Using NMR Chemical Shifts and Cryo-EM Density Restraints in Iterative Rosetta-MD Protein Structure Refinement
Using NMR Chemical Shifts and Cryo-EM Density Restraints in Iterative Rosetta-MD Protein Structure Refinement
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DOI：
10.1021/acs.jcim.9b00932
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发表时间：
2020-05-26
影响因子：
5.6
通讯作者：
Lindert, Steffen
中科院分区：
文献类型：
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作者：
Leelananda, Sumudu P.;Lindert, Steffen
Cryo-EM已成为蛋白质结构解析的主要方法之一，经常产生近原子或中等分辨率的密度图。如果蛋白质结构不能从密度图中明确地推导出来，则需要计算结构精化工具来生成蛋白质结构模型。我们之前已经开发了一种迭代Rosetta-MDFF协议，该协议使用cryo-EM密度来细化蛋白质结构。在这里，我们表明，除了冷冻EM密度，纳入其他实验限制到Rosetta-MDFF协议进一步改善了精细结构。我们在Rosetta步骤和分子动力学（MD）模拟步骤中使用NMR化学位移（CS）数据与我们的混合协议中的cryo-EM密度集成。在所有MD轮次的18种情况中，有15种情况下，密度图和NMR化学位移数据组合使用时获得的细化结果优于仅密度图细化的结果。值得注意的是，当使用中分辨率和低分辨率密度图时，细化的改善最高。使用我们的混合方法，获得的最终模型的RMSD总是优于我们以前的协议获得的RMSD，对于中等（6.9埃）和低（9埃）分辨率的地图，只有密度细化。对于具有中等分辨率密度图（6.9埃）的所有六种测试蛋白质，混合方法的最终细化结构RMSD低于仅冷冻EM细化的最终细化结构RMSD。当我们的混合方案与4埃密度图一起使用时，最终改进的RMSD小于1.5埃。对于六种蛋白质中的四种，最终的RSD甚至小于1埃。这项研究表明，通过使用冷冻EM和NMR约束的组合，有可能将结构细化到原子分辨率，优于单一约束细化。这种混合协议将是一个有价值的工具时，只有低分辨率的冷冻EM密度数据和NMR化学位移数据可用于细化结构。
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 angstrom) and low (9 angstrom) resolution maps. For all the six test proteins with medium resolution density maps (6.9 angstrom), 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 angstrom when our hybrid protocol was used with 4 angstrom density maps. For four out of the six proteins the final RMSDs were even less than 1 angstrom. 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.