Improving NMR protein structure quality by Rosetta refinement: A molecular replacement study

Theresa A. Ramelot, Srivatsan Raman, Alexandre P. Kuzin, Rong Xiao, Li Chung Ma, Thomas B. Acton, John F. Hunt, Gaetano T. Montelione, David Baker, Michael A. Kennedy

Research output: Contribution to journalArticlepeer-review

52 Scopus citations


The structure of human protein HSPC034 has been determined by both solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. Refinement of the NMR structure ensemble, using a Rosetta protocol in the absence of NMR restraints, resulted in significant improvements not only in structure quality, but also in molecular replacement (MR) performance with the raw X-ray diffraction data using MOLREP and Phaser. This method has recently been shown to be generally applicable with improved MR performance demonstrated for eight NMR structures refined using Rosetta (Qian et al., Nature 2007;450:259-264). Additionally, NMR structures of HSPC034 calculated by standard methods that include NMR restraints have improvements in the RMSD to the crystal structure and MR performance in the order DYANA, CYANA, XPLOR-NIH, and CNS with explicit water refinement (CNSw). Further Rosetta refinement of the CNSw structures, perhaps due to more thorough conformational sampling and/or a superior force field, was capable of finding alternative low energy protein conformations that were equally consistent with the NMR data according to the Recall, Precision, and F-measure (RPF) scores. On further examination, the additional MR-performance shortfall for NMR refined structures as compared with the X-ray structure were attributed, in part, to crystal-packing effects, real structural differences, and inferior hydrogen bonding in the NMR structures. A good correlation between a decrease in the number of buried unsatisfied hydrogen-bond donors and improved MR performance demonstrates the importance of hydrogen-bond terms in the force field for improving NMR structures. The superior hydrogen-bond network in Rosetta-refined structures demonstrates that correct identification of hydrogen bonds should be a critical goal of NMR structure refinement. Inclusion of nonbivalent hydrogen bonds identified from Rosetta structures as additional restraints in the structure calculation results in NMR structures with improved MR performance.

Original languageEnglish (US)
Pages (from-to)147-167
Number of pages21
JournalProteins: Structure, Function and Bioinformatics
Issue number1
StatePublished - Apr 2009

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Biochemistry
  • Molecular Biology


  • C1orf41
  • Comparison of NMR and X-ray structures
  • HSPC034
  • Hydrogen bonding
  • Molecular replacement
  • NMR
  • NMR force field refinement
  • Northeast structural genomics consortium
  • PP25
  • Refinement methods
  • Rosetta
  • Structural genomics
  • X-ray
  • X-ray crystallography

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