In a recent issue of Nature Methods , Eluru et al. ¹ describe a microfluidic approach that integrates protein purification with cryogenic electron microscopy (cryo-EM) grid preparation to enable high-resolution single-particle cryo-EM structure determination from minute amounts of starting material.

Cryogenic electron microscopy (cryoEM) has become an indispensable technique for determining the structures of isolated biological macromolecules and imaging biomolecular structures within cells. While thin films of frozen-hydrated macromolecule suspensions can be directly prepared and imaged, these molecules must be extracted from their cellular context using existing sample preparation methods. On the other hand, cellular specimens preserve the native context, but the region of interest must be sectioned or subjected to focused ion-beam milling at cryogenic temperatures to achieve the necessary thickness for transmission electron imaging. Currently, no method exists for targeted cytoplasmic extraction of a subcellular volume from individual cells for direct vitrification and cryoEM imaging. In this study, a method is presented that addresses this gap. A system has developed that utilizes a force-sensitive microfluidic cantilever pipette to aspirate and dispense sample volumes as small as 204 fL onto conventional cryoEM sample supports, maintained at the dew point. This is followed by automatic vitrification for cryoEM imaging. Coupled with a fluorescence microscope, this setup allows for the extraction of a targeted subcellular volume from an individual cell and subsequent dispensing of the aspirated content onto an electron microscopy grid. A proof-of-concept is demonstrated by dispensing femtolitre volumes of the standard cryoEM single-particle sample, tobacco mosaic virus, and performing a subcellular biopsy from a single HeLa cell. Additionally, the challenges of manipulating such small volumes for cryoEM sample preparation are discussed, highlighting the current limitations of this approach and potential solutions for overcoming them.

Antimicrobial resistance poses a severe threat to human health and Pseudomonas aeruginosa stands out among the pathogens responsible for this emergency. The SOS response to DNA damage is crucial in bacterial evolution, influencing resistance development and adaptability in challenging environments, especially under antibiotic exposure. Recombinase A (RecA) and the transcriptional repressor LexA are the key players that orchestrate this process, determining either the silencing or the active transcription of the genes under their control. By integrating state-of-the-art structural approaches with in vitro binding and functional assays, we elucidated the molecular events activating the SOS response in P. aeruginosa, focusing on the RecA-LexA interaction. Our findings identify the conserved determinants and strength of the interactions that allow RecA to trigger LexA autocleavage and inactivation. These results provide the groundwork for designing novel antimicrobial strategies and exploring the potential translation of Escherichia coli-derived approaches, to address the implications of P. aeruginosa infections.

RNA can directly control protein activity in a process called riboregulation; only a few mechanisms of riboregulation have been described in detail, none of which have been characterized on structural grounds. Here, we present a comprehensive structural, functional, and phylogenetic analysis of riboregulation of cytosolic serine hydroxymethyltransferase (SHMT1), the enzyme interconverting serine and glycine in one-carbon metabolism. We have determined the cryoelectron microscopy (cryo-EM) structure of human SHMT1 in its free- and RNA-bound states, and we show that the RNA modulator competes with polyglutamylated folates and acts as an allosteric switch, selectively altering the enzyme's reactivity vs. serine. In addition, we identify the tetrameric assembly and a flap structural motif as key structural elements necessary for binding of RNA to eukaryotic SHMT1. The results presented here suggest that riboregulation may have played a role in evolution of eukaryotic SHMT1 and in compartmentalization of one-carbon metabolism. Our findings provide insights for RNA-based therapeutic strategies targeting this cancer-linked metabolic pathway.

Gas vesicles mediate buoyancy-based motility in aquatic bacteria and archaea and are the only protein-based structures known to enclose a gas-filled volume. Their unique physicochemical properties and ingenious architecture rank them among the most intriguing macromolecular assemblies characterised to date. This review covers the 60-year journey in quest for a high-resolution structural model of gas vesicles, first highlighting significant strides made in establishing the detailed ultrastructure of gas vesicles through transmission electron microscopy, X-ray fibre diffraction, atomic force microscopy, and NMR spectroscopy. We then survey the recent progress in cryogenic electron microscopy studies of gas vesicles, which eventually led to a comprehensive atomic model of the mature assembly. Synthesising insight from these structures, we examine possible mechanisms of gas vesicle biogenesis and growth, presenting a testable model to guide future experimental work. We conclude by discussing future directions in the structural biology of gas vesicles, particularly considering advancements in AI-driven structure prediction.

Gas vesicles are gas-filled nanocompartments that allow a diverse group of bacteria and archaea to control their buoyancy. The molecular basis of their properties and assembly remains unclear. Here, we report the 3.2 Å cryo-EM structure of the gas vesicle shell made from the structural protein GvpA that self-assembles into hollow helical cylinders closed off by cone-shaped tips. Two helical half shells connect through a characteristic arrangement of GvpA monomers, suggesting a mechanism of gas vesicle biogenesis. The fold of GvpA features a corrugated wall structure typical for force-bearing thin-walled cylinders. Small pores enable gas molecules to diffuse across the shell, while the exceptionally hydrophobic interior surface effectively repels water. Comparative structural analysis confirms the evolutionary conservation of gas vesicle assemblies and demonstrates molecular features of shell reinforcement by GvpC. Our findings will further research into gas vesicle biology and facilitate molecular engineering of gas vesicles for ultrasound imaging.

A quantitative four-dimensional scanning transmission electron microscopy (4D-STEM) imaging technique (q4STEM) for local thickness estimation across amorphous specimen such as obtained by focused ion beam (FIB)-milling of lamellae for (cryo-)TEM analysis is presented. This study is based on measuring spatially resolved diffraction patterns to obtain the angular distribution of electron scattering, or the ratio of integrated virtual dark and bright field STEM signals, and their quantitative evaluation using Monte Carlo simulations. The method is independent of signal intensity calibrations and only requires knowledge of the detector geometry, which is invariant for a given instrument. This study demonstrates that the method yields robust thickness estimates for sub-micrometer amorphous specimen using both direct detection and light conversion 2D-STEM detectors in a coincident FIB-SEM and a conventional SEM. Due to its facile implementation and minimal dose reauirements, it is anticipated that this method will find applications for in situ thickness monitoring during lamella fabrication of beam-sensitive materials.

Single molecule localization microscopy offers resolution nearly down to the molecular level with specific molecular labelling, and is thereby a promising tool for structural biology. In practice, however, the actual value to this field is limited primarily by incomplete fluorescent labelling of the structure. This missing information can be completed by merging information from many structurally identical particles in a particle fusion approach similar to cryo-EM single-particle analysis. In this paper, we present a data analysis of particle fusion results of fluorescently labelled Nup96 nucleoporins in the Nuclear Pore Complex to show that Nup96 occurs in a spatial arrangement of two rings of 8 units with two Nup96 copies per unit giving a total of 32 Nup96 copies per pore. We use Artificial Intelligence assisted modeling in Alphafold to extend the existing cryo-EM model of Nup96 to accurately pinpoint the positions of the fluorescent labels and show the accuracy of the match between fluorescent and cryo-EM data to be better than 3 nm in-plane and 5 nm out-of-plane.

Correction to: Scientific Reports, published online 16 August 2023 The original version of this Article contained an error in the upper inset of Figure 4, where the atomic model was missing. The original Figure 4 and accompanying legend appear below. (Figure presented.) Overlay of the fluorophore positions from the SMLM particle fusion data (pink) and the SNAP-tag derived from the cryo-EM data (purple). For our overall SMLM emitters (pink), the lateral distance between a unit are 9.1 nm for NR and 10.0 nm for CR. The axial distances between a unit are 2.4 nm for NR and 1.2 nm for CR. The SNAP tags (purple) have lateral distances between a unit of 11.6 nm for NR and 11.5 nm for CR as well as axial distances of 2.5 nm for NR and 2.9 nm for CR. The original Article has been corrected.

Cryogenic electron tomography (cryo-ET) combined with sub-tomogram averaging, allows in-situ visualization and structure determination of macromolecular complexes at sub-nanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in-situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident 3-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing focused ion-beam scanning electron microscope (FIB-SEM). We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope (TEM) tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

Growing microtubule ends organize end-tracking proteins into comets of mixed composition. Here using a reconstituted fission yeast system consisting of end-binding protein Mal3, kinesin Tea2 and cargo Tip1, we found that these proteins can be driven into liquid-phase droplets both in solution and at microtubule ends under crowding conditions. In the absence of crowding agents, cryo-electron tomography revealed that motor-dependent comets consist of disordered networks where multivalent interactions may facilitate non-stoichiometric accumulation of cargo Tip1. We found that two disordered protein regions in Mal3 are required for the formation of droplets and motor-dependent accumulation of Tip1, while autonomous Mal3 comet formation requires only one of them. Using theoretical modelling, we explore possible mechanisms by which motor activity and multivalent interactions may lead to the observed enrichment of Tip1 at microtubule ends. We conclude that microtubule ends may act as platforms where multivalent interactions condense microtubule-associated proteins into large multi-protein complexes.

Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.

Resolution-dependent loss of contrast in cryo-EM maps may obscure features at high resolution that are critical for map interpretation. Post-processing of cryo-EM maps can improve the interpretability by adjusting the resolution-dependence of structure factor amplitudes through map sharpening. Traditionally this has been done by rescaling the relative contribution of low and high-resolution frequencies globally. More recently, the realisation that molecular motion and heterogeneity cause non-uniformity of resolution throughout the map has inspired the development of techniques that optimise sharpening locally. We previously developed LocScale, a method that utilises the radial structure factor from a refined atomic model as a restraint for local map sharpening. While this method has proved beneficial for the interpretation of cryo-EM maps, the dependence on the availability of (partial) model information limits its general applicability. Here, we review the basic assumptions of resolution-dependent contrast loss in cryo-EM maps and propose a route towards a robust alternative for local map sharpening that utilises information on expected scattering properties of biological macromolecules, but requires no detailed knowledge of the underlying molecular structure. We examine remaining challenges for implementation and discuss possible applications.

Transformative technological advances have propelled cryogenic electron microscopy (cryo-EM) to take center stage in elucidating the intricacies of the nanoscale molecular machinery of viruses, bacteria and eukaryotic cells. Continued developments hold exciting promise for structural biophysicists to move closer to their dream of visualising atomic resolution snapshots of individual molecules at work in their native cellular environment.