PCS + ROSETTA = NMR structure determination ?

Christophe SchmitzThomas Huber groupThe University of Queensland

ECPM2009Coffs 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 ROSETTAFragment 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

Dens

ity3 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

Dens

ity1 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

Dens

ity

- 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

Dens

ity

- 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

Dens

ity

- 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

Dens

ity

- ROSETTA only- PCS only- PCS-ROSETTA

11 PCS data setsC, N and H

Calbindin75 residues

1KQV

PCS

SCO

RE

Cα RMSD

Cα RMSD

Dens

ity

- 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

Dens

ity

- 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

Dens

ity

- 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