Rosetta software package

4/10/2011
Rosetta ..
• Breif introduction to Rosetta
Ph.D. Thomas M. Frimurer
• Rosetta docking example
Novo Nordisk Foundation
Center for Potein Reseach
Center for Basic Metabilic Research
Rosetta software package
• Rosetta consists of multiple modules:
– protein folding, comparative modeling, ligand docking, protein design,
antibody/antigen interactions, etc.
• Rosetta is developed in a consortium of twelve laboratories by
• Breif introduction to Rosetta
• Rosetta docking example
around 50 developers
• Rosetta is free for academic
– user guide and tutorials are available
• PyRosetta is a python interface
– allows integration with Pymol
• FoldIt is the better video game for you and your kids
• Rosetta@home uses your computer for our RosettaCon 2009,
Leavenworth, WA, USA research
www.rosettacommons.org
Conformational sampling and
scoring of models in Rosetta
1. Rosetta Combines Conformational Sampling
~12,000,000 sequences
>Q8TDV5 GP119_HUMAN Glucose-dependent....
MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSL
CFTLNLAVADTLIGVAISGLLTDQLSSPSRPTQKTLCSLRM
AFVTSSAAASVLTVMLITFDRYLAIKQPFRYLKIMSGFVAG
ACIAGLWLVSYLIGFLPLGIPMFQQTAYKGQCSFFAVFHPH
FVLTLSCVGFFPAMLLFVFFYCDMLKIASMHSQQIRKMEHA
GAMAGGYRSPRTPSDFKALRTVSVLIGSFALSWTPFLITGI
.....
1. Scoring Function for Structure Determination
~70,000 structures
?
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Conformational sampling and
scoring of models in Rosetta
1. Rosetta Combines Conformational Sampling
1.
2.
Exchanging the backbone conformation of 9 and 3 amino
acids peptide fragments, collected from the pdb of homologous
strethes.
Metropolis Monte Carlo
Enew < Eold accept
Enew ≥ Eold accept with probability e-[ (Enew - Eold)/T ]
Sampling strategies for backbone
degrees of freedom
>Q8TDV5 GP119_HUMAN Homo sapiens
MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSLCFTLNLAVADTLIGVAISGLLTDQLSSPSR
LAVLASLIIA
PTQKTLCSLRMAFVTSSAAASVLTVMLITFDRYLAIKQPFRYLKIMSGFVAGACIAGLWLVSYLIGFLPLG
VLTLSCVGF
IPMFQQTAYKGQCSFFAVFHPHFVLTLSCVGFFPAMLLFVFFYCDMLKIASMHSQQIRKMEHAGAMAGGYR
SPRTPSDFKALRTVSVLIGSFALSWTPFLITGI.....
Approximate local interactions using the
distribution of conformations seen for
similar sequences in known protein
structures ..
2. Scoring Function for Structure Determination
1.
2.
For each sequence window, select fragments
that represent the conformations sampled
during folding
Low resolution: Reduced atom representation (centroid) Simple
energy function which Aggressively search conformational space
High resolution: Full atom more sophisticated energy function.
“Local” search of conformational (and sequence) space
Majority of conformational sampling protocols in
Rosetta use Metropolis Monte Carlo follow by
gradient based minmization
• Monte Carlo simulated annealing assembly of fragments
• Simplified protein representation
• One centroid per amino acid side chain
identify the ”best” structure
1) Remove very low contact order structures
2) Select broadest minima using cluster analysis
3) Select lowest energy structures with full atom potential
The free energy minimum
corresponds (usually) to the
native protein fold
• High resolution potential energy function
• full atom representation
Figure form Kistian W. Kaufmann et. al. 2010
Figure form Kistian W. Kaufmann et. al. 2010
Computational strategy ..
ab initio protein folding
Approximate local
interactions using the
distribution of
conformations seen for
similar sequences in
known protein structures
..
Filter conformational ensample:
Cluster models that
maintain the same overall
fold e.g. Cα rmsd < 5 Å
• Statistically-derived potential function
• Steric overlap (vdw interactions)
• Residue environment (solvation)
• Pairwise interactions (electrostatics)
• Strand pairing (hydrogen bonding)
• Compactness (solvation)
Folding Units ..
While not every protein fold is present in the protein databank,
all possible conformations of small peptides are ..
Its depth is obscured
because of the simplified
energy approximation
Rosetta begins with an extended peptide chain.
Insertions of backbone ”fragments” rapidly folds
the protein
Statistically-derived
potential function
Cluster analysis ..
Select broadest minima
Sample conformational
space using Monte Carlo
simulations
Figure form Kistian W. Kaufmann et. al. 2010
Refine models ..
Simons K. T.; Kooperberg C.; Huang E.; Baker D. (1997) J. Mol. Biol. 268, 209–225
Rohl C. A.; Strauss C. E.; Misura K. M.; Baker D. (2004) Methods Enzymol. 383, 66–93.
Simons K. T.; Ruczinski I.; Kooperberg C.; Fox B. A.; Bystroff C.; Baker D. (1999) Proteins 34, 82–95.
Bradley P.; Misura K. M.; Baker D. (2005) Science 309, 1868–1871.
Full atomic potential
energy function ..
Figure form Kistian W. Kaufmann et. al. 2010
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The resulting model undergo
atomic-detail refinement
• http://rosettadesigngroup.com
• http://robetta.bakerlab.org
Figure form Kistian W. Kaufmann et. al. 2010
http://rosettadesigngroup.com
http://robetta.bakerlab.org
GPR119 AR-231453 interaction ?
• Breif introduction to Rosetta
AR-231453
• Rosetta docking example
Mutational mapping of the AR-231453 ligand binding site in the GPR119 receptor
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Rosetta docking protocol
Modelling the GPR119 receptor
Table 1: Sequence alignment of β2-adrenergic GPR with GPR119
ID=22% pP=7.2
2rh1_a
Q8TDV5_GP119_HUMAN
2rh1_a
2rh1_a
Q8TDV5_GP119_HUMAN
2rh1_a
2rh1_a
Q8TDV5_GP119_HUMAN
2rh1_a
1
1
.E..#....I##.L#.L#I#...####.###.#.+...V..#F...LA#AD.##G#A#..#....#..........#C.#...#....#.AS#.T###I.#DRY#AI..PF+Y..
DEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQS
MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSLCFTLNLAVADTLIGVAISGLLTDQLSSPSRPTQKTLCSLRMAFVTSSAAASVLTVMLITFDRYLAIKQPFRYLK
1.
2.
1. Resulting models sorted with respect to energy
2. Best scoring model had 2.7 Å to TM domain of b2ar template
##....A.##I#.#W#VS.L..FLP#.###........#...A....C.FF....#####...#..##P########...#..#...#......................#.-#K
116 LLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYAEETCCDFFT-NQAYAIASSIVSFYVPLVIMVFVYSRVFQEAKRQLNIFE----------------FCLKEHK
116 IMSGFVAGACIAGLWLVSYLIGFLPLGIPM--------FQQTAYKGQCSFFAVFHPHFVLTLSCVGFFPAMLLFVFFYCDMLKIASMHSQQIRKMEHAGAMAGGYRSPRTPSDFK
AL+T#.###G.F.L.W.PF#I..IV.V#....##...#..#L.##G#.NS.#NPLIY#....-#R#.#..###........................................
214 ALKTLGIIMGTFTLCWLPFFIVNIVHVIQDN-LIRKEVYILLNWIGYVNSGFNPLIYC-RSPDFRIAFQELLCL--------------------------------------223 ALRTVSVLIGSFALSWTPFLITGIVQVACQECHLYLVLERYLWLLGVGNSLLNPLIYAYWQKEVRLQLYHMALGVKKVLTSFLLFLSARNCGPERPRESSCHIVTISSSEFDG
3.
4.
5.
2. Lack of sequnece similarity in loop regions ..
6.
Rosetta docking protocol
Generate initial
homology model
Produce 1000 models
using Rosetta relaxed
1000 diffeent loop conformations were generated on best model
1. Kinematic closure algorithm
2. Disulphide bridge constrain between Cys in EXL2B and Cys III:01
1. Week sequence similarity to templates ..
3. Quality of homology model is questionable ..
Generate GPR119 Homology model (b2ar template)
1000 GPR119 models were produced using Rosetta relax
7.
14 low energy ligand (AR-231453) conformations were generated
A total of 2000 docking trajectories of randomly picked ligand
conformations were performed on each of the 1000 loop models
Of the 2.000.000 combinatorial solutions – top 5 % were selected based
on total energy and receptor-ligand interaction energy
5 best docking poses were relaxed 5000 times to optimize loop structure
and receptor ligand packing
”Low resolution” docking mode
Refine best docking poses
Generate ensamle of low energy
conformations of AR-231453
In between helices
lack agreement with
experimental mapping
2000 docking trajectories
performed for each of
the 1000 loop models
2.000.000 decoys
Cluster, filter and extract
top10 best models
lack agreement with
experimental mapping
Pose 1
Pose 2
Pose 3
Pose 5
Pose 4
A total og 5000 docking trajectories were performed
for each of the 5 best docking poses to relax and
refine the receptor and ligand packing
Build and refine 100 loop
conformations for each
of the 10 best models
A total of 1000 models
Docking pose I: Refinement
Docking pose I: Refinement
Pose 1
4
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Docking pose II: Refinement
Docking pose II: Refinement
Pose 2
Docking pose III: Refinement
Docking pose III: Refinement
Pose 3
Docking pose III: Refinement
Docking pose IV: Refinement
Pose 4
5
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Docking pose V: Refinement
Docking pose V: Refinement
Pose 5
Refined GPR119 docking decoys
Docking pose III
Docking pose IV
Refined GPR119 docking decoys
Docking pose III
Docking pose IV
Proposed binding pose of AR231453
agonist to the GPR119 receptor
• http://www.rosettacommons.org
• http://robetta.bakerlab.org
• http://rosettadesigngroup.com
Binding pocket of AR231453 in GPR119. Amino acids investigation by
mutagenesis is coloured according to potency shift.
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