Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before being able to inspect a biological sample in an electron microscope, it needs to be deposited in a thin layer on a grid and rapidly frozen. The VitroJet was designed with this aim, as well as avoiding the delicate manual handling and transfer steps that occur during the conventional grid-preparation process. Since its creation, numerous technical developments have resulted in a device that is now widely utilized in multiple laboratories worldwide. It features plasma treatment, low-volume sample deposition through pin printing, optical ice-thickness measurement and cryofixation of pre-clipped Autogrids through jet vitrification. This paper presents recent technical improvements to the VitroJet and the benefits that it brings to the cryo-EM workflow. A wide variety of applications are shown: membrane proteins, nucleosomes, fatty-acid synthase, Tobacco mosaic virus, lipid nanoparticles, tick-borne encephalitis viruses and bacteriophages. These case studies illustrate the advancement of the VitroJet into an instrument that enables accurate control and reproducibility, demonstrating its suitability for time-efficient cryo-EM structure determination.

Cryo-electron microscopy (cryoEM) has become a well established technique with the potential to produce structures of large and dynamic supramolecular complexes that are not amenable to traditional approaches for studying structure and dynamics. The size and low resolution of such molecular systems often make structural modelling and molecular dynamics simulations challenging and computationally expensive. This, together with the growing wealth of structural data arising from cryoEM and other structural biology methods, has driven a trend in the computational biophysics community towards the development of new pipelines for analysing global dynamics using coarse-grained models and methods. At the centre of this trend has been a return to elastic network models, normal mode analysis (NMA) and ensemble analyses such as principal component analysis, and the growth of hybrid simulation methodologies that make use of them. Here, this field is reviewed with a focus on ProDy, the Python application programming interface for protein dynamics, which has been developed over the last decade. Two key developments in this area are highlighted: (i) ensemble NMA towards extracting and comparing the signature dynamics of homologous structures, aided by the recent SignDy pipeline, and (ii) pseudoatom fitting for more efficient global dynamics analyses of large and low-resolution supramolecular assemblies from cryoEM, revisited in the CryoDy pipeline. It is believed that such a renewal and extension of old models and methods in new pipelines will be critical for driving the field forward into the next cryoEM revolution.

Buffer-composition and sample-preparation guidelines for cryo-electron microscopy are geared towards maximizing imaging contrast and reducing electron-beam-induced motion. These pursuits often involve the minimization or the complete removal of additives that are commonly used to facilitate proper protein folding and minimize aggregation. Among these admonished additives is glycerol, a widely used osmolyte that aids protein stability. In this work, it is shown that the inclusion of glycerol does not preclude high-resolution structure determination by cryoEM, as demonstrated by an ∼2.3 Å resolution reconstruction of mouse apoferritin (∼500 kDa) and an ∼3.3 Å resolution reconstruction of rabbit muscle aldolase (∼160 kDa) in the presence of 20%(v/v) glycerol. While it was found that generating thin ice that is amenable to high-resolution imaging requires long blot times, the addition of glycerol did not result in increased beam-induced motion or an inability to pick particles. Overall, these findings indicate that glycerol should not be discounted as a cryoEM sample-buffer additive, particularly for large, fragile complexes that are prone to disassembly or aggregation upon its removal.

Experimental techniques, such as cryo-electron microscopy, require biological samples to be recovered at cryogenic temperatures (T ≈ 100 K) with water being in an amorphous ice state. However, (bulk) water can exist in two amorphous ices at P < 1 GPa, low-density amorphous (LDA) ice at low pressures and high-density amorphous ice (HDA) at high pressures; HDA is ≈20-25% denser than LDA. While fast/plunge cooling at 1 bar brings the sample into LDA, high-pressure cooling (HPC), at sufficiently high pressure, produces HDA. HDA can also be produced by isothermal compression of LDA at cryogenic temperatures. Here, we perform classical molecular dynamics simulations to study the effects of LDA, HDA, and the LDA-HDA transformation on the structure and hydration of a small peptide, polyalanine. We follow thermodynamic paths corresponding to (i) fast/plunge cooling at 1 bar, (ii) HPC at P = 400 MPa, and (iii) compression/decompression cycles at T = 80 K. While process (i) produced LDA in the system, path (iii) produces HDA. Interestingly, the amorphous ice produced in process (ii) is an intermediate amorphous ice (IA) with properties that fall in-between those of LDA and HDA. Remarkably, the structural changes in polyalanine are negligible at all conditions studied (0-2000 MPa, 80-300 K) even when water changes among the low and high-density liquid states as well as the amorphous solids LDA, IA, and HDA. The similarities and differences in the hydration of polyalanine vitrified in LDA, IA, and HDA are described. Since the studied thermodynamic paths are suitable for the cryopreservation of biomolecules, we also study the structure and hydration of polyalanine along isobaric and isochoric heating paths, which can be followed experimentally for the recovery of cryopreserved samples. Upon heating, the structure of polyalanine remains practically unchanged. We conclude with a brief discussion of the practical advantages of (a) using HDA and IA as a cryoprotectant environment (as opposed to LDA), and (b) the use of isochoric heating as a recovery process (as opposed to isobaric heating).

Stimulating broadly neutralizing antibodies (bnAbs) directly from germline remains a barrier for HIV vaccines. HIV superinfection elicits bnAbs more frequently than single infection, providing clues of how to elicit such responses. We used longitudinal antibody sequencing and structural studies to characterize bnAb development from a superinfection case. BnAb QA013.2 bound initial and superinfecting viral Env, despite its probable naive progenitor only recognizing the superinfecting strain, suggesting both viruses influenced this lineage. A 4.15 Å cryo-EM structure of QA013.2 bound to native-like trimer showed recognition of V3 signatures (N301/N332 and GDIR). QA013.2 relies less on CDRH3 and more on framework and CDRH1 for affinity and breadth compared to other V3/glycan-specific bnAbs. Antigenic profiling revealed that viral escape was achieved by changes in the structurally-defined epitope and by mutations in V1. These results highlight shared and novel properties of QA013.2 relative to other V3/glycan-specific bnAbs in the setting of sequential, diverse antigens.

The challenges associated with operating electron microscopes (EM) in biosafety level 3 and 4 containment facilities have slowed progress of cryo-EM studies of high consequence viruses. We address this gap in a case study of Venezuelan Equine Encephalitis Virus (VEEV) strain TC-83. Chemical inactivation of viruses may physically distort structure, and hence to verify retention of native structure, we selected VEEV strain TC-83 to develop this methodology as this virus has a 4.8 Å resolution cryo-EM structure. In our method, amplified VEEV TC-83 was concentrated directly from supernatant through a 30 % sucrose cushion, resuspended, and chemically inactivated with 1 % glutaraldehyde. A second 30 % sucrose cushion removed any excess glutaraldehyde that might interfere with single particle analyses. A cryo-EM map of fixed, inactivated VEEV was determined to a resolution of 7.9 Å. The map retained structural features of the native virus such as the icosahedral symmetry, and the organization of the capsid core and the trimeric spikes. Our results suggest that our strategy can easily be adapted for inactivation of other enveloped, RNA viruses requiring BSL-3 or BSL-4 for cryo-EM. However, the validation of inactivation requires the oversight of Biosafety Committee for each Institution.

Historically, structural biology has been largely centered on in vitro approaches as the dominant technique to obtain indispensable high-resolution data. In situ structural biology is now poised to contribute with high-precision observations in a near-physiological context. Mass spectrometry, electron tomography, and fluorescence microscopy are opening up new opportunities for structural analysis, including the study of the protein machinery in living cells. The complementarity between studies is increasingly used to reveal biologically significant observations. Here we compare two complementary studies addressing the mechanisms of vesicle tethering with in vitro and in situ approaches. Cryoelectron microscopy and live-cell imaging assisted by anchoring platforms team up to explore elusive mechanisms of exocytosis, showing directions of future research.

Cryo-electron microscopy (cryo-EM) is emerging as a real alternative for structural elucidation. In spite of this, very few cryo-EM structures have been described so far as successful platforms for in silico drug design. Gabapentin and pregabalin are some of the most successful drugs in the treatment of epilepsy and neuropathic pain. Although both are in clinical use and are known to exert their effects by binding to the regulatory α2δ subunit of voltage gated calcium channels, their binding modes have never been characterized. We describe here the successful use of an exhaustive protein-ligand sampling algorithm on the α2δ-1 subunit of the recently published cryo-EM structure, with the goal of characterizing the ligand entry path and binding mode for gabapentin, pregabalin, and several other amino acidic α2δ-1 ligands. Our studies indicate that (i) all simulated drugs explore the same path for accessing the occluded binding site on the interior of the α2δ-1 subunit; (ii) they all roughly occupy the same pocket; (iii) the plasticity of the binding site allows the accommodation of a variety of amino acidic modulators, driven by the flexible \"capping loop\" delineated by residues Tyr426-Val435 and the floppy nature of Arg217; (iv) the predicted binding modes are in line with previously available mutagenesis data, confirming Arg217 as key for binding, with Asp428 and Asp467 highlighted as additional anchoring points for all amino acidic drugs. The study is one of the first proofs that latest-generation cryo-EM structures combined with exhaustive computational methods can be exploited in early drug discovery.

Electron cryo-microscopy (cryo-EM) of purified macromolecular complexes is now providing 3D-structures at near-atomic resolution (Kühlbrandt, 2014). Cryo-EM can tolerate heterogeneous specimens, however, high-resolution efforts demand highly optimized samples. Therefore, significant pre-screening and evaluation is essential before a final dataset can be obtained. While cryo-EM is comparably slow and requires access to expensive high-end electron microscopes, room temperature negative stain EM is fast, inexpensive and provides immediate feedback. This has made it a popular approach for sample quality control in the early phases of a project. Optimization in negative stain can be critical not only for cryo-EM, but also for X-ray crystallography, as highlighted for example by studies on GPCR complexes (Kang et al., 2015; Rasmussen et al., 2012). However, when not done carefully and interpreted correctly, negative stain can be prone to artifacts. A typical problem, which is often overlooked in the interpretation of EM data of small membrane proteins, is the background, caused by empty detergent micelles, as it can be easily confused with detergent embedded protein samples. To counteract this ubiquitous problem, we present a case study on commonly used detergents.We show that most detergents produce significant background in negative stain EM, even below nominal critical micelle concentration (CMC). Unawareness of such artefacts can lead to misinterpretation of sample quality and homogeneity. We hope that this study can serve as a template to evaluate images in the early phases of a project.

There is growing interest in the use of mammalian protein expression systems, and in the use of antibody-derived chaperones, for structural studies. Here, we describe protocols ranging from the production of recombinant membrane proteins in stable inducible cell lines to biophysical characterization of purified membrane proteins in complex with llama antibody domains. These protocols were used to solve the structure of the mouse 5-HT3 serotonin receptor but are of broad applicability for crystallization or cryo-electron microscopy projects.