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Iterative Molecular Dynamics-Rosetta Protein Structure Refinement Protocol to Improve Model Quality.

ABSTRACT: Rosetta is one of the prime tools for high resolution protein structure refinement. While its scoring function can distinguish native-like from non-native-like conformations in many cases, the method is limited by conformational sampling for larger proteins, that is, leaving a local energy minimum in which the search algorithm may get stuck. Here, we test the hypothesis that iteration of Rosetta with an orthogonal sampling and scoring strategy might facilitate exploration of conformational space. Specifically, we run short molecular dynamics (MD) simulations on models created by de novo folding of large proteins into cryoEM density maps to enable sampling of conformational space not directly accessible to Rosetta and thus provide an escape route from the conformational traps. We present a combined MD-Rosetta protein structure refinement protocol that can overcome some of these sampling limitations. Two of four benchmark proteins showed incremental improvement through all three rounds of the iterative refinement protocol. Molecular dynamics is most efficient in applying subtle but important rearrangements within secondary structure elements and is thus highly complementary to the Rosetta refinement, which focuses on side chains and loop regions.

SUBMITTER: Lindert S

PROVIDER: S-EPMC3744128 | biostudies-other | 2013 Aug

REPOSITORIES: biostudies-other

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Project description:Knowing atomistic details of proteins is essential not only for the understanding of protein function but also for the development of drugs. Experimental methods such as X-ray crystallography, NMR, and cryo-electron microscopy (cryo-EM) are the preferred forms of protein structure determination and have achieved great success over the most recent decades. Computational methods may be an alternative when experimental techniques fail. However, computational methods are severely limited when it comes to predicting larger macromolecule structures with little sequence similarity to known structures. The incorporation of experimental restraints in computational methods is becoming increasingly important to more reliably predict protein structure. One such experimental input used in structure prediction and refinement is cryo-EM densities. Recent advances in cryo-EM have arguably revolutionized the field of structural biology. Our previously developed cryo-EM-guided Rosetta-MD protocol has shown great promise in the refinement of soluble protein structures. In this study, we extended cryo-EM density-guided iterative Rosetta-MD to membrane proteins. We also improved the methodology in general by picking models based on a combination of their score and fit-to-density during the Rosetta model selection. By doing so, we have been able to pick models superior to those with the previous selection based on Rosetta score only and we have been able to further improve our previously refined models of soluble proteins. The method was tested with five membrane spanning protein structures. By applying density-guided Rosetta-MD iteratively we were able to refine the predicted structures of these membrane proteins to atomic resolutions. We also showed that the resolution of the density maps determines the improvement and quality of the refined models. By incorporating high-resolution density maps (∼4 Å), we were able to more significantly improve the quality of the models than when medium-resolution maps (6.9 Å) were used. Beginning from an average starting structure root mean square deviation (RMSD) to native of 4.66 Å, our protocol was able to refine the structures to bring the average refined structure RMSD to 1.66 Å when 4 Å density maps were used. The protocol also successfully refined the HIV-1 CTD guided by an experimental 5 Å density map.

Project description: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.