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A New Protocol for Atomic-Level Protein Structure Modeling and Refinement Using Low-to-Medium Resolution Cryo-EM Density Maps.

ABSTRACT: The rapid progress of cryo-electron microscopy (cryo-EM) in structural biology has raised an urgent need for robust methods to create and refine atomic-level structural models using low-resolution EM density maps. We propose a new protocol to create initial models using I-TASSER protein structure prediction, followed by EM density map-based rigid-body structure fitting, flexible fragment adjustment and atomic-level structure refinement simulations. The protocol was tested on a large set of 285 non-homologous proteins and generated structural models with correct folds for 260 proteins, where 28% had RMSDs below 2 Å. Compared to other state-of-the-art methods, the major advantage of the proposed pipeline lies in the uniform structure prediction and refinement protocol, as well as the extensive structural re-assembly simulations, which allow for low-to-medium resolution EM density map-guided structure modeling starting from amino acid sequences. Interestingly, the quality of both the image fitting and subsequent structure refinement was found to be strongly correlated with the correctness of the initial I-TASSER models; this is mainly due to the different correlation patterns observed between force field and structural quality for the models with template modeling score (or TM-score, a metric quantifying the similarity of models to the native) above and below a threshold of 0.5. Overall, the results demonstrate a new avenue that is ready to use for large-scale cryo-EM-based structure modeling and atomic-level density map-guided structure refinement.

SUBMITTER: Zhang B

PROVIDER: S-EPMC7502543 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Recent advances in instrumentation and software have resulted in cryo-EM rapidly becoming the method of choice for structural biologists, especially for those studying the three-dimensional structures of very large macromolecular complexes. In this contribution, the tools available for macromolecular structure refinement into cryo-EM reconstructions that are available via CCP-EM are reviewed, specifically focusing on REFMAC5 and related tools. Whilst originally designed with a view to refinement against X-ray diffraction data, some of these tools have been able to be repurposed for cryo-EM owing to the same principles being applicable to refinement against cryo-EM maps. Since both techniques are used to elucidate macromolecular structures, tools encapsulating prior knowledge about macromolecules can easily be transferred. However, there are some significant qualitative differences that must be acknowledged and accounted for; relevant differences between these techniques are highlighted. The importance of phases is considered and the potential utility of replacing inaccurate amplitudes with their expectations is justified. More pragmatically, an upper bound on the correlation between observed and calculated Fourier coefficients, expressed in terms of the Fourier shell correlation between half-maps, is demonstrated. The importance of selecting appropriate levels of map blurring/sharpening is emphasized, which may be facilitated by considering the behaviour of the average map amplitude at different resolutions, as well as the utility of simultaneously viewing multiple blurred/sharpened maps. Features that are important for the purposes of computational efficiency are discussed, notably the Divide and Conquer pipeline for the parallel refinement of large macromolecular complexes. Techniques that have recently been developed or improved in Coot to facilitate and expedite the building, fitting and refinement of atomic models into cryo-EM maps are summarized. Finally, a tool for symmetry identification from a given map or coordinate set, ProSHADE, which can identify the point group of a map and thus may be used during deposition as well as during molecular visualization, is introduced.

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A New Protocol for Atomic-Level Protein Structure Modeling and Refinement Using Low-to-Medium Resolution Cryo-EM Density Maps.

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