Smart data collection for CryoEM.

This report provides an overview of the discussions, presentations, and consensus thinking from the Workshop on Smart Data Collection for CryoEM held at the New York Structural Biology Center on April 6-7, 2022. The goal of the workshop was to address next generation data collection strategies that integrate machine learning and real-time processing into the workflow to reduce or eliminate the need for operator intervention.

Montage electron tomography of vitrified specimens.

Cryo-electron tomography provides detailed views of macromolecules in situ. However, imaging a large field of view to provide more cellular context requires reducing magnification during data collection, which in turn restricts the resolution. To circumvent this trade-off between field of view and resolution, we have developed a montage data collection scheme that uniformly distributes the dose throughout the specimen. In this approach, sets of slightly overlapping circular tiles are collected at high magnification and stitched to form a composite projection image at each tilt angle. These montage tilt-series are then reconstructed into massive tomograms with a small pixel size but a large field of view. For proof-of-principle, we applied this method to the thin edge of HeLa cells. Thon rings to better than 10 Å were detected in the montaged tilt-series, and diverse cellular features were observed in the resulting tomograms. These results indicate that the additional dose required by this technique is not prohibitive to performing structural analysis to intermediate resolution across a large field of view. We anticipate that montage tomography will prove particularly useful for lamellae, increase the likelihood of imaging rare cellular events, and facilitate visual proteomics.

Ensemble Learning-Based Pulse Signal Recognition: Classification Model Development Study.

BACKGROUND: In pulse signal analysis and identification, time domain and time frequency domain analysis methods can obtain interpretable structured data and build classification models using traditional machine learning methods. Unstructured data, such as pulse signals, contain rich information about the state of the cardiovascular system, and local features of unstructured data can be extracted and classified using deep learning. OBJECTIVE: The objective of this paper was to comprehensively use machine learning and deep learning classification methods to fully exploit the information about pulse signals. METHODS: Structured data were obtained by using time domain and time frequency domain analysis methods. A classification model was built using a support vector machine (SVM), a deep convolutional neural network (DCNN) kernel was used to extract local features of the unstructured data, and the stacking method was used to fuse the above classification results for decision making. RESULTS: The highest average accuracy of 0.7914 was obtained using only a single classifier, while the average accuracy obtained using the ensemble learning approach was 0.8330. CONCLUSIONS: Ensemble learning can effectively use information from structured and unstructured data to improve classification accuracy through decision-level fusion. This study provides a new idea and method for pulse signal classification, which is of practical value for pulse diagnosis objectification.

Rapid tilt-series method for cryo-electron tomography: Characterizing stage behavior during FISE acquisition.

We and others recently developed rapid tilt-series acquisition methods for cryo-electron tomography on a Titan Krios G3i equipped with a single axis holder and a K-series direct electron detector and showed that one of these, the fast-incremental single exposure (FISE) method, significantly accelerates tilt-series acquisition when compared to traditional methods while preserving the quality of the images. Here, we characterize the behavior of our single axis holder in detail during a FISE experiment to optimally balance data quality with speed. We explain our methodology in detail so others can characterize their own stages, and conclude with recommendations for projects with different resolution goals.

Measuring gas vesicle dimensions by electron microscopy.

Gas vesicles (GVs) are cylindrical or spindle-shaped protein nanostructures filled with air and used for flotation by various cyanobacteria, heterotrophic bacteria, and Archaea. Recently, GVs have gained interest in biotechnology applications due to their ability to serve as imaging agents and actuators for ultrasound, magnetic resonance and several optical techniques. The diameter of GVs is a crucial parameter contributing to their mechanical stability, buoyancy function and evolution in host cells, as well as their properties in imaging applications. Despite its importance, reported diameters for the same types of GV differ depending on the method used for its assessment. Here, we provide an explanation for these discrepancies and utilize electron microscopy (EM) techniques to accurately estimate the diameter of the most commonly studied types of GVs. We show that during air drying on the EM grid, GVs flatten, leading to a ~1.5-fold increase in their apparent diameter. We demonstrate that GVs' diameter can be accurately determined by direct measurements from cryo-EM samples or alternatively indirectly derived from widths of flat collapsed and negatively stained GVs. Our findings help explain the inconsistency in previously reported data and provide accurate methods to measure GVs dimensions.

Repurposing a chemosensory macromolecular machine.

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identify ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we trace the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost.

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.

Electron cryotomography of Mycoplasma pneumoniae mutants correlates terminal organelle architectural features and function.

The Mycoplasma pneumoniae terminal organelle functions in adherence and gliding motility and is comprised of at least eleven substructures. We used electron cryotomography to correlate impaired gliding and adherence function with changes in architecture in diverse terminal organelle mutants. All eleven substructures were accounted for in the prkC, prpC and P200 mutants, and variably so for the HMW3 mutant. Conversely, no terminal organelle substructures were evident in HMW1 and HMW2 mutants. The P41 mutant exhibits a terminal organelle detachment phenotype and lacked the bowl element normally present at the terminal organelle base. Complementation restored this substructure, establishing P41 as either a component of the bowl element or required for its assembly or stability, and that this bowl element is essential to anchor the terminal organelle but not for leverage in gliding. Mutants II-3, III-4 and topJ exhibited a visibly lower density of protein knobs on the terminal organelle surface. Mutants II-3 and III-4 lack accessory proteins required for a functional adhesin complex, while the topJ mutant lacks a DnaJ-like co-chaperone essential for its assembly. Taken together, these observations expand our understanding of the roles of certain terminal organelle proteins in the architecture and function of this complex structure.

Best practices for managing large CryoEM facilities.

This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.

Uncharacterized Bacterial Structures Revealed by Electron Cryotomography.

Electron cryotomography (ECT) can reveal the native structure and arrangement of macromolecular complexes inside intact cells. This technique has greatly advanced our understanding of the ultrastructure of bacterial cells. We now view bacteria as structurally complex assemblies of macromolecular machines rather than as undifferentiated bags of enzymes. To date, our group has applied ECT to nearly 90 different bacterial species, collecting more than 15,000 cryotomograms. In addition to known structures, we have observed, to our knowledge, several uncharacterized features in these tomograms. Some are completely novel structures; others expand the features or species range of known structure types. Here, we present a survey of these uncharacterized bacterial structures in the hopes of accelerating their identification and study, and furthering our understanding of the structural complexity of bacterial cells.IMPORTANCE Bacteria are more structurally complex than is commonly appreciated. Here we present a survey of previously uncharacterized structures that we observed in bacterial cells by electron cryotomography, structures that will initiate new lines of research investigating their identities and roles.

Broadly Neutralizing Antibody 8ANC195 Recognizes Closed and Open States of HIV-1 Env.

The HIV-1 envelope (Env) spike contains limited epitopes for broadly neutralizing antibodies (bNAbs); thus, most neutralizing antibodies are strain specific. The 8ANC195 epitope, defined by crystal and electron microscopy (EM) structures of bNAb 8ANC195 complexed with monomeric gp120 and trimeric Env, respectively, spans the gp120 and gp41 Env subunits. To investigate 8ANC195's gp41 epitope at higher resolution, we solved a 3.58 Å crystal structure of 8ANC195 complexed with fully glycosylated Env trimer, revealing 8ANC195 insertion into a glycan shield gap to contact gp120 and gp41 glycans and protein residues. To determine whether 8ANC195 recognizes the CD4-bound open Env conformation that leads to co-receptor binding and fusion, one of several known conformations of virion-associated Env, we solved EM structures of an Env/CD4/CD4-induced antibody/8ANC195 complex. 8ANC195 binding partially closed the CD4-bound trimer, confirming structural plasticity of Env by revealing a previously unseen conformation. 8ANC195's ability to bind different Env conformations suggests advantages for potential therapeutic applications.

Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography.

Cryo-electron tomography (CET) produces three-dimensional images of cells in a near-native state at macromolecular resolution, but identifying structures of interest can be challenging. Here we describe a correlated cryo-PALM (photoactivated localization microscopy)-CET method for localizing objects within cryo-tomograms to beyond the diffraction limit of the light microscope. Using cryo-PALM-CET, we identified multiple and new conformations of the dynamic type VI secretion system in the crowded interior of Myxococcus xanthus.

Structural diversity of bacterial flagellar motors.

The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species.

Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria.

How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (> 80 nm) helical filaments exist near or along either surface of the inner membrane. We also use correlated cryo-fluorescent light microscopy (cryo-fLM) and electron cryo-tomography (ECT) to identify cytoplasmic bundles of MreB, showing that MreB filaments are detectable by ECT. In light of these results, the structure and function of MreB must be reconsidered: instead of acting as a large, rigid scaffold that localizes cell-wall synthetic machinery, moving MreB complexes may apply tension to growing peptidoglycan strands to ensure their orderly, linear insertion.

Correlated light and electron cryo-microscopy.

Light and electron cryo-microscopy have each proven to be powerful tools to study biological structures in a near-native state. Light microscopy provides important localization information, while electron microscopy provides the resolution necessary to resolve fine structural details. Imaging the same sample by both light and electron cryo-microscopy is a powerful new approach that combines the strengths of both techniques to provide novel insights into cellular ultrastructure. In this chapter, the methods and instrumentation currently used to correlate light and electron cryo-microscopy are described in detail.

Electron cryotomography of bacterial cells.

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy. Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (approximately 4nm).(1, 2) In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (< -180 degrees C). For slender cells (up to approximately 500 nm in thickness(3)), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.(1, 2) In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.

Universal architecture of bacterial chemoreceptor arrays.

Chemoreceptors are key components of the high-performance signal transduction system that controls bacterial chemotaxis. Chemoreceptors are typically localized in a cluster at the cell pole, where interactions among the receptors in the cluster are thought to contribute to the high sensitivity, wide dynamic range, and precise adaptation of the signaling system. Previous structural and genomic studies have produced conflicting models, however, for the arrangement of the chemoreceptors in the clusters. Using whole-cell electron cryo-tomography, here we show that chemoreceptors of different classes and in many different species representing several major bacterial phyla are all arranged into a highly conserved, 12-nm hexagonal array consistent with the proposed "trimer of dimers" organization. The various observed lengths of the receptors confirm current models for the methylation, flexible bundle, signaling, and linker sub-domains in vivo. Our results suggest that the basic mechanism and function of receptor clustering is universal among bacterial species and was thus conserved during evolution.

Molecular organization of Gram-negative peptidoglycan.

The stress-bearing component of the bacterial cell wall--a multi-gigadalton bag-like molecule called the sacculus--is synthesized from peptidoglycan. Whereas the chemical composition and the 3-dimensional structure of the peptidoglycan subunit (in at least one conformation) are known, the architecture of the assembled sacculus is not. Four decades' worth of biochemical and electron microscopy experiments have resulted in two leading 3-D peptidoglycan models: "Layered" and "Scaffold", in which the glycan strands are parallel and perpendicular to the cell surface, respectively. Here we resolved the basic architecture of purified, frozen-hydrated sacculi through electron cryotomography. In the Gram-negative sacculus, a single layer of glycans lie parallel to the cell surface, roughly perpendicular to the long axis of the cell, encircling the cell in a disorganized hoop-like fashion.

Atomic-scale dynamical structures of fatty acid bilayers observed by ultrafast electron crystallography.

The structure and dynamics of a biological model bilayer are reported with atomic-scale resolution by using ultrafast electron crystallography. The bilayer was deposited as a Langmuir-Blodgett structure of arachidic (eicosanoic) fatty acids with the two chains containing 40 carbon atoms (approximately = 50 angstroms), on a hydrophobic substrate, the hydrogen terminated silicon(111) surface. We determined the structure of the 2D assembly, establishing the orientation of the chains and the subunit cell of the CH2 distances: a0 = 4.7 angstroms, b0 = 8.0 angstroms, and c0 = 2.54 angstroms. For structural dynamics, the diffraction frames were taken every 1 picosecond after a femtosecond temperature jump. The observed motions, with sub-angstroms resolution and monolayer sensitivity, clearly indicate the coherent anisotropic expansion of the bilayer solely along the aliphatic chains, followed by nonequilibrium contraction and restructuring at longer times. This motion is indicative of a nonlinear behavior among the anharmonically coupled bonds on the ultrashort time scale and energy redistribution and diffusion on the longer time scale. The ability to observe such atomic motions of complex structures and at interfaces is a significant leap forward for the determination of macromolecular dynamical structures by using ultrafast electron crystallography.