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Improved cryoEM-Guided Iterative Molecular Dynamics--Rosetta Protein Structure Refinement Protocol for High Precision Protein Structure Prediction.

ABSTRACT: Many excellent methods exist that incorporate cryo-electron microscopy (cryoEM) data to constrain computational protein structure prediction and refinement. Previously, it was shown that iteration of two such orthogonal sampling and scoring methods – Rosetta and molecular dynamics (MD) simulations – facilitated exploration of conformational space in principle. Here, we go beyond a proof-of-concept study and address significant remaining limitations of the iterative MD–Rosetta protein structure refinement protocol. Specifically, all parts of the iterative refinement protocol are now guided by medium-resolution cryoEM density maps, and previous knowledge about the native structure of the protein is no longer necessary. Models are identified solely based on score or simulation time. All four benchmark proteins showed substantial improvement through three rounds of the iterative refinement protocol. The best-scoring final models of two proteins had sub-Ångstrom RMSD to the native structure over residues in secondary structure elements. Molecular dynamics was most efficient in refining secondary structure elements and was thus highly complementary to the Rosetta refinement which is most powerful in refining side chains and loop regions.

SUBMITTER: Lindert S

PROVIDER: S-EPMC4393324 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Improved cryoEM-Guided Iterative Molecular Dynamics--Rosetta Protein Structure Refinement Protocol for High Precision Protein Structure Prediction.

Lindert Steffen S McCammon J Andrew JA

Journal of chemical theory and computation 20150301 3

Many excellent methods exist that incorporate cryo-electron microscopy (cryoEM) data to constrain computational protein structure prediction and refinement. Previously, it was shown that iteration of two such orthogonal sampling and scoring methods – Rosetta and molecular dynamics (MD) simulations – facilitated exploration of conformational space in principle. Here, we go beyond a proof-of-concept study and address significant remaining limitations of the iterative MD–Rosetta protein structure r ...[more]

PMID: 25883538

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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:Cryo-EM has become one of the prime methods for protein structure elucidation, frequently yielding density maps with near-atomic or medium resolution. If protein structures cannot be deduced unambiguously from the density maps, computational structure refinement tools are needed to generate protein structural models. We have previously developed an iterative Rosetta-MDFF protocol that used cryo-EM densities to refine protein structures. Here we show that, in addition to cryo-EM densities, incorporation of other experimental restraints into the Rosetta-MDFF protocol further improved refined structures. We used NMR chemical shift (CS) data integrated with cryo-EM densities in our hybrid protocol in both the Rosetta step and the molecular dynamics (MD) simulations step. In 15 out of 18 cases for all MD rounds, the refinement results obtained when density maps and NMR chemical shift data were used in combination outperformed those of density map-only refinement. Notably, the improvement in refinement was highest when medium and low-resolution density maps were used. With our hybrid method, the RMSDs of final models obtained were always better than the RMSDs obtained by our previous protocol with just density refinement for both medium (6.9 Å) and low (9 Å) resolution maps. For all the six test proteins with medium resolution density maps (6.9 Å), the final refined structure RMSDs were lower for the hybrid method than for the cryo-EM only refinement. The final refined RMSDs were less than 1.5 Å when our hybrid protocol was used with 4 Å density maps. For four out of the six proteins the final RMSDs were even less than 1 Å. This study demonstrates that by using a combination of cryo-EM and NMR restraints, it is possible to refine structures to atomic resolution, outperforming single restraint refinement. This hybrid protocol will be a valuable tool when only low-resolution cryo-EM density data and NMR chemical shift data are available to refine structures.

Project description:When good structural templates can be identified, template-based modeling is the most reliable way to predict the tertiary structure of proteins. In this study, we combine template-based modeling with a realistic coarse-grained force field, AWSEM, that has been optimized using the principles of energy landscape theory. The Associative memory, Water mediated, Structure and Energy Model (AWSEM) is a coarse-grained force field having both transferable tertiary interactions and knowledge-based local-in-sequence interaction terms. We incorporate template information into AWSEM by introducing soft collective biases to the template structures, resulting in a model that we call AWSEM-Template. Structure prediction tests on eight targets, four of which are in the low sequence identity "twilight zone" of homology modeling, show that AWSEM-Template can achieve high-resolution structure prediction. Our results also confirm that using a combination of AWSEM and a template-guided potential leads to more accurate prediction of protein structures than simply using a template-guided potential alone. Free energy profile analyses demonstrate that the soft collective biases to the template effectively increase funneling toward native-like structures while still allowing significant flexibility so as to allow for correction of discrepancies between the target structure and the template. A further stage of refinement using all-atom molecular dynamics augmented with soft collective biases to the structures predicted by AWSEM-Template leads to a further improvement of both backbone and side-chain accuracy by maintaining sufficient flexibility but at the same time discouraging unproductive unfolding events often seen in unrestrained all-atom refinement simulations. The all-atom refinement simulations also reduce patches of frustration of the initial predictions. Some of the backbones found among the structures produced during the initial coarse-grained prediction step already have CE-RMSD values of less than 3 Å with 90% or more of the residues aligned to the experimentally solved structure for all targets. All-atom structures generated during the following all-atom refinement simulations, which started from coarse-grained structures that were chosen without reference to any knowledge about the native structure, have CE-RMSD values of less than 2.5 Å with 90% or more of the residues aligned for 6 out of 8 targets. Clustering low energy structures generated during the initial coarse-grained annealing picks out reliably structures that are within 1 Å of the best sampled structures in 5 out of 8 cases. After the all-atom refinement, structures that are within 1 Å of the best sampled structures can be selected using a simple algorithm based on energetic features alone in 7 out of 8 cases.

Dataset Information

Improved cryoEM-Guided Iterative Molecular Dynamics--Rosetta Protein Structure Refinement Protocol for High Precision Protein Structure Prediction.

Publications

Improved cryoEM-Guided Iterative Molecular Dynamics--Rosetta Protein Structure Refinement Protocol for High Precision Protein Structure Prediction.

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