Download - Christophe Schmitz Thomas Huber group The University of Queensland ECPM2009 Coffs Harbour
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PCS + ROSETTA = NMR structure determination ?
Christophe Schmitz
Thomas Huber group
The University of Queensland
ECPM2009
Coffs Harbour
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1. NMR effect induced by a paramagnetic lanthanide.
2. Measured on active spin (1H, 15N, 13C…)
3. Long range effect.
4. Effect described within the Δχ-tensor.
5. Combine distance and angular dependence.
40 Å
The Pseudocontact shift (PCS)
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What is PCS used for?Structure refinement
Allegrozzi, M.; Bertini, I.; Janik, M. B. L.; Lee, Y. M.; Lin, G. H.; Luchinat, C. JACS 2000, 122, (17), 4154-4161.
Chemical shift assignment
Schmitz, C.; John, M.; Park, A. Y.; Dixon, N. E.; Otting, G.; Pintacuda, G.; Huber, T., JBNMR 2006, 35, 79-87.
Protein-protein complex
Pintacuda, G.; Park, A. Y.; Keniry, M. A.; Dixon, N. E.; Otting, G. JACS 2006, 128, (11), 3696-3702.
What about structure determination?
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PCS for structure determination?
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Rosetta for structure predictionhttp://www.rosettacommons.org/software/index.html
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Fragment picking
…GFMDYEFSL………….. RQIVLDTET…………..TGMNQIGAH…
High resolution refinement
Abinitio
Rosetta structure prediction protocol
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ψ
ϕ
Fragment picking
Yang Shen, Oliver Lange, Frank Delaglio, et al.PNAS, (2008) 105, 4685-4690
• Rosetta Fragments
• CS-Rosetta Fragments
• Native fragment
ϕ
ψ
Fragment picking with CS-ROSETTA
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PCS into ROSETTA
Fragment picking
High resolution refinement
Abinitio
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List of protein targets
ArgN repressor78 residues
1AOY
Calbindin75 residues
1KQV
Myoglobin153 residues
1BZR
Protein G56 residues
3GB1
Calmodulin146 residues
2K61
N-t Calmodulin79 residues
1SW8
Thioredoxin108 residues
1XOA
ε subunit186 residues
1J54
Parvalbumin110 residues
1RJV
θ subunit76 residues
2AE9
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PCS
SCO
RE
Cα RMSD
• 1000 decoys from low to high RMSD • Native structure
ArgN repressor78 residues
1AOY
Cα RMSD
Den
sity
3 PCS data setsN and H
Example 1 - ROSETTA only- PCS only- PCS-ROSETTA
■ Native ■ Best PCS-ROSETTA decoy
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PCS
SCO
RE
Cα RMSD
Cα RMSD
Den
sity
1 PCS data setsH
Parvalbumin110 residues
1RJV
• 1000 decoys from low to high RMSD • Native structure
Example 2 (it doesn’t work well, but we sort of know why) - ROSETTA only
- PCS only- PCS-ROSETTA
■ Native ■ Best PCS-ROSETTA decoy
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PCS
SCO
RE
Cα RMSD
Cα RMSD
Den
sity
- ROSETTA only- PCS only- PCS-ROSETTA
4 PCS data setsH
Calmodulin146 residues
2K61
• 1000 decoys from low to high RMSD • Native structure
Example 3 (it works and it’s cool because it’s a big one)
■ Native ■ Best PCS-ROSETTA decoy
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PCS
SCO
RE
Cα RMSD
Cα RMSD
Den
sity
- ROSETTA only- PCS only- PCS-ROSETTA
5 PCS data setsC, N and H
ε subunit186 residues
1J54
• 1000 decoys from low to high RMSD• Native structure
Example 5 (it doesn't' work yet, but it really should, and we are going to keep working hard on that one)
Coming soon…
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ArgN repressor78 residues
1AOY
Calbindin75 residues
1KQV
Myoglobin153 residues
1BZR
Protein G56 residues
3GB1
Calmodulin146 residues
2K61
N-t Calmodulin79 residues
1SW8
Thioredoxin108 residues
1XOA
ε subunit186 residues
1J54
Parvalbumin110 residues
1RJV
Theta76 residues
2AE9
3 11 32 2
1 1 4 2 5
List of protein targets
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Conclusion
1. New road for structure determination
2. PCS and Rosetta work hand in hand
3. PCS are awesome
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Acknowledgments
GSRTG
Dr Thomas Huber (UQ)
Prof. David Baker (UW)
Robert Vernon (UW)Dr Guido Pintacuda(ENS-Lyon)
Dr Xun-Cheng Su (ANU)
Dr Michael John (GAU)
Prof. Gottfried Otting (ANU)
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PCS
SCO
RE
Cα RMSD
Cα RMSD
Den
sity
- ROSETTA only- PCS only- PCS-ROSETTA
θ subunit76 residues
2AE9
■ θ ■ ε
Example 4: it works and it’s a good surprise because we thought it wouldn’t.
2 PCS data setsH
• 1000 decoys from low to high RMSD• Native structure
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• 1000 decoys from low to high RMSD• Native structure
PCS
SCO
RE
Cα RMSD
Cα RMSD
Den
sity
- ROSETTA only- PCS only- PCS-ROSETTA
11 PCS data setsC, N and H
Calbindin75 residues
1KQV
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PCS
SCO
RE
Cα RMSD
Cα RMSD
Den
sity
- ROSETTA only- PCS only- PCS-ROSETTA
3 PCS data setsH
Protein G56 residues
3GB1
• 1000 decoys from low to high RMSD• Native structure
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PCS
SCO
RE
Cα RMSD
Cα RMSD
Den
sity
- ROSETTA only- PCS only- PCS-ROSETTA
1 PCS data setsH and N
Thioredoxin108 residues
1XOA
• 1000 decoys from low to high RMSD• Native structure
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PCS
SCO
RE
Cα RMSD
Cα RMSD
Den
sity
- ROSETTA only- PCS only- PCS-ROSETTA
2 PCS data setsH
Myoglobin153 residues
1BZR
• 1000 decoys from low to high RMSD• Native structure
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Fragment picking
High resolution refinement
Abinitio
PCS into ROSETTA