Department of Chemistry and Biochemistry, The University of Arizona, 1041 E. Lowell Street, Tucson, AZ 85721, USA.
J Struct Biol. 2012 Feb;177(2):561-70. doi: 10.1016/j.jsb.2011.10.002. Epub 2011 Oct 13.
Cryo-elecron microscopy (cryo-EM) can provide important structural information of large macromolecular assemblies in different conformational states. Recent years have seen an increase in structures deposited in the Protein Data Bank (PDB) by fitting a high-resolution structure into its low-resolution cryo-EM map. A commonly used protocol for accommodating the conformational changes between the X-ray structure and the cryo-EM map is rigid body fitting of individual domains. With the emergence of different flexible fitting approaches, there is a need to compare and revise these different protocols for the fitting. We have applied three diverse automated flexible fitting approaches on a protein dataset for which rigid domain fitting (RDF) models have been deposited in the PDB. In general, a consensus is observed in the conformations, which indicates a convergence from these theoretically different approaches to the most probable solution corresponding to the cryo-EM map. However, the result shows that the convergence might not be observed for proteins with complex conformational changes or with missing densities in cryo-EM map. In contrast, RDF structures deposited in the PDB can represent conformations that not only differ from the consensus obtained by flexible fitting but also from X-ray crystallography. Thus, this study emphasizes that a "consensus" achieved by the use of several automated flexible fitting approaches can provide a higher level of confidence in the modeled configurations. Following this protocol not only increases the confidence level of fitting, but also highlights protein regions with uncertain fitting. Hence, this protocol can lead to better interpretation of cryo-EM data.
冷冻电子显微镜（cryo-EM）可以提供不同构象状态下的大型大分子组装体的重要结构信息。近年来，通过将高分辨率结构拟合到其低分辨率冷冻电子显微镜图谱中，在蛋白质数据库（PDB）中沉积的结构数量有所增加。通常用于适应 X 射线结构和冷冻电子显微镜图谱之间构象变化的方法是将各个结构域进行刚体拟合。随着不同的灵活拟合方法的出现，需要比较和修改这些不同的拟合协议。我们已经在一个蛋白质数据集上应用了三种不同的自动灵活拟合方法，该数据集已经在 PDB 中存储了刚体拟合（RDF）模型。一般来说，在构象中观察到一致，这表明这些理论上不同的方法收敛到最可能的解决方案，对应于冷冻电子显微镜图谱。然而，结果表明，对于具有复杂构象变化或冷冻电子显微镜图谱中存在缺失密度的蛋白质，可能不会观察到收敛。相比之下，在 PDB 中存储的 RDF 结构不仅代表与灵活拟合获得的共识不同的构象，而且还代表与 X 射线晶体学不同的构象。因此，这项研究强调，使用几种自动灵活拟合方法获得的“共识”可以为模型配置提供更高水平的置信度。遵循此协议不仅可以提高拟合的置信度，还可以突出具有不确定拟合的蛋白质区域。因此，该协议可以促进对冷冻电子显微镜数据的更好解释。
