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.

Structure of the Fanconi anaemia monoubiquitin ligase complex.

The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress1,2. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2-FANCI4,5 by the FA core complex-a megadalton multiprotein E3 ubiquitin ligase6,7. Monoubiquitinated FANCD2 then recruits additional protein factors to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used cryo-electron microscopy and mass spectrometry to determine its structure. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Thus, the structure provides a mechanistic basis for the low numbers of patients with mutations in FANCB, FANCL and FAAP100. Despite a lack of sequence homology, FANCB and FAAP100 adopt similar structures. The two FANCL subunits are in different conformations at opposite ends of the complex, suggesting that each FANCL has a distinct role. This structural and functional asymmetry of dimeric RING finger domains may be a general feature of E3 ligases. The cryo-electron microscopy structure of the FA core complex provides a foundation for a detailed understanding of its E3 ubiquitin ligase activity and DNA interstrand crosslink repair.

Honeycomb gold specimen supports enabling orthogonal focussed ion beam-milling of elongated cells for cryo-ET.

Cryo-focussed ion beam (FIB)-milling is a powerful technique that opens up thick, cellular specimens to high-resolution structural analysis by electron cryotomography (cryo-ET). FIB-milled lamellae can be produced from cells on grids, or cut from thicker, high-pressure frozen specimens. However, these approaches can put geometrical constraints on the specimen that may be unhelpful, particularly when imaging structures within the cell that have a very defined orientation. For example, plunge frozen rod-shaped bacteria orient parallel to the plane of the grid, yet the Z-ring, a filamentous structure of the tubulin-like protein FtsZ and the key organiser of bacterial division, runs around the circumference of the cell such that it is perpendicular to the imaging plane. It is therefore difficult or impractical to image many complete rings with current technologies. To circumvent this problem, we have fabricated monolithic gold specimen supports with a regular array of cylindrical wells in a honeycomb geometry, which trap bacteria in a vertical orientation. These supports, which we call "honeycomb gold discs", replace standard EM grids and when combined with FIB-milling enable the production of lamellae containing cross-sections through cells. The resulting lamellae are more stable and resistant to breakage and charging than conventional lamellae. The design of the honeycomb discs can be modified according to need and so will also enable cryo-ET and cryo-EM imaging of other specimens in otherwise difficult to obtain orientations.

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.

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.

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.

Structural insights into the bacterial carbon-phosphorus lyase machinery.

Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolizes phosphonates remain unknown. Here we determine the crystal structure of the 240-kilodalton Escherichia coli C-P lyase core complex (PhnG-PhnH-PhnI-PhnJ; PhnGHIJ), and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that probably couple phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy, and show that it binds to a conserved insertion domain of PhnJ. Our results provide a structural basis for understanding microbial phosphonate breakdown.

Controlling protein adsorption on graphene for cryo-EM using low-energy hydrogen plasmas.

Despite its many favorable properties as a sample support for biological electron microscopy, graphene is not widely used because its hydrophobicity precludes reliable protein deposition. We describe a method to modify graphene with a low-energy hydrogen plasma, which reduces hydrophobicity without degrading the graphene lattice. Use of plasma-treated graphene enables better control of protein distribution in ice for electron cryo-microscopy and improves image quality by reducing radiation-induced sample motion.

Ultrastable gold substrates: Properties of a support for high-resolution electron cryomicroscopy of biological specimens.

Electron cryomicroscopy (cryo-EM) allows structure determination of a wide range of biological molecules and specimens. All-gold supports improve cryo-EM images by reducing radiation-induced motion and image blurring. Here we compare the mechanical and electrical properties of all-gold supports to amorphous carbon foils. Gold supports are more conductive, and have suspended foils that are not compressed by differential contraction when cooled to liquid nitrogen temperatures. These measurements show how the choice of support material and geometry can reduce specimen movement by more than an order of magnitude during low-dose imaging. We provide methods for fabrication of all-gold supports and preparation of vitrified specimens. We also analyse illumination geometry for optimal collection of high resolution, low-dose data. Together, the support structures and methods herein can improve the resolution and quality of images from any electron cryomicroscope.

Atom-by-atom nucleation and growth of graphene nanopores.

Graphene is an ideal thin membrane substrate for creating molecule-scale devices. Here we demonstrate a scalable method for creating extremely small structures in graphene with atomic precision. It consists of inducing defect nucleation centers with energetic ions, followed by edge-selective electron recoil sputtering. As a first application, we create graphene nanopores with radii as small as 3 Å, which corresponds to 10 atoms removed. We observe carbon atom removal from the nanopore edge in situ using an aberration-corrected electron microscope, measure the cross-section for the process, and obtain a mean edge atom displacement energy of 14.1 ± 0.1 eV. This approach does not require focused beams and allows scalable production of single nanopores and arrays of monodisperse nanopores for atomic-scale selectively permeable membranes.

Robust evaluation of 3D electron cryomicroscopy data using tilt-pairs.

Determining the structure of a protein complex using electron microscopy requires the calculation of a 3D density map from 2D images of single particles. Since the individual images are taken at low electron dose to avoid radiation damage, they are noisy and difficult to align with each other. This can result in incorrect maps, making validation essential. Pairs of electron micrographs taken at known angles to each other (tilt-pairs) can be used to measure the accuracy of assigned projection orientations and verify the soundness of calculated maps. Here we establish a statistical framework for evaluating images and density maps using tilt-pairs. The directional distribution of such angular data is modelled using a Fisher distribution on the unit sphere. This provides a simple, quantitative and easily comparable metric, the concentration parameter κ, for evaluating the quality of datasets and density maps that is independent of the data collection and analysis methods. A large κ is indicative of good agreement between the particle images and the 3D density map. For structure validation, we recommend κ>10 and a p-value <0.01. The statistical framework herein allows one to objectively answer the question: Is a reconstructed density map correct within a particular confidence interval?

Impact of brain tissue filtering on neurostimulation fields: a modeling study.

Electrical neurostimulation techniques, such as deep brain stimulation (DBS) and transcranial magnetic stimulation (TMS), are increasingly used in the neurosciences, e.g., for studying brain function, and for neurotherapeutics, e.g., for treating depression, epilepsy, and Parkinson's disease. The characterization of electrical properties of brain tissue has guided our fundamental understanding and application of these methods, from electrophysiologic theory to clinical dosing-metrics. Nonetheless, prior computational models have primarily relied on ex-vivo impedance measurements. We recorded the in-vivo impedances of brain tissues during neurosurgical procedures and used these results to construct MRI guided computational models of TMS and DBS neurostimulatory fields and conductance-based models of neurons exposed to stimulation. We demonstrated that tissues carry neurostimulation currents through frequency dependent resistive and capacitive properties not typically accounted for by past neurostimulation modeling work. We show that these fundamental brain tissue properties can have significant effects on the neurostimulatory-fields (capacitive and resistive current composition and spatial/temporal dynamics) and neural responses (stimulation threshold, ionic currents, and membrane dynamics). These findings highlight the importance of tissue impedance properties on neurostimulation and impact our understanding of the biological mechanisms and technological potential of neurostimulatory methods.

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.

Topical anesthetic cream is associated with spontaneous cutaneous abscess drainage in children.

OBJECTIVE: The objective of the study was to determine whether use of topical anesthetic cream increases spontaneous drainage of skin abscesses and reduces the need for procedural sedation. METHODS: A retrospective multicenter cohort study from 3 academic pediatric emergency departments was conducted for randomly selected children with a cutaneous abscess in 2007. Children up to 18 years of age were eligible if they had a skin abscess at presentation. Demographics, abscess characteristics, and use of a topical analgesic were obtained from medical records. RESULTS: Of 300 subjects, 58% were female and the median age was 7.8 years (interquartile range, 2-15 years). Mean abscess size was 3.5 ± 2.4 cm, most commonly located on the lower extremity (30%), buttocks (24%), and face (12%). A drainage procedure was required in 178 children, of whom 9 underwent drainage in the operating room. Of the remaining 169 children who underwent emergency department-based drainage, 110 (65%) had a topical anesthetic agent with an occlusive dressing placed on their abscess before drainage. Use of a topical anesthetic resulted in spontaneous abscess drainage in 26 patients, of whom 3 no longer required any further intervention. In the 166 patients who underwent additional manipulation, procedural sedation was required in 26 (24%) of those who had application of a topical anesthetic and in 24 (41%) of those who had no topical anesthetic (odds ratio, 0.45; 95% confidence interval, 0.23-0.89). CONCLUSIONS: Topical anesthetic cream application before drainage procedures promotes spontaneous drainage and decreases the need for procedural sedation for pediatric cutaneous abscess patients.

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.

Phase contrast imaging with inelastically scattered electrons from any layer of a thick specimen.

A controversy exists as to whether the signal in a high resolution phase contrast electron micrograph of a particle in a thick specimen is the same irrespective of the particle's position along the beam axis. Different conceptions of inelastic scattering and its effects on wave interference have led to radically different expectations about the degree of phase contrast vs. depth. Here we examine the information available from bright field phase contrast images of small crystalline particles on the top or bottom of a thick support. The support is an aluminium foil which has strong plasmon resonances that cause a large proportion of the electron beam to lose energy in transit. Phase contrast micrographs of the atomic lattice of two ensembles of platinum particles were measured in an energy loss window corresponding to the first plasmon resonance. The signal measured for particles on top was equal to that for particles on the bottom of the foil to within a 99% confidence interval, and the measurements exclude other models of depth dependent phase contrast in the literature to >5σ. These observations are consistent with quantum theory which considers dynamical effects as independent of event sequence and is distinct from the "top-bottom effect" observed in amplitude contrast. We thus confirm that phase contrast using inelastically scattered electrons can be obtained equally well from particles within any layer of a thick specimen.

Imaging biological macromolecules in thick specimens: The role of inelastic scattering in cryoEM.

We investigate potential improvements in using electron cryomicroscopy to image thick specimens with high-resolution phase contrast imaging. In particular, using model experiments, electron scattering theory, Monte Carlo and multislice simulations, we determine the potential for improving electron cryomicrographs of proteins within a cell using chromatic aberration (Cc) correction. We show that inelastically scattered electrons lose a quantifiable amount of spatial coherence as they transit the specimen, yet can be used to enhance the signal from thick biological specimens (in the 1000 to 5000 Å range) provided they are imaged close to focus with an achromatic lens. This loss of information quantified here, which we call "specimen induced decoherence", is a fundamental limit on imaging biological molecules in situ. We further show that with foreseeable advances in transmission electron microscope technology, it should be possible to directly locate and uniquely identify sub-100 kDa proteins without the need for labels, in a vitrified specimen taken from a cell.