De novo protein structure determination from near-atomic-resolution cryo-EM maps

Abstract

We present a de novo model building approach that combines predicted backbone conformations with side-chain density-fit to accurately assign sequence into density maps. We show this method yields accurate models for six experimental maps at 3.3–4.8 Å resolution, and produces a nearly complete model for an unsolved map containing a 660-residue hetero-dimeric protein. This method should enable rapid and reliable protein structure determination from near-atomic resolution cryo-EM maps.

Solid-state NMR: An emerging technique in structural biology of self-assemblies

“…High-resolution electron microscopy (EM), notably cryo-EM has developed into a powerful technique to allow for 3D reconstruction of the molecular envelope of protein assemblies, ranging from several dozens to only a few Å resolution [17][18][19][20][21]. Cryo-EM and MAS ssNMR constitute highly complementary techniques, providing on one hand density map and long-range symmetry parameters (EM) and atomic-level details such as dihedral angle and distance restraints (MAS ssNMR).…”

“…In contrast, cryo-electron microscopy (cryo-EM) and solid-state NMR (ssNMR) are complementary techniques that have the potential to investigate the 3D structure and assembly mechanisms of complex biomolecular self-assemblies in their intact state. Cryo-EM has demonstrated its power to reach nearatomic-resolution structural models based on density maps with a resolution b5 Å [17][18][19][20][21]. Solid-state NMR (ssNMR) is a technique similar to cryo-EM as it is not limited by the solubility or crystallinity of the sample, and has proven during the past decade its potential to solve complex protein assemblies [22][23][24][25][26].…”

Transmission electron microscopy and the molecular structure of icosahedral viruses

“…Availability of powerful computing clusters allows data processing with pixel and angular samplings fine enough to determine particle orientation with precision better than 0.2°. Interpretation of near atomic resolution maps required the development of new computational tools to assign secondary structure elements or folds to the different regions of the map density [53][54][55][56][57][58]. At resolutions beyond 4.5 Å, crystallographic model refinement and evaluation tools can also be used [59].…”

High-speed atomic force microscopy and its future prospects

“…The number of revealed structures is already enormous, while the number of proteins whose structure remains unsolved is steadily becoming less and less. The recent advances of cryo-EM (Wang et al 2015) and NMR are accelerating this clear trend. Thus, the focus of life sciences is changing from solving the static structure of more proteins to gaining an understanding of and using the acquired structural information.…”