Introduction to Bioinformatics Introduction to Bioinformatics

Transcription

1 Dr. rer. nat. Gong Jing Cancer Research center Medicine School of Shandong University

2 Chapter 5 Structure 2

3 Protein Structure When you study a protein, you are usually interested in its function. Function Structure Sequence Motif 3

4 Protein Structure There are four distinct levels of protein structure: Primary structure amino acid sequence Secondary structure regular sub-structures Tertiary structure 3-dimentional structure Quaternary structure complex of protein molecules 4

5 Protein Structure 5

6 Protein Structure - Primary Structure V G S A Primary structure the amino acid sequence of a protein >sp P06968 DUT_ECOLI MKKIDVKILDPRVGKEFPLPTYATSGSAGLDLRACLNDAVELAPGDTTLVPTGL AIHIADPSLAAMMLPRSGLGHKHGIVLGNLVGLIDSDYQGQLMISVWNRGQDSF TIQPGERIAQMIFVPVVQAEFNLVEDFDATDRGEGGFGHSGRQ 6

7 Protein Structure - Primary Structure >sp P06968 DUT_ECOL MKKIDVKILDPRVGKEFPL PTYATSGSAGLDLRACLND AVELAPGDTTLVPTGLAIH IADPSLAAMMLPRSGLGHK HGIVLGNLVGLIDSDYQGQ LMISVWNRGQDSFTIQPGE RIAQMIFVPVVQAEFNLVE DFDATDRGEGGFGHSGRQ Chapter 2 Chapter 3 sequence search sequence analysis 7

8 Protein Structure - Secondary Structure 90 o Helices: Where residues seem to be following the shape of a spring. The most common are the so-called alpha helices. 90 o Beta-strands: Where residues are in line and almost completely extended. Beta-sheets consist of beta-strands connected laterally, ming a generally twisted, pleated sheet. Random coils (loop): When the amino-acid chain is neither helices nor Beta-strands. Turn: in those cases where the chain makes a sharp turn (90 or more). 8

9 How to get secondary structures? 1. from PDB/DSSP structure known proteins The DSSP (Definition of Secondary Structure of Proteins) program was designed to standardize secondary structure assignment. DSSP is a database of secondary structure assignments all protein entries in the Protein Data Bank (PDB). DSSP does not predict secondary structure. Home page : alpha-helix Protein Structure - Secondary Structure coil beta-sheet 9

10 How to get secondary structures? 1. from PDB/DSSP structure known proteins Protein Structure - Secondary Structure secondary structures part of a DSSP file 10

11 How to get secondary structures? 1. from PDB/DSSP structure known proteins Protein Structure - Secondary Structure secondary structures H = alpha helix B = residue in isolated beta-bridge E = extended strand, participates in beta ladder G = 3-helix (3/10 helix) I = 5 helix (pi helix) T = hydrogen bonded turn S = bend Blank = loop or irregular part of a DSSP file 11

12 Protein Structure - Secondary Structure How to get secondary structures? 1. from PDB/DSSP structure known proteins

13 Protein Structure - Secondary Structure How to get secondary structures? 1. from PDB/DSSP structure known proteins

14 How to get secondary structures? 1. from PDB/DSSP structure known proteins Protein Structure - Secondary Structure 14

15 Protein Structure - Secondary Structure How to get secondary structures? 1. from PDB/DSSP structure known proteins primary structure secondary structure 15

16 Protein Structure - Secondary Structure How to get secondary structures? 1. from PDB/DSSP structure known proteins

17 Protein Structure - Secondary Structure How to get secondary structures? 1. from PDB/DSSP structure known proteins PDB ID in lower case 17

18 How to get secondary structures? 1. from PDB/DSSP structure known proteins Protein Structure - Secondary Structure A FASTA matted file ("ss.txt") generated using DSSP with sequences and secondary structures of all entries in PDB is available at: (compressed) 18

19 How to get secondary structures? Protein Structure - Secondary Structure 2. from predictions structure unknown proteins Predicting the secondary structure of proteins was one of the hottest goals of the 1990s. Nowadays, fairly good servers are available to accurately predict the secondary structure of any protein that may interest you. If your protein has enough homologues in the current databases, you can expect its secondary structure prediction to be close to 80% accurate. However, bear in mind that this is only a prediction; as with all predictions, it can be more or less inaccurate. PSIPRED - one of the most accurate secondary structure prediction servers. 19

20 How to get secondary structures? Protein Structure - Secondary Structure 2. from predictions structure unknown proteins

21 How to get secondary structures? Protein Structure - Secondary Structure 2. from predictions structure unknown proteins 3cig.fasta This server returns its results by , usually in less than 30 minutes. Give a name if you need help remembering what the analysis was all about. 21

22 Protein Structure - Secondary Structure How to get secondary structures? Prediction (Pred) : the 2. from predictions structure unknown proteins predicted conmation each residue. Confidence (Conf) : the reliability of the prediction each position

23 How to get secondary structures? Protein Structure - Secondary Structure 2. from predictions structure unknown proteins The result will be sent to the address you provided. Note : The server does not accept free, Web-based e- mail addresses (such as those from MSN s Hotmail or Google s Gmail). No results will be returned. 23

24 Protein Structure - Secondary Structure How to get secondary structures? DSSP 2. from predictions structure unknown proteins PSIPRED 51 LAILDAGFNSISKLEPELCQILPLLKVLNLQHNELSQISDQTFVFCTNLT CSEEECCSSCCCCCCTHHHHHSTTCCEEECTTCCCCCCCTTSTTSCCSCC CCEEECCCCCCCCCCHHHHCCCCCCCEEECCCCCCCCCCCCCCCCCCCCC 101 ELDLMSNSIHKIKSNPFKNQKNLIKLDLSHNGLSSTKLGTGVQLENLQEL EEECTTSCCCCCCSCTTTTCTTCCEEECCSSCCCCCCCCSSSCCTTCCEE EEEEECCCCCCCCCCCCCCCCCCCEEECCCCCCCCCCCCCCCCCCCCCEE 151 LLAKNKILALRSEELEFLGNSSLRKLDLSSNPLKEFSPGCFQTIGKLFAL ECCSSCCCEECSGGGGGGTTCEESEEECCSCCCCEECTTTTTTSSEECEE EEECCCCCCCCCCCCCCCCCCCCCEEECCCCCCCCCCCCCCCCCCCCCEE over 90% correctly predicted positions 24

25 This level of structure describes how regions of secondary structure fold together - the 3D arrangement of a polypeptide chain, including alpha-helices, beta-sheets, and any other loops and folds. Tertiary structure results from interactions between side chains, or between side chains and the polypeptide backbone, which are often distant in sequence. The tertiary structure of a protein is defined by its atomic coordinates

26 How to get tertiary structures? 1. Search 3D Structure in PDB e.g. PDB ID, protein name, author 26 26

27 How to get tertiary structures? 1. Search 3D Structure in PDB 27

28 How to get tertiary structures? 1. Search 3D Structure in PDB 28

29 How to get tertiary structures? 1. Search 3D Structure in PDB 29

30 How to get tertiary structures? 2. Search PDB Structure in Dali helsinki.fi/dali_server PDB File 30 30

31 How to get tertiary structures? 2. Search PDB Structure in Dali helsinki.fi/dali_server 31

32 How to get tertiary structures? 2. Search PDB Structure in Dali helsinki.fi/dali_server 32 32

33 How to get tertiary structures? 1. Search PDB Structure in Dali helsinki.fi/dali_server 33 33

34 How to get tertiary structures? 1. Search PDB Structure in Dali helsinki.fi/dali_server 34 34

35 How to get tertiary structures? 1. Search PDB Structure in Dali helsinki.fi/dali_server Superimposed structures shown by Jmol Applet mol1a 3CIG 1ZIW 35

36 How to get tertiary structures? 3. 3D Molecular Viewer Jmol online (PDB) 36

37 How to get tertiary structures? 1. Experimental Methods Search Structure in PDB Press left button Press left button to rotate the molecule Roll middle button to zoom in/out Click right button to access Jmol menu 37

38 How to get tertiary structures? 3. 3D Molecular Viewer Jmol online (PDB) Cartoon Secondary Structure None Backbone By chain None 38

39 How to get tertiary structures? 3. 3D Molecular Viewer VMD Maestro 3H6X.pdb Pymol 39

40 How to get tertiary structures? 3. 3D Molecular Viewer --- Jmol 40

41 How to get tertiary structures? 3. 3D Molecular Viewer --- RasMol

42 How to get tertiary structures? 3. 3D Molecular Viewer --- CanVasMol 42

43 How to get tertiary structures? 3. 3D Molecular Viewer --- Cn3D Structure/CN3D/cn3d.shtml 43 43

44 How to get tertiary structures? 3. 3D Molecular Viewer --- Molegro mmv-product.php 44

45 How to get tertiary structures? 3. 3D Molecular Viewer --- MGL Tools

46 How to get tertiary structures? 3. 3D Molecular Viewer --- Maestro products/14/

47 How to get tertiary structures? 3. 3D Molecular Viewer --- PyMOL

48 How to get tertiary structures? 3. 3D Molecular Viewer --- PyMOL

49 How to get tertiary structures? 3. 3D Molecular Viewer --- Swiss-PDBViewer

50 How to get tertiary structures? 3. 3D Molecular Viewer --- JMV 50

51 How to get tertiary structures? 3. 3D Molecular Viewer --- VMD 51

52 How to get tertiary structures? 3. 3D Molecular Viewer --- VMD Free! 52

53 How to get tertiary structures? 3. 3D Molecular Viewer --- VMD More details under VMD tutorial: Tutorials/vmd/tutorialhtml/index.html 53

54 How to get tertiary structures? 4. Experimental Methods The first 3D structure of a protein was determined in 1958 by Drs. Kendrew and Perutz using X-ray crystallography X-ray Crystallography 7929 Max Ferdinand Perutz ( ) nobel prize 1962 John Cowdery Kendrew ( ) nobel prize 1962 Nuclear Magnetic Resonance (NMR) 54

55 How to get tertiary structures? 4. Experimental Methods X-ray Crystallography Crystallography can solve structures of relatively large molecules. X-ray crystallography is now used routinely by scientists to determine how a pharmaceutical drug interacts with its protein target and what changes might improve it. However, intrinsic membrane proteins remain challenging to crystallize because they require detergents or other means to solubilize them in isolation, and such detergents often interfere with crystallization. Such membrane proteins are a large component of the genome and include many proteins of great physiological importance, such as ion channels and receptors. 55

56 How to get tertiary structures? 4. Experimental Methods Why X-ray, not Ultraviolet or other? When using light to measure an object, the wavelength of the light needs to be similar to the size of the object. X-rays, with wavelengths of approximately 0.5 to 1.5 angstroms, can measure the distance between atoms. Visible light, with a wavelength of 4,000 to 7,000 angstroms, is used in ordinary light microscopes because it can measure objects with the size of cellular components. 56

57 How to get tertiary structures? 4. Experimental Methods Solution-state NMR is restricted to relatively small ones (less than 70 kda). It is used to obtain inmation about the structure and dynamics of proteins. Protein NMR techniques are continually being used and improved in both academia and the biotech industry. Not one, but many structures $ 60,0000 Nuclear Magnetic Resonance (NMR) 57

58 How to get tertiary structures? 4. Experimental Methods SDU Experts in X-ray Crystallography Prof. SUN Jinpeng Ph.D. Institute of Biochemistry and Molecular Biology School of Medicine, SDU Prof. GU Lichuan Ph.D. State Key Laboratory of Microbial Technology School of Life Sciences, SDU 58

59 How to get tertiary structures? 4. Experimental Methods Advantages & Disadvantages The crystal structure is necessary, only that proteins which can be crystallized are examinable. (disadvantage of X-ray) (advantage of NMR) There is no chance direct determination of secondary structures and especially domain movements. (disadvantage of X-ray) (advantage of NMR) The hydrogen in the molecules are not examinable since it has only one electron. (disadvantage of X-ray) (advantage of NMR) The highest molecular mass which was examined successfully is just a 70kDa protein-complex. (disadvantage of NMR) (advantage of X-ray) The cost of the experimental implementation is increasing with the higher strength and the complexity of the determination. (disadvantage of X-ray) (disadvantage of NMR) 59

60 How to get tertiary structures? 4. Experimental Methods Advantages & Disadvantages The crystal structure is necessary, only that proteins which can be crystallized are examinable. (disadvantage of X-ray) (advantage of NMR) Time-consuming There is no chance direct determination of secondary structures and especially domain movements. (disadvantage of X-ray) (advantage of NMR) Labor-consuming The hydrogen in the molecules are not examinable since it has only one electron. (disadvantage of X-ray) (advantage of NMR) Expensive The highest molecular mass which was examined successfully is just a 70kDa protein-complex. (disadvantage of NMR) (advantage of X-ray) The cost of the experimental implementation is increasing with the higher strength and the complexity of the determination. (disadvantage of X-ray) (disadvantage of NMR) 60

61 How to get tertiary structures? 5. Computational Methods MEAKIVKVLDSSRCEDGFGKKRKRAASYAAYVTGV SCAKLQNVPPPNGQCQIPDKRRRLEGENKLSAYE NRSGKALVRYYTYFKKTGIAKRVMMYENGEWNDL PEHVICAIQNELEEKSAAIEFKLCGHSFILDFLHMQR LDMETGAKTPLAWIDNAGKCFFPEIYESDERTNYC HHKCVEDPKQNAPHDIKLRLEIDVNGGETPRLNLE ECSDESGDNMMDDVPLAQRSSNEHYDEATEDSC SRKLEAAVSKWDETDAIVVSGAKLTGSEVLDKDAV KKMFAVGTASLGHVPVLDVGRFSSEIAEARLALFQ KQVEITKKHRGDANVRYAWLPAKREVLSAVMMQG LGVGGAFIRKSIYGVGIHLTAADCPYFSARYCDVDE NGVRYMVLCRVIMGNMELLRGDKAQFFSGGEEYD NGVDDIESPKNYIVWNINMNTHIFPEFVVRFKLSNL PNAEGNLIAKRDNSGVTLEGPKDLPPQLESNQGAR GSGSANSVGSSTTRPKSPWMPFPTLFAAISHKVAE NDMLLINADYQQLRDKKMTRAEFVRKLRVIVGDDL LRSTITTLQNQPKSKEIPGSIRDHEEGAGGL input output 61

62 How to get tertiary structures? 5. Computational Methods AB initio Homology Modeling Threading Ensemble 62

63 How to get tertiary structures? 5. Computational Methods AB initio predicts structures based on physical principles. Indeed, the protein amino acid sequence already contains all inmation needed to create a correctly folded protein [Anfinsen, 1937, Science]. All the energetic conmations involved in the protein folding process are calculated by energy functions to find the structure with the lowest free energy. 63

64 How to get tertiary structures? 5. Computational Methods --- AB initio : QUARK ed.umich.edu/quark quark.fasta It takes 1-2 days. 64

65 How to get tertiary structures? 5. Computational Methods --- AB initio : QUARK ed.umich.edu/quark 65

66 How to get tertiary structures? 5. Computational Methods --- AB initio : QUARK ed.umich.edu/quark 66

67 How to get tertiary structures? 5. Computational Methods --- AB initio : QUARK ed.umich.edu/quark Homology Modeling is based on the assumption that similar sequences among evolutionarily related proteins share an overall structural similarity [Marti-Renom, 2000, Annu Rev Biophys Biomol Struct].? 67

68 How to get tertiary structures? 5. Computational Methods Homology Modeling is based on the assumption that similar sequences among evolutionarily related proteins share an overall structural similarity [Marti-Renom, 2000, Annu Rev Biophys Biomol Struct]. 68

69 How to get tertiary structures? 5. Computational Methods --- Homology Modeling 1. Identification of a homologue(s) (sequence identity >= 30%) of known structure as template(s) modeling the target sequence. 69

70 How to get tertiary structures? 5. Computational Methods --- Homology Modeling 2. Generating and improving an alignment of the target sequence with the template sequence(s). Usually the knowledge based manual adjustment is necessary. 70

71 How to get tertiary structures? 5. Computational Methods --- Homology Modeling 3. Building coordinates of the three-dimensional model through automatic modeling programs, based on the alignment. 71

72 How to get tertiary structures? 5. Computational Methods --- Homology Modeling 4. Evaluation of the model and then to repeat the previous steps according the evaluation results until the model becomes satisfactory. 72

73 How to get tertiary structures? 5. Computational Methods --- Homology Modeling : SWISS-MODEL 73 73

74 How to get tertiary structures? 5. Computational Methods --- Homology Modeling : SWISS-MODEL swiss_model.fasta 74

75 How to get tertiary structures? 5. Computational Methods --- Homology Modeling : SWISS-MODEL 75

76 How to get tertiary structures? 5. Computational Methods --- Homology Modeling Homology modeling is most accurate when the target and template have similar sequences. 76

77 How to get tertiary structures? 5. Computational Methods --- Homology Modeling Two proteins with a high level of sequence identity will nevertheless have not exactly identical backbone conmations. [Gong et.al. BMC Struct Biol, 2012, 12:23] 77

78 How to get tertiary structures? 5. Computational Methods --- Homology Modeling Homology modeling cannot be used when the sequence identity between the target and template is < 30%. 78

79 How to get tertiary structures? 5. Computational Methods Threading (fold recognition) The prediction is made by "threading" (i.e. placing, aligning) each amino acid in the target sequence to a position in the template structure, and evaluating how well the target fits the template. After the best-fit template is selected, the structural model of the target is built based on the alignment along with the threading path within the chosen template. 79

80 How to get tertiary structures? 5. Computational Methods Threading (fold recognition) Protein threading is based on two basic observations: that the number of different folds in nature is fairly small (approximately 1000); and that 90% of the new structures submitted to the PDB in the past three years have similar structural folds to ones already in the PDB. Threading method doesn t depends on the sequence identity between target and template. 80

81 How to get tertiary structures? 5. Computational Methods --- Threading ed.umich.edu/i-tasser 81

82 How to get tertiary structures? 5. Computational Methods --- Threading ed.umich.edu/i-tasser itasser.fasta Free Registration 82

83 How to get tertiary structures? 5. Computational Methods --- Threading ed.umich.edu/i-tasser 83

84 How to get tertiary structures? 5. Computational Methods --- Threading ed.umich.edu/i-tasser 84

85 How to get tertiary structures? 5. Computational Methods --- Threading ed.umich.edu/i-tasser Part I 85

86 How to get tertiary structures? 5. Computational Methods --- Threading ed.umich.edu/i-tasser Part II 86

87 How to get tertiary structures? 5. Computational Methods --- Threading ed.umich.edu/i-tasser Part III 87

88 How to get tertiary structures? 5. Computational Methods --- Threading ed.umich.edu/i-tasser Part IV 88

89 How to get tertiary structures? 5. Computational Methods --- Homology Modeling / Threading Input Modeller is a partly automated protein structure prediction program. Output 89 89

90 How to get tertiary structures? 5. Computational Methods --- AB Initio + Homology Modeling Robetta provides both ab initio and homology modeling models of protein domains. Domains without a detectable PDB homolog are modeled with the Rosetta de novo protocol. The procedure is fully automated

91 How to get tertiary structures? 5. Computational Methods Homologues in PDB (>30%)? Best-fit template (energy function)? < 200 AA? 91

92 How to get tertiary structures? 5. Computational Methods --- MQAPs When building a protein model, with or without the aid of experimental inmation, it is often necessary to use an independent measure to evaluate the correctness of the model. This is the role of Model Quality Assessment programs (MQAPs). Different types of MQAPs have been developed during the last decades. A model on hand, finish? a random model (a low quality model) = maybe 92

93 How to get tertiary structures? 5. Computational Methods --- MQAPs I-TASSER Swiss-Model 93

94 How to get tertiary structures? 5. Computational Methods --- MQAPs I-TASSER (C-score) : C-score is typically in the range of [-5,2], where a C- score of higher value signifies a model with a high confidence and vice-versa. Swiss-Model (QMEAN4) : QMEAN4 is a reliability score the whole model which can be used in order to compare and rank alternative models of the same target. The quality estimate ranges between 0 and 1 with higher values better models. Quark (TM-score) : TM-score is a recently proposed scale measuring the structural similarity between two structures. A TM-score >0.5 indicates a model of correct topology and a TM-score<0.17 means a random similarity. 94

95 How to get tertiary structures? 5. Computational Methods --- MQAPs : SAVES 95

96 How to get tertiary structures? 5. Computational Methods --- MQAPs : SAVES >85% of the residues have an averaged 3D- 1D score > 0.2 means a good model. 96

97 How to get tertiary structures? 5. Computational Methods --- MQAPs : ProQ predicted secondary structure required 97

98 How to get tertiary structures? 5. Computational Methods --- MQAPs : MetaMQAPII 98

99 How to get tertiary structures? 5. Computational Methods --- MQAPs : ModFOLD One job takes minutes. 99

100 How to get tertiary structures? 5. Computational Methods --- Model Refinement : ModLoop freely available academic users one loop between residues 15 and 22, no chain identifier, another loop between residues 98 and 103 in chain B

101 How to get tertiary structures? 5. Computational Methods --- Model Refinement : MDS Molecular Dynamic Simulation (MDS) is a computer simulation of physical movements of atoms and molecules. Free! Free!

102 How to get tertiary structures? 5. Computational Methods --- Model Refinement : MDS Molecular Dynamic Simulation (MDS) is a computer simulation of physical movements of atoms and molecules. 500-aa protein, 1 ns (10-9 s), 120 Cores : 5 hours a meaningful simulation > 20ns 102

103 Protein Structure - Quaternary Structure How to get quaternary structures? 1. Experimentally determined molecular complexes Many proteins are actually assemblies of more than one polypeptide chain. Quaternary structure can play important functional roles multi-subunit proteins. More recently, people refer to protein-protein interaction when discussing quaternary structure of proteins and consider all assemblies of proteins as protein complexes. crystal structure of the mouse TLR3 ECD- dsrna-2:1 complex (PDB ID: 3CIY) crystal structure of the human TLR4 ECD-human MD2-E.coli LPS complex (PDB ID: 3FIX) 103

104 Protein Structure - Quaternary Structure How to get quaternary structures? 2. Protein-Protein interaction databases DIP (the Database of Interacting Proteins) : experimentally determined interactions between proteins BioGRID (the Biological General Repository Interaction Datasets) : collections of protein and genetic interactions from major model organism species STRING - Known and Predicted Protein-Protein Interactions etc. 104

105 Protein Structure - Quaternary Structure How to get quaternary structures? 3. Protein-Protein docking Macromolecular docking describes the way in which two large molecules approach and interact with each other. Docking between molecules may be likened to docking a ship at a wharf. Docking programs try all possible conmations and use scoring functions to rank them. Scoring functions including: shape complementarily residue contacts desolvation hydrophobicity electrostatics energy are usually exploited in ranking the docking conmations. 105

106 Protein Structure - Quaternary Structure How to get quaternary structures? 3. Protein-Protein docking Two kinds of protein-protein docking: Rigid Docking 刚性对接 relate to the molecules as rigid objects that cannot change their spatial shape during the docking process. When substantial conmational change occurs within the components at the time of complex mation, rigid-body docking is inadequate. Flexible Docking 柔性对接 model changes in internal geometry of the interacting partners that may occur when a complex is med. However, scoring all possible conmational changes is prohibitively expensive in computing time. 106

107 Protein Structure - Quaternary Structure How to get quaternary structures? 3. Protein-Ligand docking This problems involves a large molecule (the protein - also called the receptor ) and a small molecule (the ligand) and is very useful in developing medicines. Rigid Docking - the ligand will be kept completely rigid during the orientation step. Flexible Docking the ligand will be allowed to be flexible. This type of docking allows the ligand to structurally rearrange in response to the receptor. 107

108 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- HADDOCK 108

109 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- AutoDock 109

110 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- AutoDock Vina 110

111 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- AutoDock

112 112

113 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- AutoDock More details under AutoDock Tutorials: s.edu/faqs-help/tutorial 113

114 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- ZDOCK Almighty Online Server Almighty Online Server zdock1.pdb zdock2.pdb gongj@inmatik.u Uploading the receptor and ligand PDB files to the ZDOCK Server Please note that you must use an academic account as the ZDOCK Server is only available to academic users. 114

115 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- ZDOCK Choosing residues to block from or ce into the binding site Suppose, we had inmation from the literature that residues 732 in zdock1 is crucial binding. So this example we use this inmation to choose residue 732 and ce it into the binding site. 115

116 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- ZDOCK Confirm residues selected 116

117 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- ZDOCK The result page contains 4 links. The first three are download links the ZDOCK output file, ligand file and receptor file. To automatically create the predicted complex files, download these three files into the same directory. Then click the fourth link, "Launch Create Complexes!" which is a Java program that will create the complexes you. 117

118 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- ZDOCK A new window will appear named "Create Complexes. To create a subset (e.g. top 10), you need to specify the start and end number and then click the "Create Subset" button. "Create All" would return all max predicted complexes, which may take a few minutes to complete. In the Create Complexes window, click the "ZDOCK Output File..." button to browse the ZDOCK output file you downloaded earlier. 118

119 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- ZDOCK Now you can find the top 10 complexes in the identical directory, and you can now compare them. 119

120 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- ZDOCK 120

121 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- ZDOCK 121

122 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- PDBePISA complex1.pdb

123 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- PDBePISA 123

124 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- PDBePISA 124

125 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- PDBePISA

126 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- PDBePISA

127 Protein Structure - Quaternary Structure How to get quaternary structures? 4. Docking Program --- PDBePISA 127

128 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening Virtual screening (VS) is a computational technique used in drug discovery research. It involves the rapid in silico assessment of large libraries of chemical structures in order to identify those structures which are most likely to bind to a drug target, typically a protein receptor or enzyme. Library of chemical compounds VS focuses on questions like how can we filter down the enormous chemical space of numerous conceivable compounds to a manageable number that can be synthesized, purchased, and tested. Virtual screening 128

129 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening Commercial Free! 129

130 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening --- ZINC

131 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening --- ZINC

132 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening --- ZINC

133 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening --- ZINC The simplified molecular input line entry specification (SMILES) is a specification in m of a line notation describing the structure of chemical molecules using short ASCII strings

134 Protein Structure - Quaternary Structure 134

135 Protein Structure - Quaternary Structure

136 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening --- ZINC

137 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening --- ZINC

138 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening --- ZINC Benzoic Acid 192 similar compounds with identity > 90% 138

139 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening --- Preparing Input Files Ligands benzoic.mol2 split transm Programming required *.mol2 x 192 *.pdbqt x

140 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening --- Preparing Input Files Receptor receptor.pdb receptor.pdbqt

141 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening --- Docking with AutoDock 192 compounds automatically, programming required X 192 docking receptor 8 cores : 1.2 hours Dell T , docking results 141

142 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening --- Ranking the Docking Results Ranked by Free Energy 1.Rank Benzoic Acid: positive control 142

143 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening --- Visualization of Complex 143

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145 Protein Structure - Quaternary Structure How to get quaternary structures? 5. Virtual Screening --- Testing the Binding Affinity by Experiments 145

146 >exercise_seq AVLAGEFSDIQACSAAWKADGVCSTVAGSRPEN VRKNRYKDVLPYDQTRVILSLLQEEGHSDYING NFIRGVDGSLAYIATQGPLPHTLLDFWRLVWEF GVKVILMACREIENGRKRCERYWAQEQEPLQTG LFCITLIKEKWLNEDIMLRTLKVTFQKESRSVY QLQYMSWPDRGVPSSPDHMLAMVEEARRLQGSG PEPLCVHCSAGCGRTGVLCTVDYVRQLLLTQMI PPDFSLFDVVLKMRKQRPAAVQTEEQYRFLYHT VAQMFC exercise.fasta What kind of chemical structure is most likely to be active against this protein target? 146

147 >exercise_seq AVLAGEFSDIQACSAAWKADGVCSTVAGSRPEN VRKNRYKDVLPYDQTRVILSLLQEEGHSDYING NFIRGVDGSLAYIATQGPLPHTLLDFWRLVWEF GVKVILMACREIENGRKRCERYWAQEQEPLQTG LFCITLIKEKWLNEDIMLRTLKVTFQKESRSVY QLQYMSWPDRGVPSSPDHMLAMVEEARRLQGSG PEPLCVHCSAGCGRTGVLCTVDYVRQLLLTQMI PPDFSLFDVVLKMRKQRPAAVQTEEQYRFLYHT VAQMFC Know more about this protein: protein tyrosine phosphatase non-receptor type 18 is an enzyme that Download compounds Refine model: in the low quality regions < 24 hours Virtual screening Find homologue: 2QCT vs PTP_N18 Identity: > 48% MQAPs: ModFOLD, ProQ, QMEAN, etc. Construct 3D model: homology modeling 147