.4. How to cite ModRefiner? If you are using the ModRefiner program, you can cite: D Xu, Y Zhang. Improving the Physical Realism and Structural Accuracy of Protein Models by a Two-step Atomic-level Energy Minimization. Biophysical Journal, 101: 2525-2534 (2011) ####################################################### # # # 14. Installation and implementation of NWalign # # # ####################################################### 14.1. Introduction of NWalign NW-align is simple and robust alignment program for protein sequence-to-sequence alignments based on the standard Needleman-Wunsch dynamic programming algorithm. The mutation matrix is from BLOSUM62 with gap opening penalty=-11 and gap extension penalty=-1. 14.2. How to install NWalign program? When you unpack the C-I-TASSER Suite, NWalign program is already installed at $pkgdir/bin/align. 14.3. How to use NWalign program? > align F1.fasta F2.fasta (align two sequences in fasta file) > align F1.pdb F2.pdb 1 (align two sequences in PDB file) > align F1.fasta F2.pdb 2 (align Sequence 1 in fasta and 2 in pdb) > align GKDGL EVADELVSE 3 (align sequences typed by keyboard) > align GKDGL F.fasta 4 (align Seq-1 by keyboard and 2 in fasta) > align GKDGL F.pdb 5 (align Seq-1 by keyboard and 2 in pdb) By running the program itself, it will print out the usage options of the program. 14.4. How to cite NWalign? There is no published paper associated with this program. If you are using the NWalign program, you can cite it as Y Zhang, http://zhanglab.dcmb.med.umich.edu/NW-align ####################################################### # # # 15. Installation and implementation of PSSpred # # # ####################################################### 15.1 Introduction of PSSpred PSSpred (Protein Secondary Structure PREDiction) is a simple neural network training algorithm for accurate protein secondary structure prediction. It first collects multiple sequence alignments using PSI-BLAST. Amino-acid frequency and log-odds data with Henikoff weights are then used to train secondary structure, separately, based on the Rumelhart error back propagation method. The final secondary structure prediction result is a combination of 7 neural network predictors from different profile data and parameters. 15.2 How to install PSSpred program? When you unpack the C-I-TASSER Suite, NWalign program is already installed at $pkgdir/PSSpred 15.3 How to use PSSpred program? $pkgdir/PSSpred/mPSSpred.pl seq.txt $pkgdir $libdir Please note that 'seq.txt' should be in current directory and the script will generate two files 'seq.dat' and 'seq.dat.ss' in the current folder. Here, $pkgdir is the root path of C-I-TASSER package. 15.4 How to cite PSSpred? If you are using the PSSpred program, you can cite: http://zhanglab.dcmb.med.umich.edu/PSSpred ####################################################### # # # 16. Installation and implementation of COFACTOR # # # ####################################################### 16.1 Introduction of COFACTOR COFACTOR is a structure-based method for biological function annotation of protein molecules. COFACTOR threads the structure through three comprehensive function libraries by local and global structure matches to identify functional sites and homology. Functional insights, including ligand-binding site, gene-ontology terms and enzyme classification, will be derived from the best functional homology template. The COFACTOR algorithm was ranked as the best method for function prediction in the community-wide CASP9 experiments. 16.2 How to install COFACTOR program? When you unpack the C-I-TASSER Suite, COFACTOR program is already installed at $pkgdir/COFACTOR 16.3 How to use COFACTOR program? $pkgdir/I-TASSERmod/runCOFACTOR.pl 16.4 How to interpret the results If your input data is at $datadir/model1.pdb, the output of COFACTOR will be at $datadir/model1/cofactor: (1)List of similar structures in PDB: similarpdb_model1.lst. The columns are (PDB_ID, TM-score, RMSD, Cov, Seq_id) (2)Ligand-binding sites: BSITE_model1/Bsites_model1.dat. The columns are (Rank, C-score, PDB_ID, TM-score, RMSD, Seq_id, Cov, Lig_name, SITE_num, BS-score, LTM, BS_ID, BS_cov,BS_err, BS_ID1,BS_ID2, Binding residues) (3)EC number: ECsearchresult_model1.dat The columns are (PDB_ID, TM-score, RMSD, Seq_ID, Cov, EC-score, EC number, Active site residues) (4)GO terms: GOsearchresult_model1.dat. The columns are (PDB_ID, TM-score, RMSD, Seq_ID, Cov, GO-score, GO terms) 16.5 How to cite COFACTOR? If you are using the COFACTOR program, you can cite: 1. A Roy, J Yang, Y Zhang. COFACTOR: An accurate comparative algorithm for structure-based protein function annotation. Nucleic Acids Research, 40:W471-W477 (2012). 2. J Yang, A Roy, Y Zhang. BioLiP: a semi-manually curated database for biologically relevant ligand-protein interactions. Nucleic Acids Research, 41: D1096-D1103 (2013). ####################################################### # # # 17. Installation and implementation of COACH # # # ####################################################### 17.1 Introduction of COACH COACH is a meta-server approach to protein function annotations. Starting from given structure of target proteins, COACH will generate complementary ligand binding site predictions using two comparative methods: TM-SITE and S-SITE, which recognize ligand-binding templates from the BioLiP protein function database by binding-specific substructure and sequence profile comparisons. These predictions will be combined with results from COFACTOR to generate multiple function annotations, including ligand-binding sites, enzyme commission and gene ontology terms. 17.2 How to install COACH program? When you unpack the C-I-TASSER Suite, COACH program is already installed at $pkgdir/COACH 17.3 How to use COACH program? $pkgdir/I-TASSERmod/runCOACH.pl 17.4 How to interpret the results If your input data is at $datadir/model1.pdb, the output of COACH will be at $datadir/model1/coach: (1) Ligand-binding sites: Bsites.dat. The columns are (C-score, cluster_densitiy, product_of_top_templates_zscore, Binding residues) (2) Detailed clustering information: Bsites.inf, Bsites.clr, which list the templates used in the cluster that generates the prediction in (1). (3) Ligand-protein complex structures are with name: CH_complex*.pdb (4) Predicions from COFACTOR, TM-SITE, and S-SITE are at, respectively: $datadir/model1/cofactor $datadir/model1/tmsite $datadir/ssite 17.5 How to cite COACH? If you are using the COACH program, you can cite: 1. J Yang, A Roy, Y Zhang. Protein-ligand binding site recognition using complementary binding-specific substructure comparison and sequence profile alignment. Bioinformatics, 29:2588-2595 (2013). 2. J Yang, A Roy, Y Zhang. BioLiP: a semi-manually curated database for biologically relevant ligand-protein interactions. Nucleic Acids Research, 41: D1096-D1103 (2013). ####################################################### # # # 18. Installation and implementation of TM-SITE # # # ####################################################### 18.1 Introduction of TM-SITE TM-SITE is a structure-based approach to protein-ligand binding site prediction. Structure alignment between query and BioLiP templates is performed on binding-specific substructure using TM-align. The final ligand-binding sites are collected based on the clustering of multiple templates. 18.2 How to install TM-SITE program? When you unpack the C-I-TASSER Suite, TM-SITE program is already installed at $pkgdir/COACH 18.3 How to use TM-SITE program? $pkgdir/I-TASSERmod/runTMSITE.pl 18.4 How to interpret the results If your input data is at $datadir/model1.pdb, the output of TM-SITE will be at $datadir/model1/tmsite: (1)Ligand-binding sites: Bsites.dat. The columns are (C-score, top_templates_zscore, JSD_score, cluster_density, Binding residues) (2)Detailed clustering information: Bsites.inf, Bsites_lig.clr, which lists the templates used in the cluster that generates the prediction in (1). (3)Ligand-protein complex structures are with name: complex*.pdb 18.5 How to cite TM-SITE? If you are using the TM-SITE program, you can cite: 1. J Yang, A Roy, Y Zhang. Protein-ligand binding site recognition using complementary binding-specific substructure comparison and sequence profile alignment. Bioinformatics, 29:2588-2595 (2013). 2. J Yang, A Roy, Y Zhang. BioLiP: a semi-manually curated database for biologically relevant ligand-protein interactions. Nucleic Acids Research, 41: D1096-D1103 (2013). ####################################################### # # # 19. Installation and implementation of S-SITE # # # ####################################################### 19.1 Introduction of S-SITE S-SITE is a sequence-based approach to protein-ligand binding site prediction. Binding-specific sequence profile-profile alignment is used to recognize homologous templates in BioLiP. The ligand-binding sites predictions are collected from the clustering of multiple homologous templates. 19.2 How to install S-SITE program? When you unpack the C-I-TASSER Suite, S-SITE program is already installed at $pkgdir/COACH 19.3 How to use S-SITE program? $pkgdir/I-TASSERmod/runSSITE.pl 19.4 How to interpret the results If your input data is at $datadir/seq.fasta, then the output of S-SITE will be at $datadir/ssite: (1)Ligand-binding sites: Bsites_fpt.dat. The columns are (C-score, top_templates_zscore, cluster_density, cluster_density1, JSD_score, Binding residues) (2)Detailed clustering information: Bsites_fpt.clr, which list the templates used in the cluster that generates the prediction in (1). 19.5 How to cite S-SITE? If you are using the S-SITE program, you can cite: 1. J Yang, A Roy, Y Zhang. Protein-ligand binding site recognition using complementary binding-specific substructure comparison and sequence profile alignment. Bioinformatics, 29:2588-2595 (2013). 2. J Yang, A Roy, Y Zhang. BioLiP: a semi-manually curated database for biologically relevant ligand-protein interactions. Nucleic Acids Research, 41: D1096-D1103 (2013). ####################################################### # # # 20. Installation and implementation of ResQ # # # ####################################################### 20.1 Introduction of ResQ ResQ is a method for estimating B-factor and residue-level quality in protein structure prediction, based on local variations of modelling simulations and the uncertainty of homologous alignments. Given a protein structure model, ResQ first identifies a set of homologous and/or analogous templates from the PDB by threading and structure alignment techniques. The residue-level modeling errors are then derived by support vector regression that was trained on the local structural and alignment variations of the templates, with the B-factor of each residue deduced from the experimental records of the top homologous proteins. 20.2 How to install ResQ program? When you unpack the C-I-TASSER Suite, ResQ program is already installed at $pkgdir/ResQ. 20.3 How to use ResQ program? There are two methods to run ResQ depending on how your models were generated. 1) If your models were generated by C-I-TASSER, you can run the script of $pkgdir/ResQ/runResQ_IT.pl to predict B-factor and local structure errors. The only argument required is the directory of the C-I-TASSER decoys. You can read more at the head of this script to get more information about its input. 2) If your models were not generated by C-I-TASSER, you can run the script $pkgdir/ResQ/runResQ.pl to predict B-factor and local structure errors. It will automatically run LOMETS2 to generate the threading alignment file 'init.dat'. LOMETS2 is included in this package. 20.4 What is the output of ResQ? For C-I-TASSER models, the output of ResQ is: rsq_bfp_new.dat For other models, the output of ResQ is: 1) global.txt for global accuracy estiamtion 2) local.txt for local error and B-factor estimation 20.4 How to cite ResQ? If you are using the ResQ program, you can cite: 1. J Yang, Y Wang, Y Zhang. ResQ: Approach to unified estimation of B-factor and residue-specific error in protein structure prediction, Journal of Molecular Biology, 428: 693-701 (2016).