PCS + ROSETTA = NMR structure determination ?
Christophe Schmitz
Thomas Huber group
The University of Queensland
ECPM2009
Coffs Harbour
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)
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?
PCS for structure determination?
Rosetta for structure predictionhttp://www.rosettacommons.org/software/index.html
Fragment picking
…GFMDYEFSL………….. RQIVLDTET…………..TGMNQIGAH…
High resolution refinement
Abinitio
Rosetta structure prediction protocol
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
PCS into ROSETTA
Fragment picking
High resolution refinement
Abinitio
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
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
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
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
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…
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
Conclusion
1. New road for structure determination
2. PCS and Rosetta work hand in hand
3. PCS are awesome
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)
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
• 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
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
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
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
Fragment picking
High resolution refinement
Abinitio
PCS into ROSETTA
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