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4/10/2011
1
Rosetta ..
Ph.D. Thomas M. Frimurer
Novo Nordisk FoundationCenter for Potein Reseach
Center for Basic Metabilic Research
• Breif introduction to Rosetta
• Rosetta docking example
• Breif introduction to Rosetta
• Rosetta docking example
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 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
1. Rosetta Combines Conformational Sampling
1. Scoring Function for Structure Determination
Conformational sampling and scoring of models in Rosetta
~12,000,000 sequences
>Q8TDV5 GP119_HUMAN Glucose-dependent....
MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSL
CFTLNLAVADTLIGVAISGLLTDQLSSPSRPTQKTLCSLRM
AFVTSSAAASVLTVMLITFDRYLAIKQPFRYLKIMSGFVAG
ACIAGLWLVSYLIGFLPLGIPMFQQTAYKGQCSFFAVFHPH
FVLTLSCVGFFPAMLLFVFFYCDMLKIASMHSQQIRKMEHA
GAMAGGYRSPRTPSDFKALRTVSVLIGSFALSWTPFLITGI
.....
~70,000 structures?
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Conformational sampling and scoring of models in Rosetta
1. Rosetta Combines Conformational Sampling 1. Exchanging the backbone conformation of 9 and 3 amino
acids peptide fragments, collected from the pdb of homologous
strethes.
2. Metropolis Monte Carlo
Enew < Eold accept
Enew ≥ Eold accept with probability e-[ (Enew - Eold)/T ]
2. Scoring Function for Structure Determination1. Low resolution: Reduced atom representation (centroid) Simple
energy function which Aggressively search conformational space
2. High resolution: Full atom more sophisticated energy function.
“Local” search of conformational (and sequence) space
Sampling strategies for backbonedegrees of freedom
Approximate local interactions using the distribution of conformations seen for
similar sequences in known protein structures ..
While not every protein fold is present in the protein databank,
all possible conformations of small peptides are ..
For each sequence window, select fragments
that represent the conformations sampled
during folding
>Q8TDV5 GP119_HUMAN Homo sapiens
MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSLCFTLNLAVADTLIGVAISGLLTDQLSSPSR
PTQKTLCSLRMAFVTSSAAASVLTVMLITFDRYLAIKQPFRYLKIMSGFVAGACIAGLWLVSYLIGFLPLG
IPMFQQTAYKGQCSFFAVFHPHFVLTLSCVGFFPAMLLFVFFYCDMLKIASMHSQQIRKMEHAGAMAGGYR
SPRTPSDFKALRTVSVLIGSFALSWTPFLITGI.....
VLTLSCVGF
LAVLASLIIA
Majority of conformational sampling protocols in
Rosetta use Metropolis Monte Carlo follow by gradient based minmization
• Monte Carlo simulated annealing assembly of fragments
• Statistically-derived potential function
• Steric overlap (vdw interactions)• Residue environment (solvation)• Pairwise interactions (electrostatics)• Strand pairing (hydrogen bonding)• Compactness (solvation)
• Simplified protein representation
• One centroid per amino acid side chain
Figure form Kistian W. Kaufmann et. al. 2010
• High resolution potential energy function
• full atom representation
Filter conformational ensample: identify the ”best” structure1) Remove very low contact order structures2) Select broadest minima using cluster analysis3) Select lowest energy structures with full atom potential
The free energy minimum corresponds (usually) to the
native protein fold
Its depth is obscuredbecause of the simplified
energy approximationFigure form Kistian W. Kaufmann et. al. 2010
Cluster models that maintain the same overall
fold e.g. Cα rmsd < 5 Å
Computational strategy ..ab initio protein folding
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.
Folding Units .. Approximate local
interactions using the
distribution of
conformations seen for
similar sequences in
known protein structures
..
Sample conformational space using Monte Carlo simulations
Cluster analysis ..
Select broadest minima
Statistically-derived
potential function
Figure form Kistian W. Kaufmann et. al. 2010
Refine models ..
Full atomic potential energy function ..
Rosetta begins with an extended peptide chain.
Insertions of backbone ”fragments” rapidly folds
the protein
Figure form Kistian W. Kaufmann et. al. 2010
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The resulting model undergo atomic-detail refinement
Figure form Kistian W. Kaufmann et. al. 2010
• http://rosettadesigngroup.com
• http://robetta.bakerlab.org
http://rosettadesigngroup.com http://robetta.bakerlab.org
• Breif introduction to Rosetta
• Rosetta docking example
GPR119 AR-231453 interaction ?
AR-231453
Mutational mapping of the AR-231453 ligand binding site in the GPR119 receptor
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Modelling the GPR119 receptor
ID=22% pP=7.2 .E..#....I##.L#.L#I#...####.###.#.+...V..#F...LA#AD.##G#A#..#....#..........#C.#...#....#.AS#.T###I.#DRY#AI..PF+Y..
2rh1_a 1 DEVWVVGMGIVMSLIVLAIVFGNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAAHILMKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQS
Q8TDV5_GP119_HUMAN 1 MESSFSFGVILAVLASLIIATNTLVAVAVLLLIHKNDGVSLCFTLNLAVADTLIGVAISGLLTDQLSSPSRPTQKTLCSLRMAFVTSSAAASVLTVMLITFDRYLAIKQPFRYLK
2rh1_a
##....A.##I#.#W#VS.L..FLP#.###........#...A....C.FF....#####...#..##P########...#..#...#......................#.-#K
2rh1_a 116 LLTKNKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYAEETCCDFFT-NQAYAIASSIVSFYVPLVIMVFVYSRVFQEAKRQLNIFE----------------FCLKEHK
Q8TDV5_GP119_HUMAN 116 IMSGFVAGACIAGLWLVSYLIGFLPLGIPM--------FQQTAYKGQCSFFAVFHPHFVLTLSCVGFFPAMLLFVFFYCDMLKIASMHSQQIRKMEHAGAMAGGYRSPRTPSDFK
2rh1_a
AL+T#.###G.F.L.W.PF#I..IV.V#....##...#..#L.##G#.NS.#NPLIY#....-#R#.#..###........................................
2rh1_a 214 ALKTLGIIMGTFTLCWLPFFIVNIVHVIQDN-LIRKEVYILLNWIGYVNSGFNPLIYC-RSPDFRIAFQELLCL---------------------------------------
Q8TDV5_GP119_HUMAN 223 ALRTVSVLIGSFALSWTPFLITGIVQVACQECHLYLVLERYLWLLGVGNSLLNPLIYAYWQKEVRLQLYHMALGVKKVLTSFLLFLSARNCGPERPRESSCHIVTISSSEFDG
2rh1_a
Table 1: Sequence alignment of β2-adrenergic GPR with GPR119
1. Week sequence similarity to templates ..
2. Lack of sequnece similarity in loop regions ..
3. Quality of homology model is questionable ..
Rosetta docking protocol
1. Generate GPR119 Homology model (b2ar template)
2. 1000 GPR119 models were produced using Rosetta relax1. Resulting models sorted with respect to energy
2. Best scoring model had 2.7 Å to TM domain of b2ar template
3. 1000 diffeent loop conformations were generated on best model1. Kinematic closure algorithm
2. Disulphide bridge constrain between Cys in EXL2B and Cys III:01
4. 14 low energy ligand (AR-231453) conformations were generated
5. A total of 2000 docking trajectories of randomly picked ligand conformations were performed on each of the 1000 loop models
6. Of the 2.000.000 combinatorial solutions – top 5 % were selected based on total energy and receptor-ligand interaction energy
7. 5 best docking poses were relaxed 5000 times to optimize loop structure and receptor ligand packing
Rosetta docking protocol
Generate initial homology model
Produce 1000 modelsusing Rosetta relaxed
Cluster, filter and extract top10 best models
Build and refine 100 loop conformations for each
of the 10 best models
A total of 1000 models
2000 docking trajectoriesperformed for each of
the 1000 loop models2.000.000 decoys
Generate ensamle of low energyconformations of AR-231453
”Low resolution” docking mode Refine best docking poses
Pose 5
Pose 4
Pose 3Pose 2
Pose 1
In between heliceslack agreement with
experimental mapping
lack agreement withexperimental mapping
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
Docking pose I: Refinement
Pose 1
Docking pose I: Refinement
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Docking pose II: Refinement
Pose 2
Docking pose II: Refinement
Docking pose III: Refinement
Pose 3
Docking pose III: Refinement
Docking pose III: Refinement
Pose 4
Docking pose IV: Refinement
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Docking pose V: Refinement
Pose 5
Docking pose V: Refinement
Refined GPR119 docking decoys
Docking pose IVDocking pose III
Refined GPR119 docking decoys
Docking pose IVDocking pose III
Proposed binding pose of AR231453 agonist to the GPR119 receptor
Binding pocket of AR231453 in GPR119. Amino acids investigation by mutagenesis is coloured according to potency shift.
• http://www.rosettacommons.org
• http://robetta.bakerlab.org
• http://rosettadesigngroup.com
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