Accurate magnification determination for cryoEM using gold.

Determining the correct magnified pixel size of single-particle cryoEM micrographs is necessary to maximize resolution and enable accurate model building. Here we describe a simple and rapid procedure for determining the absolute magnification in an electron cryomicroscope to a precision of <0.5%. We show how to use the atomic lattice spacings of crystals of thin and readily available test specimens, such as gold, as an absolute reference to determine magnification for both room temperature and cryogenic imaging. We compare this method to other commonly used methods, and show that it provides comparable accuracy in spite of its simplicity. This magnification calibration method provides a definitive reference quantity for data analysis and processing, simplifies the combination of multiple datasets from different microscopes and detectors, and improves the accuracy with which the contrast transfer function of the microscope can be determined. We also provide an open source program, magCalEM, which can be used to accurately estimate the magnified pixel size of a cryoEM dataset ex post facto.

Structure determination by cryoEM at 100 keV.

Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems.

Cryomicroscopy in situ: what is the smallest molecule that can be directly identified without labels in a cell?

Electron cryomicroscopy (cryoEM) has made great strides in the last decade, such that the atomic structure of most biological macromolecules can, at least in principle, be determined. Major technological advances - in electron imaging hardware, data analysis software, and cryogenic specimen preparation technology - continue at pace and contribute to the exponential growth in the number of atomic structures determined by cryoEM. It is now conceivable that within the next decade we will have structures for hundreds of thousands of unique protein and nucleic acid molecular complexes. But the answers to many important questions in biology would become obvious if we could identify these structures precisely inside cells with quantifiable error. In the context of an abundance of known structures, it is appropriate to consider the current state of electron cryomicroscopy for frozen specimens prepared directly from cells, and try to answer to the question of the title, both now and in the foreseeable future.

On the reduction in the effects of radiation damage to two-dimensional crystals of organic and biological molecules at liquid-helium temperature.

We have studied the fading of electron diffraction spots from two-dimensional (2D) crystals of paraffin (C44H90), purple membrane (bacteriorhodopsin) and aquaporin 4 (AQP4) at stage temperatures between 4K and 100K. We observed that the diffraction spots at resolutions between 3 Å and 20 Å fade more slowly at liquid-helium temperatures compared to liquid-nitrogen temperatures, by a factor of between 1.2 and 1.8, depending on the specimens. If the reduction in the effective rate of radiation damage for 2D crystals at liquid-helium temperature (as measured by spot fading) can be shown to extend to macromolecular assemblies embedded in amorphous ice, this would suggest that valuable improvements to electron cryomicroscopy (cryoEM) of biological specimens could be made by reducing the temperature of the specimens under irradiation below what is obtainable using standard liquid-nitrogen cryostats.

Differential assembly diversifies GABAA receptor structures and signalling.

Type A γ-aminobutyric acid receptors (GABAARs) are pentameric ligand-gated chloride channels that mediate fast inhibitory signalling in neural circuits1,2 and can be modulated by essential medicines including general anaesthetics and benzodiazepines3. Human GABAAR subunits are encoded by 19 paralogous genes that can, in theory, give rise to 495,235 receptor types. However, the principles that govern the formation of pentamers, the permutational landscape of receptors that may emerge from a subunit set and the effect that this has on GABAergic signalling remain largely unknown. Here we use cryogenic electron microscopy to determine the structures of extrasynaptic GABAARs assembled from α4, ß3 and δ subunits, and their counterparts incorporating γ2 instead of δ subunits. In each case, we identified two receptor subtypes with distinct stoichiometries and arrangements, all four differing from those previously observed for synaptic, α1-containing receptors4-7. This, in turn, affects receptor responses to physiological and synthetic modulators by creating or eliminating ligand-binding sites at subunit interfaces. We provide structural and functional evidence that selected GABAAR arrangements can act as coincidence detectors, simultaneously responding to two neurotransmitters: GABA and histamine. Using assembly simulations and single-cell RNA sequencing data8,9, we calculated the upper bounds for receptor diversity in recombinant systems and in vivo. We propose that differential assembly is a pervasive mechanism for regulating the physiology and pharmacology of GABAARs.

2.4-Å structure of the double-ring Gemmatimonas phototrophica photosystem.

Phototrophic Gemmatimonadetes evolved the ability to use solar energy following horizontal transfer of photosynthesis-related genes from an ancient phototrophic proteobacterium. The electron cryo-microscopy structure of the Gemmatimonas phototrophica photosystem at 2.4 Å reveals a unique, double-ring complex. Two unique membrane-extrinsic polypeptides, RC-S and RC-U, hold the central type 2 reaction center (RC) within an inner 16-subunit light-harvesting 1 (LH1) ring, which is encircled by an outer 24-subunit antenna ring (LHh) that adds light-gathering capacity. Femtosecond kinetics reveal the flow of energy within the RC-dLH complex, from the outer LHh ring to LH1 and then to the RC. This structural and functional study shows that G. phototrophica has independently evolved its own compact, robust, and highly effective architecture for harvesting and trapping solar energy.

Integrated wafer-scale manufacturing of electron cryomicroscopy specimen supports.

We present a process for the manufacture of electron cryomicroscopy (cryoEM) specimen supports with an integrated foil-grid structure, using cryogenic vacuum evaporation (cryoEvap) and patterned electroplating on a silicon wafer substrate. The process is designed to produce a pattern of nanometre scale holes in a thin metal foil, which is attached to a pattern of micrometre scale grid bars that support it and allow handling of the millimetre scale device. All steps are carried out on a single 4 inch (100 mm) silicon wafer, without any need to handle individual grids during processing, and yield about 600 supports per wafer. The approach is generally applicable to the problem of creating a thin foil with nanometre scale features and a micrometre scale support structure; here it is used to make an all gold, HexAuFoil type design. It also allows for the addition of custom fiducial markers and patterns which aid in locating and identifying particular regions of a grid at several length scales: by eye, in an optical microscope, and in the electron microscope. Implemented at scale, this manufacturing process can supply ample grids to support the continued growth of cryoEM for determining the structure of biological molecules.

Structure of the SARS-CoV-2 RNA-dependent RNA polymerase in the presence of favipiravir-RTP.

The RNA polymerase inhibitor favipiravir is currently in clinical trials as a treatment for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 Å. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, nonproductive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp, which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.

The 2.4 Å cryo-EM structure of a heptameric light-harvesting 2 complex reveals two carotenoid energy transfer pathways.

We report the 2.4 Ångström resolution structure of the light-harvesting 2 (LH2) complex from Marichromatium (Mch.) purpuratum determined by cryogenic electron microscopy. The structure contains a heptameric ring that is unique among all known LH2 structures, explaining the unusual spectroscopic properties of this bacterial antenna complex. We identify two sets of distinct carotenoids in the structure and describe a network of energy transfer pathways from the carotenoids to bacteriochlorophyll a molecules. The geometry imposed by the heptameric ring controls the resonant coupling of the long-wavelength energy absorption band. Together, these details reveal key aspects of the assembly and oligomeric form of purple bacterial LH2 complexes that were previously inaccessible by any technique.

Cryo-EM with sub-1 Å specimen movement.

Most information loss in cryogenic electron microscopy (cryo-EM) stems from particle movement during imaging, which remains poorly understood. We show that this movement is caused by buckling and subsequent deformation of the suspended ice, with a threshold that depends directly on the shape of the frozen water layer set by the support foil. We describe a specimen support design that eliminates buckling and reduces electron beam-induced particle movement to less than 1 angstrom. The design allows precise foil tracking during imaging with high-speed detectors, thereby lessening demands on cryostage precision and stability. It includes a maximal density of holes, which increases throughput in automated cryo-EM without degrading data quality. Movement-free imaging allows extrapolation to a three-dimensional map of the specimen at zero electron exposure, before the onset of radiation damage.

Multifunctional graphene supports for electron cryomicroscopy.

With recent technological advances, the atomic resolution structure of any purified biomolecular complex can, in principle, be determined by single-particle electron cryomicroscopy (cryoEM). In practice, the primary barrier to structure determination is the preparation of a frozen specimen suitable for high-resolution imaging. To address this, we present a multifunctional specimen support for cryoEM, comprising large-crystal monolayer graphene suspended across the surface of an ultrastable gold specimen support. Using a low-energy plasma surface modification system, we tune the surface of this support to the specimen by patterning a range of covalent functionalizations across the graphene layer on a single grid. This support design reduces specimen movement during imaging, improves image quality, and allows high-resolution structure determination with a minimum of material and data.

Measuring the effects of particle orientation to improve the efficiency of electron cryomicroscopy.

The orientation distribution of a single-particle electron cryomicroscopy specimen limits the resolution of the reconstructed density map. Here we define a statistical quantity, the efficiency, E od, which characterises the orientation distribution via its corresponding point spread function. The efficiency measures the ability of the distribution to provide uniform information and resolution in all directions of the reconstruction, independent of other factors. This metric allows rapid and rigorous evaluation of specimen preparation methods, assisting structure determination to high resolution with minimal data.A number of parameters influence the resolution of a cryo-EM structure. Here the authors investigate the effects of specimen orientation in single particle cryo-EM and present open-source software for rapidly assessing orientation distributions to improve data collection.