Tertiary Structure Prediction Methods

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Tertiary Structure Prediction Methods
Any given protein sequence
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Why Homology modelling ?

• X-ray Diffraction
• Only a small number of proteins can be made to
  form crystals.
• A crystal is not the proteins native
  environment.
• Very time consuming.
• NMR Distance Measurement
• Not all proteins are found in solution.
• This method generally looks at isolated
  proteins rather
• than protein complexes.
• Very time consuming

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Homology ModelingPrinciples, tools and
techniques

• 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 constraints
  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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Database approach to homology modelling
As of June 2000, 12,500 protein structures have
been deposited into the Protein Data Bank (PDB)
and 86,500 protein sequence entries were
contained in SwissProt protein sequence
database. This is a 17 ratio relatively few
structures are known. The number of sequence
will increase much faster than the number of
structures due to advances in sequencing.
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Sequence similarity methods
These methods can be very accurate if there is
gt 50 sequence similarity. They are rarely
accurate if the sequence similarity lt 30.
They use similar methods as used for sequence
alignment such as the dynamic programming
algorithm, hidden markov models, and clustering
algorithms.
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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,
Fold-recognition methods (FUGUE)
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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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 Joy (www-cryst.bioc.cam.ac.uk/
  joy/) to view secondary structure along the
  alignment and use this information as criteria
  for adjustments
• Avoid gaps in secondary structure elements

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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

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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
• Implemented 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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Modeller

• Main input are restraints on the spatial
  structure of AA and ligands to be modeled.
• Output is a 3D structure that satisfies these
  restraints
• Restraints are obtained from related protein
  structures (homology modeling) - obtained
  automatically, NMR structures, secondary struture
  packing and other experimental data

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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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What are the Restraints ?

• distances, angles, dihedral angles, pairs of
  dihedral angles and some other spatial features
  defined by atoms or pseudo atoms.

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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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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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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