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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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Restrained Rosetta refined structures are more similar to their corresponding X-ray crystalstructures than PDB NMR structures. GDT.TS values of PDB NMR structures to correspondingX-ray structures are plotted on the X-axis, and GDT.TS values of both unrestrained Rosettarefined structures (R3, represented by red solid triangle) and restrained Rosetta refinedstructures (R3rst, represented by blue solid rectangles) to their corresponding X-raystructures are plotted on the Y-axis. Data are summarized for 39 NESG NMR/X-ray pairs. Thetwo green dash lines indicate GDT.TS of PDB NMR structures equal to 0.7 and 0.85respectively. The black dash line indicates y = x, and the two gray dash lines indicate y = x+ 0.05 and y = x – 0.05 respectively.
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fig4: Restrained Rosetta refined structures are more similar to their corresponding X-ray crystalstructures than PDB NMR structures. GDT.TS values of PDB NMR structures to correspondingX-ray structures are plotted on the X-axis, and GDT.TS values of both unrestrained Rosettarefined structures (R3, represented by red solid triangle) and restrained Rosetta refinedstructures (R3rst, represented by blue solid rectangles) to their corresponding X-raystructures are plotted on the Y-axis. Data are summarized for 39 NESG NMR/X-ray pairs. Thetwo green dash lines indicate GDT.TS of PDB NMR structures equal to 0.7 and 0.85respectively. The black dash line indicates y = x, and the two gray dash lines indicate y = x+ 0.05 and y = x – 0.05 respectively.

Mentions: For this assessment, we calculated the GDT.TS between (i) PDB NMR structures, (ii)unrestrained Rosetta refined structures, and (iii) restrained Rosetta refined structures, withtheir corresponding X-ray structures. NESG target DrR147D was left out of this analysis becauseits solution NMR structure is a monomer solved at pH 4.5, while its X-ray structure is a dimersolved at pH 6.0, and NMR studies demonstrate a significant structural change over this pHrange (data not shown). These results for the remaining 39 NESG NMR/X-ray pairs are summarizedin a GDT.TS scatterplot (Figure 4), with the GDT.TS of PDB NMR structuresrelative to the corresponding X-ray crystal structure on the x-axis and GDT.TSof the unrestrained or restrained Rosetta refined structures on the y-axis.Based on observations of previous studies30,31 done with a much small number (i.e., 1 or 2) of protein targets,we expected unrestrained Rosetta refinement would generally move NMR structures closer to theirX-ray counterparts. However, as illustrated in Figure 4, using this largerdata set of 39 NMR/X-ray pairs, we observed that this is not the case. After unrestrainedRosetta refinement (R3), only 17 of 39 targets exhibit higher GDT.TS values, 6 targets remainabout the same, and 16 of the protein ensembles have lower GDT.TS values than the NMRstructures refined by conventional methods and deposited in the PDB. On average, unrestrainedRosetta refinement improved the GDT.TS by only 0.4%. On the other hand, as illustrated inFigure 4, restrained Rosetta refinement (R3rst) generally improved the GDT.TSscore to the X-ray crystal structure, compared with the NMR structure deposited in the PDB; 32of 39 targets have better GDT.TS values, 4 targets remain about the same, and only 3 targetshave slightly lower GDT.TS values. On average, restrained Rosetta refinement improved theGDT.TS scores of the PDB NMR structures by 2.5%, with some increasing by as much as 10%.


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)

Restrained Rosetta refined structures are more similar to their corresponding X-ray crystalstructures than PDB NMR structures. GDT.TS values of PDB NMR structures to correspondingX-ray structures are plotted on the X-axis, and GDT.TS values of both unrestrained Rosettarefined structures (R3, represented by red solid triangle) and restrained Rosetta refinedstructures (R3rst, represented by blue solid rectangles) to their corresponding X-raystructures are plotted on the Y-axis. Data are summarized for 39 NESG NMR/X-ray pairs. Thetwo green dash lines indicate GDT.TS of PDB NMR structures equal to 0.7 and 0.85respectively. The black dash line indicates y = x, and the two gray dash lines indicate y = x+ 0.05 and y = x – 0.05 respectively.
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Related In: Results  -  Collection

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fig4: Restrained Rosetta refined structures are more similar to their corresponding X-ray crystalstructures than PDB NMR structures. GDT.TS values of PDB NMR structures to correspondingX-ray structures are plotted on the X-axis, and GDT.TS values of both unrestrained Rosettarefined structures (R3, represented by red solid triangle) and restrained Rosetta refinedstructures (R3rst, represented by blue solid rectangles) to their corresponding X-raystructures are plotted on the Y-axis. Data are summarized for 39 NESG NMR/X-ray pairs. Thetwo green dash lines indicate GDT.TS of PDB NMR structures equal to 0.7 and 0.85respectively. The black dash line indicates y = x, and the two gray dash lines indicate y = x+ 0.05 and y = x – 0.05 respectively.
Mentions: For this assessment, we calculated the GDT.TS between (i) PDB NMR structures, (ii)unrestrained Rosetta refined structures, and (iii) restrained Rosetta refined structures, withtheir corresponding X-ray structures. NESG target DrR147D was left out of this analysis becauseits solution NMR structure is a monomer solved at pH 4.5, while its X-ray structure is a dimersolved at pH 6.0, and NMR studies demonstrate a significant structural change over this pHrange (data not shown). These results for the remaining 39 NESG NMR/X-ray pairs are summarizedin a GDT.TS scatterplot (Figure 4), with the GDT.TS of PDB NMR structuresrelative to the corresponding X-ray crystal structure on the x-axis and GDT.TSof the unrestrained or restrained Rosetta refined structures on the y-axis.Based on observations of previous studies30,31 done with a much small number (i.e., 1 or 2) of protein targets,we expected unrestrained Rosetta refinement would generally move NMR structures closer to theirX-ray counterparts. However, as illustrated in Figure 4, using this largerdata set of 39 NMR/X-ray pairs, we observed that this is not the case. After unrestrainedRosetta refinement (R3), only 17 of 39 targets exhibit higher GDT.TS values, 6 targets remainabout the same, and 16 of the protein ensembles have lower GDT.TS values than the NMRstructures refined by conventional methods and deposited in the PDB. On average, unrestrainedRosetta refinement improved the GDT.TS by only 0.4%. On the other hand, as illustrated inFigure 4, restrained Rosetta refinement (R3rst) generally improved the GDT.TSscore to the X-ray crystal structure, compared with the NMR structure deposited in the PDB; 32of 39 targets have better GDT.TS values, 4 targets remain about the same, and only 3 targetshave slightly lower GDT.TS values. On average, restrained Rosetta refinement improved theGDT.TS scores of the PDB NMR structures by 2.5%, with some increasing by as much as 10%.

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