Automated structure refinement of macromolecular assemblies from cryo-EM maps using Rosetta

Abstract

Cryo-EM has revealed the structures of many challenging yet exciting macromolecular assemblies at near-atomic resolution (3-4.5Å ), providing biological phenomena with molecular descriptions. However, at these resolutions, accurately positioning individual atoms remains challenging and error-prone. Manually refining thousands of amino acids -typical in a macromolecular assembly -is tedious and time-consuming. We present an automated method that can improve the atomic details in models that are manually built in near-atomic-resolution cryo-EM maps. Applying the method to three systems recently solved by cryo-EM, we are able to improve model geometry while maintaining the fit-to-density. Backbone placement errors are automatically detected and corrected, and the refinement shows a large radius of convergence. The results demonstrate that the method is amenable to structures with symmetry, of very large size, and containing RNA as well as covalently bound ligands. The method should streamline the cryo-EM structure determination process, providing accurate and unbiased atomic structure interpretation of such maps.

Lis1 Has Two Opposing Modes of Regulating Cytoplasmic Dynein

“…We used a monomeric dynein construct lacking GST (Dyn wt-M ), which is otherwise identical to the dimeric dynein constructs used above. We solved a 7.7Å resolution structure of Dyn wt-M bound to Lis1 in the presence of ATP and built an atomic model into the density using Rosetta (Figures 2B, 2C, S2, and Table S2) (Wang et al, 2016). …”

Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein

“…The crystal structure of GLP-1R NTD:GLP-1 complex (PDB ID: 3IOL) and β2AR:Gs complex (PDB ID: 3SN6) were used as initial models for NTD-hGLP-1 and Gs heterotrimer, respectively. All models were docked into the EM density map using Chimera 52 , followed by iterative manual adjustment and real-space refinement using COOT 53 and fragment-based refinement with Rosetta 54 . Sequence assignment was guided by bulky amino acid residues such as Phe, Tyr, Trp and Arg.…”

Structural basis of mitochondrial receptor binding and constriction by DRP1

“…Multiple rounds of refinement were done for each component against one half map (training map), and the other half map (validation map) was used to monitor overfitting according to the detailed procedure described in Wang et al 68 .…”