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

Abstract

We report the solution structure of Escherichia coli β-galactosidase (∼465 kDa), solved at ∼3.2-Å resolution by using single-particle cryo-electron microscopy (cryo-EM). Densities for most side chains, including those of residues in the active site, and a catalytic Mg 2+ ion can be discerned in the map obtained by cryo-EM. The atomic model derived from our cryo-EM analysis closely matches the 1.7-Å crystal structure with a global rmsd of ∼0.66 Å. There are significant local differences throughout the protein, with clear evidence for conformational changes resulting from contact zones in the crystal lattice. Inspection of the map reveals that although densities for residues with positively charged and neutral side chains are well resolved, systematically weaker densities are observed for residues with negatively charged side chains. We show that the weaker densities for negatively charged residues arise from their greater sensitivity to radiation damage from electron irradiation as determined by comparison of density maps obtained by using electron doses ranging from 10 to 30 e − /Å 2 . In summary, we establish that it is feasible to use cryo-EM to determine near-atomic resolution structures of protein complexes (<500 kDa) with low symmetry, and that the residue-specific radiation damage that occurs with increasing electron dose can be monitored by using dose fractionation tools available with direct electron detector technology.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 (1-10). 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 (13-15). These developments signal an important change in the way cryo-EM can now be used in structural biology. Rather than simply using cryo-EM maps, typically in the 6-to 20-Å resolution range, as an envelope in which to fit structures obtained by X-ray crystallography, there is the exciting prospect of using cryo-EM to derive de novo, high-resolution structural models of proteins in one or multiple functional conformational states.The preparation of specimens for cryo-EM involves rapid plunge freezing of a thin aqueous suspension into liquid ethane cooled by liquid nitrogen. The speed of freezing is estimated to be ∼10 7 K/s (16), enabling the rapid cooling of the aqueous medium into a glass-like state. This rapid cooling traps the macromolecular components in a near-native state, capturing the conformational distribution and spa...

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

“…With these improvements allowing higher resolution structures to be obtained, there have already been a number of structures published in complex with ligands and inhibitors (Fig. 3), from large soluble complexes such as the proteasome and -galactosidase to smaller membrane proteins including TRP channels and gamma secretase (Bartesaghi et al, 2014;Bai et al, 2015;Paulsen et al, 2015;Gao et al, 2016;Li et al, 2016).…”

“…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.…”

GENFIRE: A Generalized Fourier Iterative Reconstruction Algorithm for High-Resolution 3D Electron and X-ray Imaging

“…single-particle cryo-electron microscopy (EM) has been applied to achieve near atomic resolution of purified protein complexes 2,7,[14][15][16] , and cryo-electron tomography allows for 3D imaging of pleomorphic samples such as viral infection mechanisms of cells with resolutions on the order of a few nanometers [17][18][19] . These advances are not limited to electron tomography.…”

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.…”

“…Dynamo (CastanoDiez, Kudryashev et al 2012), PyTom (Hrabe, Chen et al 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 smaller than 500kDa (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.…”