Download Introduction to 3D-Structure Visualization and Homology Modeling

Introduction to 3D-Structure Visualization and
Homology Modeling using
the Swiss-Model Workspace
Lorenza Bordoli
Biozentrum of the University of Basel and
Swiss Institute of Bioinformatics
Lecture 3
May 2009
Lecture 3: Outline
• Homology modeling using the SwissModel Workspace:
– Target sequence feature annotation
– Template identification
– Target-template alignment
– Homology Modeling by template based
fragment assembly
– Model quality estimation
Homology (Comparative)
Modeling
Evolution of Protein Structures
Protein structure is better conserved than sequence
Similar Sequence Î Similar Structure
Homology modeling = Comparative protein modeling
Idea:
Using experimental 3D-structures of related
family members (templates) to calculate a
model for a new sequence (target).
Comparative Protein Structure Modeling
General Workflow
Known Structures
(Templates)
Target
Sequence
Template Selection
Alignment
Template - Target
Structure Evaluation &
Assessment
Structure modeling
Homology
Model(s)
Protein structure homology modeling using
SWISS-MODEL workspace
•
Freely accessible: http://swissmodel.expasy.org/workspace
Comparative Protein Structure Modeling
Protein
Domain
annotation
(InterPro)
Target
Sequence
Known Structures
(Templates)
Template Selection
Alignment
Template - Target
Structure Evaluation &
Assessment
Structure modeling
Homology
Model(s)
Functional annotation of multi-domain proteins
MSA
MSA
Model (HMM, PSSM,…) for
Model for
Model for
DNA bdg. Function
Activation Function 1
MSA
Activation Function 2
InterPro http://www.ebi.ac.uk/interpro/
InterPro is a database protein families,
domains, regions, repeats and sites in which
identifiable features found in known proteins
can be applied to new protein sequences.
Member Databases:
–
–
–
–
–
–
–
–
–
Pfam (HMMs)
PRINTS (PSSMs)
PROSITE (Patterns & Profiles)
ProDom (Motifs from PsiBlast)
SMART (HMMs)
TIGRFAMs (HMMs)
PANTHER (HMMs)
Superfamily (HMMs from SCOP)
UniProt (Sequences)
InterPro
1. Family:
An InterPro family is a group of evolutionarily related proteins that share similar
domain (or repeat) architecture.
2. Domain: An InterPro domain is an independent structural unit, which can be found alone or in
conjunction with other domains or repeats. Domains are evolutionarily related.
3. Repeat:
An InterPro repeat is a region that is not expected to fold into a globular domain on its
own. For example 6-8 copies of the WD40 repeat are needed to form a single
globular domain.
4. PTM site: A post-translational modification modifies the primary protein structure. This
modification may be necessary for activation or de-activation of function. Examples
include glycosylation, phosphorylation, and sulphation, splicing etc.
5. Binding site: An InterPro Binding site binds chemical compounds, which themselves are not
substrates for a reaction. The compound, which is bound, may be a required co-factor
for a chemical reaction, be involved in electron transport or be involved in protein
structure modification.
6. Active site: Active sites are best known as the catalytic pockets of enzymes where a substrate is
bound and converted to a product, which is then released. Distant parts of a protein's
primary structure may be involved in the formation of the catalytic pocket. Therefore,
to describe an active site, different signatures will be needed to cover the active site
residues.
Functional and structural domain annotation
• Individual structural domains of multidomain
proteins often correspond to units of distinct
molecular function => you can compare the
functional domain prediction with the functional
annotations of detected templates.
• The sensitivity of profile-based template
detection methods can be enhanced when the
search is performed at the domain level rather
than searching the whole protein sequence.
Comparative Protein Structure Modeling
Sequence feature
annotation: 1) prediction of
secondary structure
elements, 2) disorder and 3)
Known
transmembrane
(TM)Structures
regions
(Templates)
Target
Sequence
Template Selection
Alignment
Template - Target
Structure Evaluation &
Assessment
Structure modeling
Homology
Model(s)
Target sequence feature annotation
• In the twilight zone of sequence alignments,
applying secondary structure prediction to the
protein of interest may help deciding whether a
putative template shares essential structural
features.
• The sensitivity of profile-based template
detection methods can be enhanced when the
search is performed at the domain level rather
than searching the whole protein sequence.
Target sequence feature annotation
• Intrinsically unstructured (disordered) regions
in proteins have been associated with
numerous important biological cellular
functions (e.g. cell signaling, transcriptional
regulation,…)
• Prediction of disordered and transmembrane
regions therefore complement the analysis of
protein domain boundaries and functional
annotation of the target protein.
Swiss-Model Workspace: Sequence feature
annotations
Swiss-Model Workspace: Sequence feature
annotations
Human Rhodopsin
Protein Data BankModeling
PDB
Comparative Protein• Structure
http://www.pdb.org =>
Known Structures
(Templates)
Target
Sequence
•
•
•
Database of templates
Separate into single chains
Remove bad structures
Create BLASTable database
or fold library (profiles,
HMMs)
Template Selection
Alignment
Template - Target
Structure Evaluation &
Assessment
Structure modeling
Homology
Model(s)
Swiss-Model Workspace: Template Library
Swiss-Model Workspace: Template Library
Comparative Protein Structure Modeling
Known Structures
(Templates)
Target
Sequence
Template Selection
Template selection:
• Sequence Similarity (e.g.
Alignment
Blast, Psi-Blast,
HMMs-Evaluation &
Structure
Template - Target HMMs alignment) Assessment
• Structure quality (resolution,
experimental method)
Structure modeling • Experimental conditions
Homology
(ligands and cofactors)
Model(s)
Template identification: Blast, PSI-Blast
PDB: database of protein structures
unknown
structure ?
Template detection:
Similarity Search
comparative
modeling
Alignment to similar protein
with known structure
Template identification using fold libraries
Example : Leucin Zipper DNA binding Domain of Transcription Factors.
Human c-fos/c-jun
Structural superposition
Yeast bZIP
MSA
…
Model (HMM, PSSM,…) for
Structural domains
(e.g. TF DNA binding domain)
=> Library of folds
Structural Alignments
•
Protein structure is better conserved than sequence
•
Structural alignments establish equivalences between amino acid
residues based on the 3D structures of two or more proteins.
•
Structure alignments therefore provide information not available
from sequence alignment methods alone.
Template identification: HMMs, Profile
libraries
Library of HMMs/Profiles for protein structures
unknown
structure ?
Template detection:
Similarity Search
comparative
modelling
Alignment to HMMs/Profiles
of known structures
Swiss-Model Workspace: Template identification
Swiss-Model Workspace: Template identification
Swiss-Model Workspace: Template identification
Swiss-Model Workspace: Template identification
Swiss-Model Workspace: DeepView Project file
Comparative Protein Structure Modeling
• Pair wise sequence
alignments (Blast, PSIBLAST)
Known Structures
• MSA (T-coffee) or
(Templates)
• HMMs/Profile based
tools (HHpred)
Target
Sequence
Template Selection
Alignment
Template - Target
Structure Evaluation &
Assessment
Structure modeling
Homology
Model(s)
Swiss-Model Workspace: target-template
alignment
• The target–template sequence alignments generated by
the different template database search techniques can
be used as the basis for the subsequent model creation.
The alignments can be downloaded as DeepView project
file, which contains the target sequence aligned to the
template structure.
• The program DeepView allows you to display and
analyze the alignment in the structural context of the
template to manually adjust misaligned regions.
• Once you have finished editing the alignment, save the
project file on the local disk and submit it to the ‘Project
Mode’ of the Modeling session for model building
DeepView: “Manual Modeling”
[ http://www.expasy.org/spdbv/ ]
Swiss-Model Workspace: Modeling
Swiss-Model Workspace: target-template
alignment
• You might also want to apply alternative
sequence alignment methods (see Lecture 2) by
using multiple sequence alignment programs to
align the target and the template sequences and
family related proteins.
• The obtained sequence alignment between
target and template (and additional homologous
proteins) can be submitted to the ‘Alignment
mode’ of the modeling session for model
building.
Swiss-Model Workspace: Modeling
Comparative Protein Structure Modeling
Known Structures
(Templates)
Target
Sequence
1. Template based
fragment assembly
(Composer, SWISSTemplate Selection
MODEL)
2. Satisfaction of spatial
restraints (Modeller)
Alignment
Structure Evaluation &
Template - Target
Assessment
Structure modeling
Homology
Model(s)
Swiss-Model: Template based fragment assembly
•
Find structurally conserved core regions
Swiss-Model: Template based fragment assembly
•
Build model core
– … by averaging core template backbone atoms (weighted by local
sequence similarity with the target sequence). Leave non-conserved
regions (loops) for later …
Swiss-Model: Template based fragment assembly
•
Loop (insertion) modeling
– Use the “spare part” algorithm to find compatible fragments in a Loop-Database,
or “ab-initio” rebuilding (e.g. Monte Carlo, MD, GA, etc.) to build missing loops.
Swiss-Model: Template based fragment assembly
•
Side Chain placement
– Find the most probable side chain conformation, using
• homologues structure information
• back-bone dependent rotamer libraries
• energetic and packing criteria
Swiss-Model: Template based fragment assembly
•
Rotamer Libraries
– Only a small fraction of all possible side chain
conformations is observed in experimental structures
– Rotamer libraries provide an ensemble of likely conformations
– The propensity of rotamers depends on the backbone geometry:
Swiss-Model Workspace: Modeling results
Swiss-Model Workspace: Modeling results
Comparative ProteinErrors
Structure
Modeling
in template selection or
alignment result in bad models:
Î iterative cycles of alignment,
modeling and evaluation
Known Structures Î Built many models, choose
(Templates)
best.
Target
Sequence
Template Selection
Alignment
Template - Target
Structure Evaluation &
Assessment
Structure modeling
Homology
Model(s)
Comparative ProteinQuality
Structure
Modeling
estimation:
•
•
Known Structures •
(Templates)
Target
Sequence
Stereochemistry check
Global model quality
estimation (statistical potential,
e.g. DFIRE)
Local model quality estimation
(statistical potential, e.g.
ANOLEA)
Template Selection
Alignment
Template - Target
Structure Evaluation &
Assessment
Structure modeling
Homology
Model(s)
Model quality estimation
•
Ramachandran Plot of backbone angles (ϕ,ψ)
– favored regions
– generously allowed regions
– disallowed regions
– Amino acids with special properties:
• PRO: ϕ = 60º
• GLY ( )
•
Similar plots for χ-angle distributions
Î Useful to identify regions with errors in geometry
ANOLEA : (Atomic Non-Local Environment Assessment)
•
http://protein.bio.puc.cl/cardex/servers/anolea/
•
http://swissmodel.expasy.org/anolea/
ANOLEA
Correct Structure:
PDB: 1GES
Î Detects local packing errors
Î Errors in alignments
Model with wrong
alignment:
Swiss-Model Workspace: Quality estimation
All checking tools are happy, so can I believe it now?
Models are not experimental facts !
Models can be partially inaccurate or sometimes
completely wrong !
A model is a tool that helps to interpret biochemical data.
References and further reading:
References and further reading: