Atomic model of the F420-reducing [NiFe] hydrogenase by electron cryo-microscopy using a direct electron detector

Abstract

The introduction of direct electron detectors with higher detective quantum efficiency and fast read-out marks the beginning of a new era in electron cryo-microscopy. Using the FEI Falcon II direct electron detector in video mode, we have reconstructed a map at 3.36 Å resolution of the 1.2 MDa F420-reducing hydrogenase (Frh) from methanogenic archaea from only 320,000 asymmetric units. Videos frames were aligned by a combination of image and particle alignment procedures to overcome the effects of beam-induced motion. The reconstructed density map shows all secondary structure as well as clear side chain densities for most residues. The full coordination of all cofactors in the electron transfer chain (a [NiFe] center, four [4Fe4S] clusters and an FAD) is clearly visible along with a well-defined substrate access channel. From the rigidity of the complex we conclude that catalysis is diffusion-limited and does not depend on protein flexibility or conformational changes.DOI: http://dx.doi.org/10.7554/eLife.01963.001

Structure of β-galactosidase at 3.2-Å resolution obtained by cryo-electron microscopy

“…By collecting dose fractionated images, the total exposure can be divided into a large number of images, each at a very small dose, enabling correction for drift resulting from beaminduced movement and also from thermal drifts of the specimen stage. The emerging consensus from a number of recent studies (10,11,15,40), which is confirmed in our present analysis, is that, on average, the largest movements tend to occur early in the exposure (Fig. S3); these early frames can be either discarded or down-weighted during image processing.…”

“…S12, which shows that increasing the dose from 10 e − /Å 2 to 45 e − /Å 2 results in an ∼twofold increase in preferential damage of Asp and Glu residues compared with Asn and Gln residues, respectively. The trends we observe for preferential radiation damage of Asp and Glu residues can also be demonstrated by similar analysis of map value variations in a recently reported cryo-EM density map for Frh (10), an unrelated protein, at a resolution of 3.36 Å (Fig. S12A).…”

“…single-particle EM | frame alignment | CTF determination | 3D reconstruction | structure refinement R apid advances in technology for single-particle cryo-electron microscopy (cryo-EM) over the last few years have made it possible to determine high-resolution structures of large and well-ordered macromolecular assemblies such as 2D protein crystals, helical lattices, icosahedral viruses, and protein complexes with high symmetry (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). More recently, continuing developments in microscope hardware and image processing software have yielded near-atomic resolution information for two smallersized complexes with low symmetry, the 700-kDa proteasome (11) and a 300-kDa mammalian ion channel (12), and for larger assemblies with no symmetry such as ribosome complexes (13)(14)(15).…”

The potential use of single-particle electron microscopy as a tool for structure-based inhibitor design

“…Radiation damage can also have a more subtle effect aside from the degradation of resolution, where damage causes protein side chains to shift, which could lead to subtle differences in the inhibitor binding position. Previous studies have shown that negative side chains appear to suffer radiation damage preferentially (Allegretti et al, 2014;Bartesaghi et al, 2014;Grant & Grigorieff, 2015), so if there are key negatively charged residues in the binding pocket then this may induce a significant movement of the inhibitor or show a binding mode which is not physiologically relevant. However, it should be noted that weak density for negative side chains can also be a feature of the electric potential map generated in the electron-microscopy experiment (Wang & Moore, 2017) and is not in all cases radiation damage.…”

Developing 3D novel edge detection and particle picking tools for electron tomography

“…Many software packages have been developed for single particle reconstruction, including Spider (Shaikh, Gao et al 2008, Baniulis, Yamashita et al 2009), IMAGIC (Forster and Hegerl 2007), Xmipp (Sorzano, Marabini et al 2004), BSoft Belnap 2007, Heymann, Cardone et al 2008), SPARX (Hohn, Tang et al 2007) EMAN (Heymann and Belnap 2007), EMAN2 (Tang, Peng et al 2007), Relion (Scheres 2012 Furthermore state-of-the-art direct electron detectors greatly reduce the impacts of sample charging and drift on the captured image, increasingly making atomic resolution structure determination by single particle analysis (Allegretti, Mills et al 2014) a reality not only for viruses and macromolecular assemblies but also for membrane proteins in the 500kDa range (Li, Mooney et al 2013, Liao, Cao et al 2013, Lyumkis, Julien et al 2013, Bartesaghi, Matthies et al 2014, Lu, Bai et al 2014, Campbell, Veesler et al 2015 and below.…”