Rosetta - CBSblicher/Courses/10_What_Rosetta_can_do_for_you.… · • Rosetta is developed in a consortium of twelve laboratories by around 50 developers • Rosetta is free for

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Rosetta ..

Ph.D. Thomas M. Frimurer

Novo Nordisk FoundationCenter for Potein Reseach

Center for Basic Metabilic Research

• 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


Refine models ..

Full atomic potential energy function ..

Rosetta begins with an extended peptide chain.

Insertions of backbone ”fragments” rapidly folds

the protein


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The resulting model undergo atomic-detail refinement


• http://rosettadesigngroup.com

• http://robetta.bakerlab.org

http://rosettadesigngroup.com http://robetta.bakerlab.org



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



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

Documents

Rosetta - CBSblicher/Courses/10_What_Rosetta_can_do_for_you.… · • Rosetta is developed in a consortium of twelve laboratories by around 50 developers • Rosetta is free for