PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons
E-Collection

Related Articles
Automating NMR Structures
April 2015
Protein Folding and Misfolding: A TRiC-ster that Follows the Rules
March 2015
Virology: Making Sensitive Magic
March 2014
Microbiome: Solid-State NMR, Crystallized
September 2013
Membrane Proteome: Making DNA Nanotubes for NMR Structure Determination
August 2013
Protein-Nucleic Acid Interaction: Inhibition Through Allostery
July 2013
Cell-Cell Interaction: Magic Structure from Microcrystals
March 2013
Membrane Proteome: Soft Sampling
December 2012
Membrane Proteome: Specific vs. Non-specific weak interactions
November 2012
Automatic NMR
September 2012
NMR structure test
September 2012
To structure, faster
August 2012
S is for solubility
June 2012
Blind faith
April 2012
Follow the RNA leader
December 2011
Making invisible proteins visible
October 2011
A fragmented approach to membrane protein structures
September 2011
Molecular replacement by magnetic resonance
August 2011
Solutions in the solution
June 2011
No more labeled lipids
May 2011
Capsid assembly in motion
April 2011
NMR challenges current protein hydration dogma
March 2011
Solving homodimeric structures with NMR
November 2010
CASD-NMR: assessing automated structure determination by NMR
June 2010
Peptidoglycan binding: Calcium-free killing
June 2010
Removing the NMR bottleneck
April 2010
NMR has its wiki way
March 2010
Extremely salty
February 2010
The future of NMR
September 2009
Tips for crystallizing membrane proteins
June 2009
Faster solid-state NMR
May 2009
Powerful NMR
April 2009
Activating BAX
December 2008

Automatic NMR

SBKB [doi:10.1038/sbkb.2011.99]
Technical Highlight - September 2012
Short description: A new protocol for automated NMR protein structure determination minimizes instrument time and efficiently produces high-quality structures.

Ensemble stereo view of the NMR "Structure A" of YP_926445.1 determined in the first phase of the J-UNIO protocol. α-helices and β-sheets are colored red and green, respectively. Figure courtesy of Pedro Serrano.

NMR structure determination of proteins with 150 residues or less has become increasingly automated, and several distinct protocols are now available. Compared to the traditional interactive approach, automated protocols are less biased and more efficient, yet often require some manual input during the data flow. Bottlenecks are typically encountered during either chemical shift assignment or compilation of distance restraints from nuclear Overhauser enhancement (NOE) data.

Wüthrich and colleagues (PSI JCSG) have developed a streamlined protocol, J-UNIO, based on automated projection spectroscopy (APSY) and NOE experiments for data acquisition and the modular UNIO software for resonance assignment and structure calculation. The authors selected the 115-residue protein YP_926445.1 from the Gram-negative bacterium Shewanella amazonensis to illustrate the J-UNIO procedure. Assessment of an NMR profile of signal intensities from a microscale sample indicated that the APSY experiments would succeed, and a UNIO module was used to assign the backbone chemical shifts. The authors next used another UNIO module to generate side-chain assignments from the same 1H-1H NOE data sets that provide the distance constraints for automated calculation of an intermediate “Structure A” for interactive validation. Further NOE assignments and distance restraints were then obtained, allowing calculation of a high-resolution “Structure V” ensemble which was rigorously validated using several quality criteria. The YP_926445.1 protein contains a novel fold used to generate a new Pfam family, which now includes members from over 50 bacterial species.

The authors applied the J-UNIO protocol to 17 targets from the JCSG pipeline ranging in size from 67 to 149 residues. To maximize success rates, the authors were relatively generous with instrument time and acquired high-resolution APSY data resulting in backbone assignments that were generally between 81–96% complete and contained very few errors. For the set of 17 proteins, extensions of the assignments to near completion was achieved in only a few hours of interactive analysis. The authors caution that while up to 5% erroneous NOE-based side chain assignments are possible, these are permissive with respect to calculating the correct fold. They further note that the J-UNIO protocol starts from the raw data, unlike most other automated procedures. Further comparisons of automated protocols will determine which will find broad applicability in the protein NMR community.

Michael A. Durney

References

  1. P. Serrano et al. The J-UNIO protocol for automated protein structure determination by NMR in solution.
    J. Biomol. NMR. 53, 341-354 (2012). doi:10.1007/s10858-012-9645-2

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health