Molecular architecture of the HerA–NurA DNA double-strand break resection complex

DNA double-strand breaks can be repaired by homologous recombination, during which the DNA ends are long-range resected by helicase–nuclease systems to generate 3′ single strand tails. In archaea, this requires the Mre11–Rad50 complex and the ATP-dependent helicase–nuclease complex HerA–NurA. We report the cryo-EM structure of Sulfolobus solfataricus HerA–NurA at 7.4 Å resolution and present the pseudo-atomic model of the complex. HerA forms an ASCE hexamer that tightly interacts with a NurA dimer, with each NurA protomer binding three adjacent HerA HAS domains. Entry to NurA’s nuclease active sites requires dsDNA to pass through a 23 Å wide channel in the HerA hexamer. The structure suggests that HerA is a dsDNA translocase that feeds DNA into the NurA nuclease sites.     

Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells

Cryo-electron tomography (cryo-ET) allows for the visualization of biological material in a close-to-native state, in three dimensions and with nanometer scale resolution. However, due to the low signal-to-noise ratio inherent to imaging of the radiation-sensitive frozen-hydrated samples, it appears oftentimes impossible to localize structures within heterogeneous samples. Because a major potential for cryo-ET is thereby left unused, we set out to combine cryo-ET with cryo-fluorescence microscopy (cryo-FM), in order to facilitate the search for structures of interest. We describe a cryo-FM setup and workflow for correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) that can be easily implemented. Cells are grown on finder grids, vitally labeled with one or two fluorescent dyes, and vitrified. After a structure is located by cryo-FM (with 0.4 μm resolution), its image coordinates are translated to cryo-ET stage coordinates via a home-built software routine. We tested our workflow on whole mount primary human umbilical vein endothelial cells. The correlative routine enabled us to investigate mitochondrial ultrastructure for the first time on intact human mitochondria, and led us to find mitochondrial cristae that were connected to the intermembrane space via large slits, which challenges the current view that such connections are established exclusively via small circular pores. Taken together, this study emphasizes that cryo-CLEM can be a routinely used technique that opens up exciting new possibilities for cryo-ET.     

Rapid increase of near atomic resolution virus capsid structures determined by cryo-electron microscopy

The recent technological advances in electron microscopes, detectors, as well as image processing and reconstruction software have brought single particle cryo-electron microscopy (cryo-EM) into prominence for determining structures of bio-molecules at near atomic resolution. This has been particularly true for virus capsids, ribosomes, and other large assemblies, which have been the ideal specimens for structural studies by cryo-EM approaches. An analysis of time series metadata of virus structures on the methods of structure determination, resolution of the structures, and size of the virus particles revealed a rapid increase in the virus structures determined by cryo-EM at near atomic resolution since 2010. In addition, the data highlight the median resolution (～3.0?Å) and size (～310.0?Å in diameter) of the virus particles determined by X-ray crystallography while no such limits exist for cryo-EM structures, which have a median diameter of 508?Å. Notably, cryo-EM virus structures in the last four years have a median resolution of 3.9?Å. Taken together with minimal sample requirements, not needing diffraction quality crystals, and being able to achieve similar resolutions of the crystal structures makes cryo-EM the method of choice for current and future virus capsid structure determinations.     

High-resolution cryo-EM performance comparison of two latest-generation cryo electron microscopes on the human ribosome

Recent technological advances in cryo electron microscopy (cryo-EM) have led to new opportunities in the structural biology field. Here we benchmark the performance of two 300 kV latest-generation cryo electron microscopes, Titan Krios G4 from Thermofisher Scientific and CRYO ARM 300 from Jeol, with regards to achieving high resolution single particle reconstructions on a real case sample. We compare potentially limiting factors such as drift rates, astigmatism & coma aberrations and performance during image processing and show that both microscopes, while comprising rather different technical setups & parameter settings and equipped with different types of energy filters & cameras, achieve a resolution of around 2 Å on the human ribosome, a non-symmetric object which constitutes a key drug target. Astigmatism correction, CTF refinement and correction of higher order aberrations through refinement in separate optics groups helped to account for astigmatism/coma caused by beam tilting during multi-spot and multi-hole acquisition in neighbouring holes without stage movement. The obtained maps resolve Mg²⁺ ions, water molecules, inhibitors and side-chains including chemical modifications. The fact that both instruments can resolve such detailed features will greatly facilitate understanding molecular mechanisms of various targets and helps in cryo-EM structure based drug design. The methods and analysis tools used here will be useful also to characterize existing instruments and optimize data acquisition settings and are applicable broadly to other drug targets in structural biology.     

Recent advances and current trends in cryo-electron microscopy

All steps of cryogenic electron-microscopy (cryo-EM) workflows have rapidly evolved over the last decade. Advances in both single-particle analysis (SPA) cryo-EM and cryo-electron tomography (cryo-ET) have facilitated the determination of high-resolution biomolecular structures that are not tractable with other methods. However, challenges remain. For SPA, these include improved resolution in an additional dimension: time. For cryo-ET, these include accessing difficult-to-image areas of a cell and finding rare molecules. Finally, there is a need for automated and faster workflows, as many projects are limited by throughput. Here, we review current developments in SPA cryo-EM and cryo-ET that push these boundaries. Collectively, these advances are poised to propel our spatial and temporal understanding of macromolecular processes.     

Native Immunogold Labeling of Cell Surface Proteins and Viral Glycoproteins for Cryo-Electron Microscopy and Cryo-Electron Tomography Applications

Numerous methods have been developed for immunogold labeling of thick, cryo-preserved biological specimens. However, most of the methods are permutations of chemical fixation and sample sectioning, which select and isolate the immunolabeled region of interest. We describe a method for combining immunogold labeling with cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) of the surface proteins of intact mammalian cells or the surface glycoproteins of assembling and budding viruses in the context of virus-infected mammalian cells cultured on EM grids. In this method, the cells were maintained in culture media at physiologically relevant temperatures while sequentially incubated with the primary and secondary antibodies. Subsequently, the immunogold-labeled specimens were vitrified and observed under cryo-conditions in the transmission electron microscope. Cryo-EM and cryo-ET examination of the immunogold-labeled cells revealed the association of immunogold particles with the target antigens. Additionally, the cellular structure was unaltered by pre-immunolabeling chemical fixation and retained well-preserved plasma membranes, cytoskeletal elements, and macromolecular complexes. We think this technique will be of interest to cell biologists for cryo-EM and conventional studies of native cells and pathogen-infected cells.     

Structure of the nuclear pore complex goes atomic

The nuclear pore complex (NPC) is a supra-molecular assembly that mediates substance and information flow across the nuclear envelope (NE). Due to its extraordinary size and complexity, the NPC remains one of the most challenging tasks in structural elucidation at atomic resolution. Recent breakthroughs in cryo-electron microscopy (cryo-EM) reconstruction, Machine Learning empowered structure prediction and biochemical reconstitution have combined to yield molecular models of the NPC at unprecedented accuracy. Furthermore, in cellulo cryo-electron tomography (cryo-ET) structures reveal substantial structural dynamics of the NPC. These advances shed light on the organizational principles and functions of the NPC.     

Preferred orientations of LDL in vitreous ice indicate a discoid shape of the lipoprotein particle

The structure of the human low-density lipoprotein (LDL) was analyzed in vitreous ice using cryo-electron microscopy (cryo-EM). In relatively thick cryo-EM preparations, random orientation of LDL particles produced various types of projections on the microscope screen, including circular projections with a high-density ring and rectangular projections with two high-density bands. However, in especially thin preparations, preferred, non-random orientations of the LDL particle produced only circular projections of the lipoprotein structure. In preparations with high LDL concentrations, ordered two-dimensional arrays, including hexagonal arrangements of circular projections and short stacks of rectangular projections, were observed. These observations are consistent with a discoid shape of the LDL particle, and suggest that surface tension forces may influence orientation of the LDL disc in thin aqueous films. Face-on orientation of LDL in especially thin cryo-EM preparations may explain earlier difficulties in identifying discoid features of the lipoprotein particle, and illustrates that some caution is warranted when attempts are made to reconstruct the three-dimensional structure of LDL from cryo-electron micrographs.     

Cryo-EM structure of monomeric photosystem II at 2.78 Å resolution reveals factors important for the formation of dimer

Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One β-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the β-carotene, SQDG and PsbO in formation of the PSII dimer.     

Capturing actin assemblies in cells using in situ cryo-electron tomography

Actin contributes to an exceptionally wide range of cellular processes through the assembly and disassembly of highly dynamic and ordered structures. Visualizing these structures in cells can help us understand how the molecular players of the actin machinery work together to produce force-generating systems. In recent years, cryo-electron tomography (cryo-ET) has become the method of choice for structural analysis of the cell interior at the molecular scale. Here we review advances in cryo-ET workflows that have enabled this transformation, especially the automation of sample preparation procedures, data collection, and processing. We discuss new structural analyses of dynamic actin assemblies in cryo-preserved cells, which have provided mechanistic insights into actin assembly and function at the nanoscale. Finally, we highlight the latest visual proteomics studies of actin filaments and their interactors reaching sub-nanometer resolutions in cells.     

Dissecting the 3-D structure of vimentin intermediate filaments by cryo-electron tomography

Vimentin polymerizes via complex lateral interactions of coiled-coil dimers into long, flexible filaments referred to as intermediate filaments (IFs). Intermediate in diameter between microtubules and microfilaments, IFs constitute the third cytoskeletal filament system of metazoan cells. Here we investigated the molecular basis of the 3-D architecture of vimentin IFs by cryo-electron microscopy (cryo-EM) as well as cryo-electron tomography (Cryo-ET) 3-D reconstruction. We demonstrate that vimentin filaments in cross-section exhibit predominantly a four-stranded protofibrilar organization with a right-handed supertwist with a helical pitch of about 96 nm. Compact filaments imaged by cryo-EM appear surprisingly straight and hence appear very stiff. In addition, IFs exhibited an increased flexibility at sites of partial unraveling. This is in strong contrast to chemically fixed, negatively stained preparations of vimentin filaments that generally exhibit smooth bending without untwisting. At some point along the filament unraveling may be triggered and propagates in a cooperative manner so that long stretches of filaments appear to have unraveled rapidly in a coordinated fashion.     

A flexible framework for multi-particle refinement in cryo-electron tomography

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) are increasingly used for macromolecular structure determination in situ. Here, we introduce a set of computational tools and resources designed to enable flexible approaches to STA through increased automation and simplified metadata handling. We create a bidirectional interface between the Dynamo software package and the Warp-Relion-M pipeline, providing a framework for ab initio and geometrical approaches to multiparticle refinement in M. We illustrate the power of working within this framework by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles (VLPs), and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we provide a comprehensive, step-by-step guide to obtaining a 3.4-Å reconstruction from EMPIAR-10164. The guide is hosted on https://teamtomo.org/, a collaborative online platform we establish for sharing knowledge about cryo-ET.

Employing optimal computational methodology in cryo-electron tomography is not always easy; this article provides a set of tools and a complete guide to obtaining high-resolution structures from cryo-ET data.     

Computational methods for ultrastructural analysis of synaptic complexes

Electron microscopy (EM) provided fundamental insights about the ultrastructure of neuronal synapses. The large amount of information present in the contemporary EM datasets precludes a thorough assessment by visual inspection alone, thus requiring computational methods for the analysis of the data. Here, I review image processing software methods ranging from membrane tracing in large volume datasets to high resolution structures of synaptic complexes. Particular attention is payed to molecular level analysis provided by recent cryo-electron microscopy and tomography methods.     

Rapid tilt-series acquisition for electron cryotomography

Using a new Titan Krios stage equipped with a single-axis holder, we developed two methods to accelerate the collection of tilt-series. We demonstrate a continuous-tilting method that can record a tilt-series in seconds, but with loss of details finer than ～4?nm. We also demonstrate a fast-incremental method that can record a tilt-series several-fold faster than current methods and with similar resolution. We characterize the utility of both methods in real biological electron cryotomography workflows. We identify opportunities for further improvements in hardware and software and speculate on the impact such advances could have on structural biology.     

High-vacuum optical platform for cryo-CLEM (HOPE): A new solution for non-integrated multiscale correlative light and electron microscopy

Cryo-correlative light and electron microscopy (cryo-CLEM) offers a unique way to analyze the high-resolution structural information of cryo-vitrified specimen by cryo-electron microscopy (cryo-EM) with the guide of the search for unique events by cryo-fluorescence microscopy (cryo-FM). To achieve cryo-FM, a trade-off must be made between the temperature and performance of objective lens. The temperature of specimen should be kept below devitrification while the distance between the objective lens and specimen should be short enough for high resolution imaging. Although special objective lens was designed in many current cryo-FM approaches, the unavoided frosting and ice contamination are still affecting the efficiency of cryo-CLEM. In addition, the correlation accuracy between cryo-FM and cryo-EM would be reduced during the current specimen transfer procedure. Here, we report an improved cryo-CLEM technique (high-vacuum optical platform for cryo-CLEM, HOPE) based on a high-vacuum optical stage and a commercial cryo-EM holder. The HOPE stage comprises of a special adapter to suit the cryo-EM holder and a high-vacuum chamber with an anti-contamination system. It provides a clean and enduring environment for cryo specimen, while the normal dry objective lens in room temperature can be used via the optical windows. The ‘touch-free’ specimen transfer via cryo-EM holder allows least specimen deformation and thus maximizes the correlation accuracy between cryo-FM and cryo-EM. Besides, we developed a software to perform semi-automatic cryo-EM acquisition of the target region localized by cryo-FM. Our work provides a new solution for cryo-CLEM and can be adapted for different commercial fluorescence microscope and electron microscope.     

Multishot tomography for high-resolution in situ subtomogram averaging

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) can resolve protein complexes at near atomic resolution, and when combined with focused ion beam (FIB) milling, macromolecules can be observed within their native context. Unlike single particle acquisition (SPA), cryo-ET can be slow, which may reduce overall project throughput. We here propose a fast, multi-position tomographic acquisition scheme based on beam-tilt corrected beam-shift imaging along the tilt axis, which yields sub-nanometer in situ STA averages.     

HIV-2 Immature Particle Morphology Provides Insights into Gag Lattice Stability and Virus Maturation

Retrovirus immature particle morphology consists of a membrane enclosed, pleomorphic, spherical and incomplete lattice of Gag hexamers. Previously, we demonstrated that human immunodeficiency virus type 2 (HIV-2) immature particles possess a distinct and extensive Gag lattice morphology. To better understand the nature of the continuously curved hexagonal Gag lattice, we have used the single particle cryo-electron microscopy method to determine the HIV-2 Gag lattice structure for immature virions. The reconstruction map at 5.5 Å resolution revealed a stable, wineglass-shaped Gag hexamer structure with structural features consistent with other lentiviral immature Gag lattice structures. Cryo-electron tomography provided evidence for nearly complete ordered Gag lattice structures in HIV-2 immature particles. We also solved a 1.98 Å resolution crystal structure of the carboxyl-terminal domain (CTD) of the HIV-2 capsid (CA) protein that identified a structured helix 12 supported via an interaction of helix 10 in the absence of the SP1 region of Gag. Residues at the helix 10–12 interface proved critical in maintaining HIV-2 particle release and infectivity. Taken together, our findings provide the first 3D organization of HIV-2 immature Gag lattice and important insights into both HIV Gag lattice stabilization and virus maturation.     

Cryo-electron microscopy targets in CASP13: Overview and evaluation of results

Structures of seven CASP13 targets were determined using cryo-electron microscopy (cryo-EM) technique with resolution between 3.0 and 4.0 Å. We provide an overview of the experimentally derived structures and describe results of the numerical evaluation of the submitted models. The evaluation is carried out by comparing coordinates of models to those of reference structures (CASP-style evaluation), as well as checking goodness-of-fit of modeled structures to the cryo-EM density maps. The performance of contributing research groups in the CASP-style evaluation is measured in terms of backbone accuracy, all-atom local geometry and similarity of inter-subunit interfaces. The results on the cryo-EM targets are compared with those on the whole set of eighty CASP13 targets. A posteriori refinement of the best models in their corresponding cryo-EM density maps resulted in structures that are very close to the reference structure, including some regions with better fit to the density.