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Protein NMR structures refined with Rosetta have higher accuracy relative to corresponding X-ray crystal structures.

Mao B, Tejero R, Baker D, Montelione GT - J. Am. Chem. Soc. (2014)

Bottom Line: We have found that refinement of protein NMR structures using Rosetta with experimental NMR restraints yields more accurate protein NMR structures than those that have been deposited in the PDB using standard refinement protocols.The protocol for restrained refinement of protein NMR structures was also compared with restrained CS-Rosetta calculations.The restrained Rosetta protocols described here can improve the accuracy of protein NMR structures and should find broad and general for studies of protein structure and function.

View Article: PubMed Central - PubMed

Affiliation: Center for Advanced Biotechnology and Medicine, and Department of Molecular Biology and Biochemistry, and Department of Biochemistry and Molecular Biology of Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey , Piscataway, New Jersey 08854, United States.

ABSTRACT
We have found that refinement of protein NMR structures using Rosetta with experimental NMR restraints yields more accurate protein NMR structures than those that have been deposited in the PDB using standard refinement protocols. Using 40 pairs of NMR and X-ray crystal structures determined by the Northeast Structural Genomics Consortium, for proteins ranging in size from 5-22 kDa, restrained Rosetta refined structures fit better to the raw experimental data, are in better agreement with their X-ray counterparts, and have better phasing power compared to conventionally determined NMR structures. For 37 proteins for which NMR ensembles were available and which had similar structures in solution and in the crystal, all of the restrained Rosetta refined NMR structures were sufficiently accurate to be used for solving the corresponding X-ray crystal structures by molecular replacement. The protocol for restrained refinement of protein NMR structures was also compared with restrained CS-Rosetta calculations. For proteins smaller than 10 kDa, restrained CS-Rosetta, starting from extended conformations, provides slightly more accurate structures, while for proteins in the size range of 10-25 kDa the less CPU intensive restrained Rosetta refinement protocols provided equally or more accurate structures. The restrained Rosetta protocols described here can improve the accuracy of protein NMR structures and should find broad and general for studies of protein structure and function.

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For Rosetta refinement of NMR structure, preserving ensemble information is beneficial forMR success. Scatterplot of Phaser47 TFZ scores (A) andDP-scores16,17 (B) fortwo different protocols for selecting models for MR. Decoy(Energy) Rosetta-refined structureensembles are composed of the 20 lowest Rosetta energy decoys from the entire pool of decoysgenerated from all the NMR conformers. Decoy(Conformer+Energy) Rosetta-refined structureensembles are composed of each lowest Rosetta energy decoy generated from each NMR conformer.The scores of structures picked by Decoy(Energy) protocol are plotted on the X-axis, and thescores of structures picked by Decoy(Conformer+Energy) protocol are plotted on the Y-axis.Unrestrained Rosetta refined structures are represented by red solid triangles and restrainedRosetta refined structures are represented by blue solid rectangles. Data are summarized for38 NESG NMR/Xray pairs used in the crystallographic MR study.
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fig6: For Rosetta refinement of NMR structure, preserving ensemble information is beneficial forMR success. Scatterplot of Phaser47 TFZ scores (A) andDP-scores16,17 (B) fortwo different protocols for selecting models for MR. Decoy(Energy) Rosetta-refined structureensembles are composed of the 20 lowest Rosetta energy decoys from the entire pool of decoysgenerated from all the NMR conformers. Decoy(Conformer+Energy) Rosetta-refined structureensembles are composed of each lowest Rosetta energy decoy generated from each NMR conformer.The scores of structures picked by Decoy(Energy) protocol are plotted on the X-axis, and thescores of structures picked by Decoy(Conformer+Energy) protocol are plotted on the Y-axis.Unrestrained Rosetta refined structures are represented by red solid triangles and restrainedRosetta refined structures are represented by blue solid rectangles. Data are summarized for38 NESG NMR/Xray pairs used in the crystallographic MR study.

Mentions: For the initial Rosetta refinement protocol, the decoys are picked solely based on Rosettaenergy, that is, we picked the top 20 decoys with the lowest Rosetta energy from the entirepool of decoys generated from all the conformers in NMR structure ensemble. It was observed,however, that frequently those 20 decoys originated from the same one or two similar conformersin the unrefined NMR ensemble; thus the structural variance information within the NMR ensembleis lost using this simple decoy picking process. In order to preserve the conformationalvariability information within the NMR ensemble, we adopted a protocol in which the onelowest-energy Rosetta decoy was selected from the ensemble of decoys generated from each NMRconformer. As shown in Figure 6A,B, the resulting Rosetta ensembles are muchbetter MR templates and also fit the NOESY peak list data better than the Rosetta ensemblesgenerated by our initial protocol, as manifested by the significantly improved TFZ and DPscores for the majority of the targets. These ensembles of Rosetta refined (with or withoutrestraints) conformers were then trimmed to exclude not-well-defined atoms using FindCore andused as templates for Phaser as described previously.31


Protein NMR structures refined with Rosetta have higher accuracy relative to corresponding X-ray crystal structures.

Mao B, Tejero R, Baker D, Montelione GT - J. Am. Chem. Soc. (2014)

For Rosetta refinement of NMR structure, preserving ensemble information is beneficial forMR success. Scatterplot of Phaser47 TFZ scores (A) andDP-scores16,17 (B) fortwo different protocols for selecting models for MR. Decoy(Energy) Rosetta-refined structureensembles are composed of the 20 lowest Rosetta energy decoys from the entire pool of decoysgenerated from all the NMR conformers. Decoy(Conformer+Energy) Rosetta-refined structureensembles are composed of each lowest Rosetta energy decoy generated from each NMR conformer.The scores of structures picked by Decoy(Energy) protocol are plotted on the X-axis, and thescores of structures picked by Decoy(Conformer+Energy) protocol are plotted on the Y-axis.Unrestrained Rosetta refined structures are represented by red solid triangles and restrainedRosetta refined structures are represented by blue solid rectangles. Data are summarized for38 NESG NMR/Xray pairs used in the crystallographic MR study.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4129517&req=5

fig6: For Rosetta refinement of NMR structure, preserving ensemble information is beneficial forMR success. Scatterplot of Phaser47 TFZ scores (A) andDP-scores16,17 (B) fortwo different protocols for selecting models for MR. Decoy(Energy) Rosetta-refined structureensembles are composed of the 20 lowest Rosetta energy decoys from the entire pool of decoysgenerated from all the NMR conformers. Decoy(Conformer+Energy) Rosetta-refined structureensembles are composed of each lowest Rosetta energy decoy generated from each NMR conformer.The scores of structures picked by Decoy(Energy) protocol are plotted on the X-axis, and thescores of structures picked by Decoy(Conformer+Energy) protocol are plotted on the Y-axis.Unrestrained Rosetta refined structures are represented by red solid triangles and restrainedRosetta refined structures are represented by blue solid rectangles. Data are summarized for38 NESG NMR/Xray pairs used in the crystallographic MR study.
Mentions: For the initial Rosetta refinement protocol, the decoys are picked solely based on Rosettaenergy, that is, we picked the top 20 decoys with the lowest Rosetta energy from the entirepool of decoys generated from all the conformers in NMR structure ensemble. It was observed,however, that frequently those 20 decoys originated from the same one or two similar conformersin the unrefined NMR ensemble; thus the structural variance information within the NMR ensembleis lost using this simple decoy picking process. In order to preserve the conformationalvariability information within the NMR ensemble, we adopted a protocol in which the onelowest-energy Rosetta decoy was selected from the ensemble of decoys generated from each NMRconformer. As shown in Figure 6A,B, the resulting Rosetta ensembles are muchbetter MR templates and also fit the NOESY peak list data better than the Rosetta ensemblesgenerated by our initial protocol, as manifested by the significantly improved TFZ and DPscores for the majority of the targets. These ensembles of Rosetta refined (with or withoutrestraints) conformers were then trimmed to exclude not-well-defined atoms using FindCore andused as templates for Phaser as described previously.31

Bottom Line: We have found that refinement of protein NMR structures using Rosetta with experimental NMR restraints yields more accurate protein NMR structures than those that have been deposited in the PDB using standard refinement protocols.The protocol for restrained refinement of protein NMR structures was also compared with restrained CS-Rosetta calculations.The restrained Rosetta protocols described here can improve the accuracy of protein NMR structures and should find broad and general for studies of protein structure and function.

View Article: PubMed Central - PubMed

Affiliation: Center for Advanced Biotechnology and Medicine, and Department of Molecular Biology and Biochemistry, and Department of Biochemistry and Molecular Biology of Robert Wood Johnson Medical School, and Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey , Piscataway, New Jersey 08854, United States.

ABSTRACT
We have found that refinement of protein NMR structures using Rosetta with experimental NMR restraints yields more accurate protein NMR structures than those that have been deposited in the PDB using standard refinement protocols. Using 40 pairs of NMR and X-ray crystal structures determined by the Northeast Structural Genomics Consortium, for proteins ranging in size from 5-22 kDa, restrained Rosetta refined structures fit better to the raw experimental data, are in better agreement with their X-ray counterparts, and have better phasing power compared to conventionally determined NMR structures. For 37 proteins for which NMR ensembles were available and which had similar structures in solution and in the crystal, all of the restrained Rosetta refined NMR structures were sufficiently accurate to be used for solving the corresponding X-ray crystal structures by molecular replacement. The protocol for restrained refinement of protein NMR structures was also compared with restrained CS-Rosetta calculations. For proteins smaller than 10 kDa, restrained CS-Rosetta, starting from extended conformations, provides slightly more accurate structures, while for proteins in the size range of 10-25 kDa the less CPU intensive restrained Rosetta refinement protocols provided equally or more accurate structures. The restrained Rosetta protocols described here can improve the accuracy of protein NMR structures and should find broad and general for studies of protein structure and function.

Show MeSH