Homology Modeling: Principles, tools and techniques

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Homology ModelingPrinciples, tools and
techniques

• Supa Hannongbua
• Department of Chemistry,
• Faculty of Science,
• Kasetsart University, Bangkok 10900, THAILAND
• fscisph_at_ku.ac.th http//kuchem.sci.ku.ac.th

Supa Hannongbua
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Introduction

• Development of molecular biology rapid
  identification, isolation and sequencing of
  genes.
• Problem time-consuming task to obtain the
  3D-structure of proteins.
• Alternative strategy in structural biology is to
  develop models of protein when the contraints
  from X-ray diffraction or NMR are not yet
  available.
• Homology modeling is the method that can be
  applied to generate reasonable models of protein
  structure.

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What is Homology Modeling?

• Predicts the three-dimensional structure of a
  given protein sequence (TARGET) based on an
  alignment to one or more known protein structures
  (TEMPLATES)
• If similarity between the TARGET sequence and the
  TEMPLATE sequence is detected, structural
  similarity can be assumed.
• In general, 30 sequence identity is required for
  generating useful models.

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Structural Prediction by Homology Modeling
Structural Databases
SeqFold,Profiles-3D, PSI-BLAST, BLAST FASTA
Reference Proteins
Ca Matrix Matching
Conserved Regions
Protein Sequence
Sequence Alignment Coordinate Assignment
Predicted Conserved Regions
Loop Searching/generation
MODELER
Initial Model
Structure Analysis
Sidechain Rotamers and/or MM/MD
WHAT IF, PROCHECK, PROSAII,..
Refined Model
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How good can homology modeling be?

• Sequence Identity
• 60-100 Comparable to medium resolution NMR
• Substrate Specificity
• 30-60 Molecular replacement in crystallography
• Support site-directed mutagenesis
• through visualization
• lt30 Serious errors

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Significance of Protein Structure

• What does a structure offer in the way of
  biological knowledge?
• Location of mutants and conserved residues
• Ligand and functional sites
• Clefts/Cavities
• Evolutionary Relationships
• Mechanisms

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The importance of the sequence alignment

• the quality of the sequence alignment is of
  crucial importance
• Misplaced gaps, representing insertions or
  deletions, will cause residues to be misplaced in
  space
• Careful inspection and adjustment on Automatic
  alignment may improve the quality of the modeling.

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Programs for Model Protein Construction

• MODELLER 4.0
• guitar.rockefeller.edu/modeller/modeller.html
• SWISS-MOD Server
• www.expasy.ch/swissmod/SWISS-MODEL.html
• SCWRL (SideChain placement With Rotamer Library)
• www.fccc.edu/research/labs/dunbrack/scwrl/

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Protein Structural Databases

• Templates can be found using the TARGET sequence
  as a query for searching using FASTA or BLAST
• PDB (http//www.rcsb.org/pdb)
• MODELLER (http//guitar.rockefeller.edu/modeller/m
  odeller.html)
• ModBase (http//pipe.rockefeller.edu/modbase/gener
  al-info.html)
• 3DCrunch (http//www.expasy.ch/swissmod/SM_3DCrunc
  h.html)

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Gaining confidence in template searching

• Once a suitable template is found, it is a good
  idea to do a literature search (PubMed) on the
  relevant fold to determine what biological
  role(s) it plays.
• Does this match the biological/biochemical
  function that you expect?

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Other factors to consider in selecting templates

• Template environment
• pH
• Ligands present?
• Resolution of the templates
• Family of proteins
• Phylogenetic tree construction can help find the
  subfamily closest to the target sequence
• Multiple templates?

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Target-Template Alignment

• No current comparative modeling method can
  recover from an incorrect alignment
• Use multiple sequence alignments as initial
  guide.
• Consider slightly alternative alignments in areas
  of uncertainty, build multiple models
• Sequence-Structure alignment programs
• Tries to put gaps in variable regions/loops
• Note sequence from database versus sequence from
  the actual PDB are not always identical

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Differences in multiple sequence alignments

• Inserting gap at ends of helix versus in the
  middle
• When gaps are placed at the ends of helices, all
  models from these alignments resulted in rmsd.
  versus actual of 1.3-1.8 Å.
• In another helical region, placing them in the
  middle results in rmsd. of 2.0 Å versus less
  than 1.0 Å for correct alignment.

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Differences in multiple sequence alignments

• Inserting gaps into the middle of helices and
  misaligning
• For residues 75-95, this caused a rmsd. between
  model and actual of 5-7 Å versus less than 1.0 Å
  for the correct alignment.
• For residues 95-115, which include a random
  section, the rmsd. is 5.0 Å versus 1.5 Å for
  correct alignment.

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Target-Multiple Template Alignment

• Alignment is prepared by superimposing all
  template structures
• Add target sequence to this alignment
• Compare with multiple sequence alignment and
  adjust

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Adjusting the alignment

• Using tools such as Genedoc (www.psc.edu/biomed/ge
  nedoc) to view secondary structure along the
  alignment and use this information as criteria
  for adjustments
• Avoid gaps in secondary structure elements
• Use MEME to find a relatively large number of
  well conserved regions

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Secondary Structure Prediction

• The Predict Protein server
• http//www.embl-heidelberg.de/predictprotein/
• Adding secondary structure prediction algorithms
  can help make decisions on whether helices should
  be shortened/extended in areas of poor sequence
  identity.
• PHD program, output can be read by Genedoc.

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Constructing Multi-domain protein models

• Building a multi-domain protein using templates
  corresponding to the individual domains
• proteinA aaaaaaaaaaaaa---------------------
• proteinB -----------------bbbbbbbbbbbbbbb
• Target aaaaaaaaaaaaabbbbbbbbbbbbbbb

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Multiple model approach

• Reminder Consider the effects of different
  substitution matrices, different gap penalties,
  and different algorithms. (Vogt et al. J. Mol.
  Biol. 1995, 249816-831.)
• Construct multiple models
• Use structural analysis programs to determine
  best model

Jaroszewski, Pawlowski and Godsik, J. Molecular
Modeling, 1998, 4294-309 Venclovas, Ginalski and
Fidelis. PROTEINS, 1999, 373-80 (Suppl)
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Model Building

• Rigid-Body Assembly
• Assembles a model from a small number of rigid
  bodies obtained from aligned protein structure
• Implememted in COMPOSER
• Segment Matching
• Satisfaction of Spatial Restraints
• MODELLER
• guitar.rockefeller.edu/modeller/modeller.html

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Initial model and procedures

• Calculate coordinates for atoms that have
  equivalent atoms in the templates as an average
  over all templates
• CHARMM internal coordinates are used for
  remaining unknown coordinates
• Generate stereochemical and homology derived
  restraints

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Spatial restraints ?

• Minimizes the objective function, F, with respect
  to the Cartesian coordinates of the protein atoms
• F(R) Sci (fi,pi)
• R are the cartesian coordinates of the atoms
• c is a restraint dependant on f,p
• f is a geometric feature of a molecule and
  include the distance, angle and dihedral values
• p are parameters to help describe some restraints

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Homology and Sterochemical Restraints

• Initial model is an average over all templates
• Stereochemical bond, angle, dihedral, improper
• Homology mainchain and sidechain dihedrals
• mainchain CA-CA distances
• sidechain-mainchain distances
• sidechain-sidechain distances
• Non-bonded pairs (on the fly)
• user-defined

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Sidechain Conformation

• Protein sidechains play a key role in molecular
  recognition and packing of hydrophobic cores of
  globular proteins
• Protein sidechain conformations tend to exist in
  a limited number of canonical shapes, usually
  called rotamers
• Rotamer libraries can be constructed where only
  3-50 conformations are taken into account for
  each side chain

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Coupling between mainchain and sidechain

• Mainchain shifts (0.2 0.5 Å) cause increased
  sidechain coordinate errors (0.1 0.8 Å ),
  torsional errors of 10-30º and exaggerated strain
  energy for overpacked mutants compared with the
  correct mutant backbones.
• Lee, C. Folding and Design, 1995, 11-12

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Sidechains on surface of protein

• Exposed sidechains on surface can be highly
  flexible without a single dominant conformation
• So ultimately if these solvent exposed sidechains
  do not form binding interactions with other
  molecules or involved in say, a catalytic
  reaction, then accuracy may not be crucialalso
  look at the B-factors
• Can refine the sidechains with molecular
  mechanics minimization
• Sampling?
• Scoring?

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Clustering the ensemble

• Cluster analysis, based on overall fold, followed
  by selection of the structure closest to the
  centroid of the largest cluster is likely to
  identify a structure more representative of the
  ensemble than the commonly used minimized average
  structure

NMRCLUST (http//neon.chem.le.ac.uk/nmrclust/prot
ocol.html)
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Errors in Homology Modeling

• a) Side chain packing b)Distortions and
  shifts c) no template

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Errors in Homology Modeling

• d) Misalignments e) incorrect
  template
• Marti-Renom et al., Ann. Rev. Biophys. Biomol.
  Struct., 2000, 29291-325.

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Detection of Errors

• First check should include a stereochemical check
  on the modeled structurePROCHECK, WHATCHECK,
  DISTAN which will show deviations from normal
  bond lengths, dihedrals, etc.
• Visualization follow the backbone trace and then
  subsequently move out to Ca-Cß orientation.

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PROCHECK
http//www.biochem.ucl.ac.uk/roman/ procheck/proc
heck.html
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Dihydrofolate Reductase (DHFR) multiple sequence
alignment
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Dihydrofolate Reductase (DHFR) alignment
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