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Homology Modeling Workshop
GHIKLSYTVNEQNLKPERFFYTSAVAIL
Outline:
• Introduction to protein structure & databases
• Structure prediction approaches– Ab-initio– Threading– Homology modeling
• Hands ON
From Sequence to StructureProtein structure is hierarchic:
• Primary – sequence of covalently attached amino acid
• Secondary – local 3D patterns (helices, sheets, loops)
• Tertiary – overall 3D fold
• Quaternary – two or more protein chains
• All information about the native structure of a protein is encoded in the amino acid sequence + its native solution environment.
• Many possible conformation still only one or few native folds are exhibited for each protein (Levinthal’s paradox)
• Protein folding is driven by various forces:– Ionic forces– Hydrogen bonds– The hydrophobic affect– . . .
From Sequence to Structure
Protein 3D StructuresA protein’s structure has a critical effect on its function:
1. Binding pockets
PDB ID 1nw7
Protein 3D StructuresA protein’s structure has a critical effect on its function:
2. Areas of specific chemical\electrical properties
Protein 3D StructuresA protein’s structure has a critical effect on its function:
3. Importance of the global fold for function
Motivation to Acquire a Structure
• Identifying active and binding sites
• Characterization of the protein’s mechanism (catalysis & interactions)
• Searching for ligand of a given binding site
• Understanding the molecular basis of diseases
• Designing mutants
• Drug design
• And more...
Determining Structure
• NMR
• X-ray diffraction
• Electron Microscopy
Why predict protein structure if we can use experimental tools to
determine it?
• Experimental methods are slow and expensive
• Some structures were failed to be solved
• A representative family structure can suffice to
deduce structures of the entire family sequences
Protein databases
Protein Sequence& Structure Databases
Some of the available databases:
• RCSB- the Protein Data Bank- all deposited structures
• UniProt- main sequence database– SwissProt– Tremble
• NCBI- lots of databases, including sequence and structures
• PDBsum- combines structural & sequence data
UniProt- Protein Sequence Database
• UniProt is a collaboration between the European Bioinformatics Institute (EBI), the Swiss Institute of Bioinformatics (SIB) and the Protein Information Resource (PIR).
• In 2002, the three institutes decided to pool their resources and expertise and formed the UniProt Consortium.
• The world's most comprehensive catalog of information on proteins
• Sequence, function & more…
• Comprised mainly of the databases:
– SwissProt –516081 entries– high quality annotation, non-redundant & cross-referenced to many other databases.
– TrEMBL – 10618387 entries – computer translation of the genetic information from the EMBL Nucleotide Sequence Database many proteins are poorly annotated since only automatic annotation is generated
UniProt- Protein Sequence Database
UniProt- Protein Sequence Database
UniProt- Protein Sequence Database
• The PDB archive contains information about experimentally-determined structures of proteins, nucleic acids, and complex assemblies.
• The structures in the archive range from tiny proteins and bits of DNA to complex molecular machines like the ribosome.
• There are currently 57013 structures deposited in the PDB. However, taking out redundant sequences (e.g. 90%) reduces the number of structures to 19988…
• Each structure receives a unique 4 letter ID
Protein Data Bank (PDB)
Protein Data Bank (PDB)http://www.rcsb.org/pdb/home/home.do
PDB ID: 3mht
Download structure
Displaystructure
Data concerning the structure -
resolution, R-value.…
The paper describingthe structure
Protein Data Bank (PDB)http://www.rcsb.org/pdb/home/home.do
Year
Protein Data Bank (PDB)
PdbSum• A database providing an overview of all biological
macromolecular structures
• Connected to UniProt find the sequence accession of a known PDB ID
• Detailed description of many structure properties, e.g.:– EC number– Chains & ligands and their interactions– Clefts– Secondary structure– FASTA sequence of structure…– …
PDB ID
Free text
Search by sequence
http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/
PdbSum
Useful tabs
UniProtaccession
Chains & ligands
PdbSum
Protein tab
Secondary structure-from the PDB
PdbSum
More Sequences Than Structures
• Discrepancy between the number of known sequences and solved structures:
5,047,807 UniRef90 entries vs.
25566 90% Non-redundant structures
Computational methods are needed to obtain more
structures
Structure prediction approaches
Structure Prediction Approaches
1. Homology (Comparative) Modeling
Based on sequence similarity with a protein for
which a structure has been solved.
2. Threading (Fold Recognition)
Requires a structure similar to a known structure
3. Ab-initio fold prediction
Not based on similarity to a sequence\structure
Ab-initioStructure prediction from “first principals”:
Given only the sequence, try to predict the structure
based on physico-chemical properties
(energy, hydrophobicity etc.)
• When all else fails works for novel folds
• Shows that we understand the process
The Force Field(energy function)
A group of mathematical expressions describing the
potential energy of a molecular system
• Each expression describes a different type of physico-
chemical interaction between atoms in the system:
• Van der Waals forces
• Covalent bonds
• Hydrogen bonds
• Charges
• Hydrophobic effects
Non-bonded terms
Approaches to Ab-initio Prediction1. Molecular Dynamics
• Simulates the forces that governs the protein within water.• Since proteins usually naturally fold, this would lead to the
native protein structure.
Problems:• Thousands of atoms• Huge number of time steps to reach folded protein
feasible only for very small proteins
Approaches to Ab-initio Prediction
2. Minimal Energy
Assumption: the folded form is the minimal energy conformation of a protein
Main principals:• Define an energy function.• Search for 3D conformation that minimize energy.
• Current methods (e.g. Rosetta) primarily utilize the fact that although we are far from observing all protein folds, we probably have seen nearly all sub-structures:
Ab-initio
Moult J. Philos. Trans. R. Soc. B. 361:453–458 (2006)
• A library of known sub-structures (fragments less than 10 residues) is created.
• A range of possible conformations for each fragment in the query protein are selected.
Ab-initio - Example
Moult J. Philos. Trans. R. Soc. B. 361:453–458 (2006)
Given a sequence and a library of folds, thread the sequence
through each fold. Take the one with the highest score.
• Method will fail if new protein does not belong to any fold in
the library.
• Score of the threading is computed based on known
physical chemistry properties & statistics of amino acids.
• In practice, fold recognition methods are often mixtures
of sequence matching and threading.
Fold Recognition (Threading):Sequence to structure matching
Input:1. sequence
H bond donorH bond acceptor
GlycinHydrophobic
2. Library of folds of known proteins
Threading: example
Structure Prediction Approaches
S=20S=5S=-2Z=5Z=1.5Z= -1
H bond donorH bond acceptorGlycineHydrophobic
Threading: example
MAHFPGFGQSLLFGYPVYVFGD...
Potential fold
...
1) ... 56) ... n)
...
-10 ... -123 ... 20.5
Fold recognition (threading)Find best fold for a protein sequence:
We need a scoring (energy) function to distinguish native structure from misfolded structures.
Ideally, each misfolded structure should have an energy higher than the native energy, i.e. :Emisfolded-Enative> 0
Fold recognition: FFAS03
•The FFAS03 server provides an interface to the third generation of the profile-profile alignment and fold recognition algorithm FFAS.
• Profile-profile alignments utilize information present in sequences of homologous proteins to amplify the sequence conservation pattern defining the family
•The result: detection of remote homologies beyond the reach of other sequence comparison methods.
Jaroszewski, L., Rychlewski, L., Li, Z., Li, W. & Godzik, A. (2005) FFAS03: a server for profile-profile sequence alignments. Nucl. Acids Res. 33, W284-W288
Fold recognition: HHPRED
0.1
0.4
0.5
0.3
0.7
0.4
0.6
0.7
0.1
0.2
0.6
Emit Amino acid
Profiles are based on Hidden Markov Models:
Söding J. (2005) Protein homology detection by HMM-HMM comparison. Bioinformatics 21, 951-960.
Fold recognition: HHPRED
• Profile Hidden Markov Models (HMMs) are similar to sequence profiles, but in addition to the amino acid frequencies they contain information about the frequency of inserts and deletions.
• Using profile HMMs in place of simple sequence profiles should therefore further improve sensitivity.
• The first to employ HMM-HMM comparison, based on a novel statistical method.
• Using HMMs both on the query and the database side greatly enhances the sensitivity and selectivity over sequence-profile based.
Söding J. (2005) Protein homology detection by HMM-HMM comparison. Bioinformatics 21, 951-960.
I-TASSER- Hybrid Approach
• In a recent wide blind experiment, I-TASSER generated the best 3D structure predictions among all automated servers.
• Based on the secondary-structure threading and the iterative implementation of the Threading ASSEmbly Refinement (TASSER) program.
I-TASSER
Homology Modeling
Homology Modeling – Basic Idea
Triophospate ismoerases44.7% sequence identity0.95 RMSD
1. A protein structure is defined by its amino acid sequence.
2. Closely related sequences adopt highly similar structures, distantly related sequences may still fold into similar structures.
3. Three-dimensional structure of
proteins from the same family is
more conserved than their
primary sequences.
Homology modeling requires handling structures & sequences
• Query- only the protein sequence is available- usually found at the UniProt database
• Template- after identification, both structural and sequence-related data should be found- UniPort (or NCBI databases), RCSB and PDBsum
Query proteinsequence
Homology modeling-
widespread technique
e.g. Fiser et al., 2004; Petrey et al., 2005; Zhang, 2008
Homologous protein-structural template
Align query & templateprotein sequences
Build model
Evaluate model
Identify Homologous protein-structural template
Align query & templateprotein sequences
General Scheme
1. Searching for structures related to the query sequence
2. Selecting templates
3. Aligning query sequence with template structures
4. Building a model for the query using information from the template structures
5. Evaluating the model
Modeller
Fiser A et al. Methods in Enzymology 374: 461-491(2004)
General Scheme
1. Searching For Structures
• Sequence search against the PDB sequences
• Sequence-profile search
• Threading: sequence-structure fitness function
1. Searching For StructuresIf BLAST search against the PDB fail to recognize adequate templates, turn to fold recognition (threading) servers:
• FFAS03- http://ffas.ljcrf.edu/ffas-cgi/cgi/ffas.pl
• HHPRED- http://toolkit.tuebingen.mpg.de/hhpred
• HMAP (available through the FUDGE pipeline)- http://wiki.c2b2.columbia.edu/honiglab_public/index.php/Software:PUDGE
• I-TASSER- http://zhang.bioinformatics.ku.edu/I-TASSER/
These servers not only find optional templates, but also suggest a pairwise alignment and in some cases even construct the 3D model.
2. Selecting TemplatesHow to select the right template?
• Higher sequence similarity - %ID
• Close subfamily - phylogenetic tree
• “Environment” similarity - solvent, pH, ligand, quaternary interactions
• The quality of the experimentally determined
structure
• Purpose of modeling - e.g. protein-ligand model vs. geometry of active site
Seq. 2
Seq. 1
Seq. 3
Seq. 4
Seq. 5Seq. 6
2. Selecting Templates
More than one template
• Two ways to combine multiple templates:
– Global model – alignment with different domain of the target with little overlap between them
– Local model – alignment with the same part of the target
More than one template
The more the merrier -
multiple structures with
the same fold:
2. Selecting Templates
2. Selecting Templates
Trial and error
• Generate a model for each candidate template and/or their combination.
• Evaluate the models by an energy or any other scoring function.(will be discussed later…)
3. Aligning query and template sequences
• All comparative modeling programs depend on a target-template alignment.
• When the sequence similarity between the template and target proteins is high, simple pairwise alignments are usually fine (e.g. Needleman-Wunsch global alignment).
• Gaps or low/medium sequence similarity indicate that we should improve the alignment...
Guidelines:
1. Create a multiple sequence alignment and extract thetemplate-query pairwise alignment.
3. Aligning query and template sequences
Pairwise alignments – not enough!
Guidelines:
1. Create a multiple sequence alignment and extract thetemplate-query pairwise alignment.
• Visual inspection of alignments - difficult to teach… a matter of experience…
TemplateQuery
3. Aligning query and template sequences
Guidelines:
1. Create a multiple sequence alignment and extract thetemplate-query pairwise alignment.
2. Use secondary structure information to improve pairwise alignment- avoid gaps in these regions!
QueryTemplate
3. Aligning query and template sequences
Guidelines:
1. Create a multiple sequence alignment and extract thetemplate-query pairwise alignment
2. Use secondary structure information to improve pairwise alignment- avoid gaps in these regions!
3. Biochemical and structural previous data
3. Aligning query and template sequences
• Where? (to find homologues)
• Structural templates- search against the PDB
• Sequence homologues- search against SwissProt or Uniprot (recommended!)- usually using BLAST
• How many?
• As many as possible, as long as the MSA looks good (next week…)
3. Aligning query and template sequences
Tips for MSA building
• How long? (length of homologues)
• Fragments- short homologues (less than 50,60% the query’s length) = bad alignment
• Ensure your sequences exhibit the wanted domain(s)
• N/C terminal tend to vary in length between homologues
• How close? (distance from query sequence)
• All too close- no information
• Too many too far- bad alignment
• Ensure that you have a balanced collection!
3. Aligning query and template sequences
Tips for MSA building
• From who? (which species the sequence belongs to)
• Don’t care, all homologues are welcome
• Orthologues/paralogues may be helpful
• Sequences from distant/close species provide different types of information
• Which alignment method?
• The best today are MUSCLE, T-Coffee and MAFFT. All available at
3. Aligning query and template sequences
Tips for MSA building
3. Aligning query and template sequences
Tips for MSA building
• Most importantly, make sure that both the query and the selected template are included in the MSA.
• Sequences which are more distant than the template are not needed to be included in the alignment.
3. Aligning query and template sequences
Query-template alignment via a profile-to-profile approach:
1. Construct an MSA for the query, serving as profiles depicting the protein family properties.
2. Align the profile to profiles of all proteins of the PDB, using, e.g., FFAS03 or HHpred.
3. Compare pairwise alignments constructed via the different methods – hope to get a consensus prediction…
3. Aligning query and template sequences
Different levels of similarity between the template & query initiate various computational approaches:
4. Building a model
Once you have an improved pairwise alignment between your query & template
Use Modeller to build your model!
A. Sali & T.L. Blundell. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779-815, 1993.
Generation and Refinement Using satisfaction of spatial restrains Can perform additional tasks:
de novo modeling of loops Optimization of models – using an objective
function Multiple alignment Comparison of protein structures
Modeller
4. Building a model
• Other spatial features, such as hydrogen bonds, and dihedral angles, are transferred from the templates to the target.
• Thus, a number of spatial restraints on its structure are obtained.
• The 3D model is obtained by satisfying all the restraints as well as possible.
Modeller
4. Building a model
• Distance and dihedral angle restraints on the target are calculated from its alignment with template.
• Restraints were obtained also from a statistical analysis of the relationships from a large database of pairs of homologous structures.
• Various correlations were obtained, e.g. correlations between Ca-Ca distances. These relationships can be used directly as spatial restraints.
• Restraints and CHARMM energy terms are then combined into an objective function, which is optimized in 3D space.
Modeller
4. Building a model
5 .Model Evaluation
• The accuracy of the model depends on its sequence identity with the template:
5 .Model EvaluationThe model can be assessed in two levels:
• Global- reliability of the model as a whole.*Useful when several models are generated and one should be chosen as the best one.*When different models were based on various templates, may help choose the best one.
• Local- assessing the reliability of the different regions, even specific residues, of the model. *Useful to detect local mistakes, that may originate in many time from alignment errors.
5 .Model EvaluationExamples of assessment approaches:
1. Assessment of the model’s stereochemistry
2. Prediction of unreliable regions of the model - “pseudo energy” profile: peaks errors
3. Consistence with experimental observations
4. Consistence with evolutionary conservation rates
Summary :
5 Basic Steps
Hands ON
The Query ProteinName: Dihydrodipicolinate reductase
Enzyme reaction:
Molecular process: Lysine biosynthesis (early stages)
Organism: E. coli
Sequence length: 273 aa
1. Searching For Structures
1. Searching For Structures
Get your sequence
<DAPB_ECOLIMHDANIRVAIAGAGGRMGRQLIQAALALEGVQLGAALEREGSSLLGSDAGELAGAGKTGVTVQSSLDAVKDDFDVFIDFTRPEGTLNHLAFCRQHGKGMVIGTTGFDEAGKQAIRDAAADIAIVFAANFSVGVNVMLKLLEKAAKVMGDYTDIEIIEAHHRHKVDAPSGTALAMGEAIAHALDKDLKDCAVYSREGHTGERVPGTIGFATVRAGDIVGEHTAMFADIGERLEITHKASSRMTFANGAVRSALWLSGKESGLFDMRDVLDLNNL
http://www.uniprot.org/
1. Searching For StructuresFind templates with significant homology:
• BLAST against the sequences in the PDB
Find also more distant templates, using profile-to-profile approach:
• FFAS03 server• HHPRED server
1. Searching For StructuresBlast against the PDB
http://www.ncbi.nlm.nih.gov/BLAST/
1. Searching For StructuresBlast against the PDB
1 .Paste sequence
2. Select the PDB database
3.
http://www.ncbi.nlm.nih.gov/BLAST/
1. Searching For StructuresBlast against the PDB
http://www.ncbi.nlm.nih.gov/BLAST/
1. Searching For StructuresUse fold recognition - FFAS03
Select the PDB database
1 .Paste sequence
Run
1. Searching For StructuresUse fold recognition - HHPRED
Select the PDB database
1 .Paste sequence
Run
http://toolkit.tuebingen.mpg.de/hhpred
2. Selecting templates
2. Selecting templatesBlast against the PDB
The real structureof our protein
Closest homologousstructure
2. Selecting templatesBlast against the PDB
http://www.ncbi.nlm.nih.gov/BLAST/
The selected template:
1VM6, chain A
2. Selecting templatesUse fold recognition - FFAS03
http://ffas.ljcrf.edu/ffas-cgi/cgi/get_mu.pl?ses=&qdb=public&tdb=PDB0408&type=re&key=221830166.3750.0000000
2. Selecting templatesUse fold recognition - FFAS03
Scores below -9.5 significant
2. Selecting templatesUse fold recognition - HHPRED
http://toolkit.tuebingen.mpg.de/hhpred/histograms/8455009
2. Selecting templatesUse fold recognition - HHPRED
2. Selecting templatesWho is our template?
www.ebi.ac.uk/thornton-srv/databases/pdbsum
PDB ID 1VM6 is UniProt entry
‘DAPB_THEMA’
3. Alignment
http://consurftest.tau.ac.il/
3. Alignment
No model yet…
We will use ConSurf to get homologues and
build and MSA
3. Alignment
Alignment method
Database;Swissprot/
uniprot/uniref90/NR
Set to max- 500
Redundancy
Min. identity
3. Alignment
Job name
3. Alignment
3. Alignment
MSA- download the file- right click on the mouse
Filtered sequences
PSIBLAST result
3. Alignment
• http://www.mbio.ncsu.edu/BioEdit/BioEdit.html
• Easy-to-use sequence alignment editor
• View and manipulate alignments up to 20,000 sequences. •Four modes of manual alignment: select and slide, dynamic grab and drag, gap insert and delete by mouse click, and on-screen typing which behaves like a text editor.
•Reads and writes Genbank, Fasta, Phylip 3.2, Phylip 4, and NBRF/PIR formats. Also reads GCG and Clustal formats
Easiest Using Bioedit
Easiest Using Bioedit
http://www.mbio.ncsu.edu/BioEdit/bioedit.html
Easiest Using Bioedit
http://www.mbio.ncsu.edu/BioEdit/bioedit.html
• Find a specific sequence: “Edit-> search -> in titles”
• Erase\add sequences: “Edit-> cut\paste\delete sequence”
• “Sequence Identity matrix” under “Alignment”- useful for a rough evaluation of distances within the alignment.
• After taking out sequences, “Minimize Alignment” under “Alignment” takes out unessential gaps.
• Can save an image using: “File -> Graphic View” & then “Edit -> Copy page as BITMAP”
1. Open: Start Phylogeny BioEdit
2. Open the alignment: file open ‘query.aln’
2. Select the template:Edit Search Find in Titles “DAPB_THEMA”
3. AlignmentExtract query-template pairwise alignment
3. AlignmentExtract query-template pairwise alignment
“DAPB_THEMA”
4. Add the query to the template selection: ctrl + ‘query’
5. Invert selection: Edit invert title selection
6. Delete other sequences: Edit Cut Sequences(s)
7. Minimize gaps: Alignment Minimize Alignment
8. Save the pairwise alignment:File Save as (Fasta format) “DAPB_ECOLI_1VM6.fas”
3. AlignmentExtract query-template pairwise alignment
3. AlignmentExtract query-template pairwise alignment
Save as “fasta” format!!!!!!!
queryDAPB_THEMA
File name
Use fold recognition - FFAS03
Scores below -9.5 significant
3. Alignment
Use fold recognition - FFAS03
3. Alignment
http://ffas.ljcrf.edu/ffas-cgi/cgi/get_mu.pl?ses=&qdb=public&tdb=PDB0408&type=re&key=221830166.3750.0000000
Use fold recognition - HHPRED
http://toolkit.tuebingen.mpg.de/hhpred/histograms/8455009
3. Alignment
Use fold recognition - HHPRED3. Alignment
• Generally speaking, in this step we would compare the pairwise alignments computed by the three approaches:• MSA-derived• FFAS03• HHPRED
• We don’t have the time/patience for that now….
• Thus, we will now edit the pairwise from the MSA- Modeller requires a specific format, which we have to manually adjust
3 .AlignmentInspect query-template pairwise alignment
>P1; DAPB_ECOLIsequence:DAPB_ECOLI:1:A:274:A ::::MHDANIRVAIAGAGGRMGRQLIQAALALEGVQLGAALEREGSSLLGSDAGELAGAGKTGVTVQSSLDAVKDDFDVFIDFTRPEGTLNHLAFCRQHGKGMVIGTTGFDEAGKQAIRDAAADIAIVFAANFSVGVNVMLKLLEKAAKVMGDYTDIEIIEAHHRHKVDAPSGTALAMGEAIAHALDKDLKDCAVYSREGHTGERVPGTIGFATVRAGDIVGEHTAMFADIGERLEITHKASSRMTFANGAVRSALWLSGKESGLFDMRDVLDLNNL*
>P1;1VM6structureX:1VM6:1:A:212:A ::::-----MKYGIVGYSGRMGQEIQKVFSE-KGHELVLKVDV------------------------NGVEEL-DSPDVVIDFSSPEALPKTVDLCKKYRAGLVLGTTALKEEHLQMLRELSKEVPVVQAYNFSIGINVLKRFLSELVKVLE-DWDVEIVETHHRFKKDAPSGTAILLESAL--------------------GK----SVPIHSLRVGGVPGDHVVVFGNIGETIEIKHRAISRTVFAIGALKAAEFLVGKDPGMYSFEEVI-----*
3. AlignmentEdit query-template pairwise alignment
The PDB file of the template (rename DAPB_THEMA)
The name of the query protein (this will be the name of the modeled PDB file)
Save as “dapb_ecoli_1vm6.pir”
Start, end and chain
4 .Model Building
from modeller import * from modeller.automodel import *
log.verbose ()env = environ ()
a = automodel(env, alnfile = 'dapb_ecoli_1vm6.pir ,'
knowns = ('1VM6') , sequence = 'DAPB_ECOLI ('
a.starting_model= 1 a.ending_model = 1
a.make ()
A script for Modeller- copy to a text file.…
4. Model Building
4. Model BuildingGet the template structure
1 .Paste the template’s PDB ID “1VM6”
2 .
http://www.rcsb.org/pdb/home/home.do
Get the template structure: 1vm6 chain A
Save as: “1VM6.pdb”
4. Model Building
Notice:case
sensitive!
4 .Model BuildingRunning modeller:
1. Put the PDB file, PIR alignment and modeller script in a specific directory, e.g. c:\test
2. Desktop Modeller:
4 .Model BuildingRunning modeller:
3. “cd c:\test”4. “mod9v7 [modeller script name]
4 .Model BuildingRunning modeller:
5. The run completed successfully:
4 .Model BuildingRunning modeller:
6. Output files:• Model, e.g. “P2RX1_HUMAN.B99990001.pdb”• Log file- very important- specifies the problems of
the run• Other, not important, files
7. Open pymol and look at your model….
8. Evaluate it- tomorrow!
Edit query-template pairwise alignment
Watch out! Modeller can fail owing to:
1.Non-matching start and end points of the template at the PIR alignment and PDB template file
2. Small discrepancies between the sequence of the template and in the PIR alignment… may have to manually edit the alignment a little…
This, and more, will be reported in the log file
4 .Model Building