Myosin forces elicit an F-actin structural landscape that mediates mechanosensitive protein recognition.

Cells mechanically interface with their surroundings through cytoskeleton-linked adhesions, allowing them to sense physical cues that instruct development and drive diseases such as cancer. Contractile forces generated by myosin motor proteins mediate these mechanical signal transduction processes through unclear protein structural mechanisms. Here, we show that myosin forces elicit structural changes in actin filaments (F-actin) that modulate binding by the mechanosensitive adhesion protein α-catenin.

CryoCycle your grids: Plunge vitrifying and reusing clipped grids to advance cryoEM democratization.

CryoEM democratization is hampered by access to costly plunge-freezing supplies. We introduce methods, called CryoCycle, for reliably blotting, vitrifying, and reusing clipped cryoEM grids. We demonstrate that vitreous ice may be produced by plunging clipped grids with purified proteins into liquid ethane and that clipped grids may be reused several times for different protein samples.

Cryo-focused ion beam for in situ structural biology: State of the art, challenges, and perspectives.

Cryogenic-focused ion beam (cryo-FIB) instruments became essential for high-resolution imaging in cryo-preserved cells and tissues. Cryo-FIBs use accelerated ions to thin samples that would otherwise be too thick for cryo-electron microscopy (cryo-EM). This allows visualizing cellular ultrastructures in near-native frozen hydrated states.

CryoCycle your grids: Plunge vitrifying and reusing clipped grids to advance cryoEM democratization.

CryoEM democratization is hampered by access to costly plunge-freezing supplies. We introduce methods, called CryoCycle, for reliably blotting, vitrifying, and reusing clipped cryoEM grids. We demonstrate that vitreous ice may be produced by plunging clipped grids with purified proteins into liquid ethane and that clipped grids may be reused several times for different protein samples.

Cryo-Electron Microscopy Screening Automation across Multiple Grids using Smart Leginon.

Advancements in cryo-electron microscopy (cryoEM) techniques over the past decade have allowed structural biologists to routinely resolve macromolecular protein complexes to near-atomic resolution. The general workflow of the entire cryoEM pipeline involves iterating between sample preparation, cryoEM grid preparation, and sample/grid screening before moving on to high-resolution data collection. Iterating between sample/grid preparation and screening is typically a major bottleneck for researchers, as every iterative experiment must optimize for sample concentration, buffer conditions, grid material, grid hole size, ice thickness, and protein particle behavior in the ice, amongst other variables.

Heterogeneous non-canonical nucleosomes predominate in yeast cells .

Nuclear processes depend on the organization of chromatin, whose basic units are cylinder-shaped complexes called nucleosomes. A subset of mammalian nucleosomes (inside cells) resembles the canonical structure determined 25 years ago. Nucleosome structure is otherwise poorly understood.

In situ cryo-FIB/SEM Specimen Preparation Using the Waffle Method.

Cryo-focused ion beam (FIB) milling of vitrified specimens is emerging as a powerful method for in situ specimen preparation. It allows for the preservation of native and near-native conditions in cells, and can reveal the molecular structure of protein complexes when combined with cryo-electron tomography (cryo-ET) and sub-tomogram averaging. Cryo-FIB milling is often performed on plunge-frozen specimens of limited thickness.

Learning to automate cryo-electron microscopy data collection with Ptolemy.

Over the past decade, cryo-electron microscopy (cryoEM) has emerged as an important method for determining near-native, near-atomic resolution 3D structures of biological macromolecules. To meet the increasing demand for cryoEM, automated methods that improve throughput and efficiency of microscope operation are needed. Currently, the targeting algorithms provided by most data-collection software require time-consuming manual tuning of parameters for each grid, and, in some cases, operators must select targets completely manually.

Fully automated multi-grid cryoEM screening using Smart Leginon.

Single-particle cryo-electron microscopy (cryoEM) is a swiftly growing method for understanding protein structure. With increasing demand for high-throughput, high-resolution cryoEM services comes greater demand for rapid and automated cryoEM grid and sample screening. During screening, optimal grids and sample conditions are identified for subsequent high-resolution data collection.

Architecture of the human erythrocyte ankyrin-1 complex.

The stability and shape of the erythrocyte membrane is provided by the ankyrin-1 complex, but how it tethers the spectrin-actin cytoskeleton to the lipid bilayer and the nature of its association with the band 3 anion exchanger and the Rhesus glycoproteins remains unknown. Here we present structures of ankyrin-1 complexes purified from human erythrocytes. We reveal the architecture of a core complex of ankyrin-1, the Rhesus proteins RhAG and RhCE, the band 3 anion exchanger, protein 4.

Waffle Method: A general and flexible approach for improving throughput in FIB-milling.

Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens with vitrification issues, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and specimens that distribute poorly across grid squares. Here we present a general approach called the 'Waffle Method' which leverages high-pressure freezing to address these challenges.

Structural basis for the mechanisms of human presequence protease conformational switch and substrate recognition.

Presequence protease (PreP), a 117 kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption.

Better, Faster, Cheaper: Recent Advances in Cryo-Electron Microscopy.

Cryo-electron microscopy (cryo-EM) continues its remarkable growth as a method for visualizing biological objects, which has been driven by advances across the entire pipeline. Developments in both single-particle analysis and in situ tomography have enabled more structures to be imaged and determined to better resolutions, at faster speeds, and with more scientists having improved access. This review highlights recent advances at each stageof the cryo-EM pipeline and provides examples of how these techniques have been used to investigate real-world problems, including antibody development against the SARS-CoV-2 spike during the recent COVID-19 pandemic.

Label-free visual proteomics: Coupling MS- and EM-based approaches in structural biology.

Combining diverse experimental structural and interactomic methods allows for the construction of comprehensible molecular encyclopedias of biological systems. Typically, this involves merging several independent approaches that provide complementary structural and functional information from multiple perspectives and at different resolution ranges. A particularly potent combination lies in coupling structural information from cryoelectron microscopy or tomography (cryo-EM or cryo-ET) with interactomic and structural information from mass spectrometry (MS)-based structural proteomics.

Topaz-Denoise: general deep denoising models for cryoEM and cryoET.

Cryo-electron microscopy (cryoEM) is becoming the preferred method for resolving protein structures. Low signal-to-noise ratio (SNR) in cryoEM images reduces the confidence and throughput of structure determination during several steps of data processing, resulting in impediments such as missing particle orientations. Denoising cryoEM images can not only improve downstream analysis but also accelerate the time-consuming data collection process by allowing lower electron dose micrographs to be used for analysis.

Family-wide Structural and Biophysical Analysis of Binding Interactions among Non-clustered δ-Protocadherins.

Non-clustered δ1- and δ2-protocadherins, close relatives of clustered protocadherins, function in cell adhesion and motility and play essential roles in neural patterning. To understand the molecular interactions underlying these functions, we used solution biophysics to characterize binding of δ1- and δ2-protocadherins, determined crystal structures of ectodomain complexes from each family, and assessed ectodomain assembly in reconstituted intermembrane junctions by cryoelectron tomography (cryo-ET). Homophilic trans (cell-cell) interactions were preferred for all δ-protocadherins, with additional weaker heterophilic interactions observed exclusively within each subfamily.

Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs.

Cryo-electron microscopy is a popular method for the determination of protein structures; however, identifying a sufficient number of particles for analysis can take months of manual effort. Current computational approaches find many false positives and require ad hoc postprocessing, especially for unusually shaped particles. To address these shortcomings, we develop Topaz, an efficient and accurate particle-picking pipeline using neural networks trained with a general-purpose positive-unlabeled learning method.

Structure of the gene therapy vector, adeno-associated virus with its cell receptor, AAVR.

Adeno-associated virus (AAV) vectors are preeminent in emerging clinical gene therapies. Generalizing beyond the most tractable genetic diseases will require modulation of cell specificity and immune neutralization. Interactions of AAV with its cellular receptor, AAVR, are key to understanding cell-entry and trafficking with the rigor needed to engineer tissue-specific vectors.

Visualization of clustered protocadherin neuronal self-recognition complexes.

Neurite self-recognition and avoidance are fundamental properties of all nervous systems. These processes facilitate dendritic arborization, prevent formation of autapses and allow free interaction among non-self neurons. Avoidance among self neurites is mediated by stochastic cell-surface expression of combinations of about 60 isoforms of α-, β- and γ-clustered protocadherin that provide mammalian neurons with single-cell identities.

Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: Bacterial RNA polymerase and CHAPSO.

Preferred particle orientation presents a major challenge for many single particle cryo-electron microscopy (cryo-EM) samples. Orientation bias limits the angular information used to generate three-dimensional maps and thus affects the reliability and interpretability of the structural models. The primary cause of preferred orientation is presumed to be due to adsorption of the particles at the air/water interface during cryo-EM grid preparation.

Mechanisms of opening and closing of the bacterial replicative helicase.

Assembly of bacterial ring-shaped hexameric replicative helicases on single-stranded (ss) DNA requires specialized loading factors. However, mechanisms implemented by these factors during opening and closing of the helicase, which enable and restrict access to an internal chamber, are not known. Here, we investigate these mechanisms in the DnaB helicase•bacteriophage λ helicase loader (λP) complex.

Reducing effects of particle adsorption to the air-water interface in cryo-EM.

Most protein particles prepared in vitreous ice for single-particle cryo-electron microscopy (cryo-EM) are adsorbed to air-water or substrate-water interfaces, which can cause the particles to adopt preferred orientations. By using a rapid plunge-freezing robot and nanowire grids, we were able to reduce some of the deleterious effects of the air-water interface by decreasing the dwell time of particles in thin liquid films. We demonstrated this by using single-particle cryo-EM and cryo-electron tomography (cryo-ET) to examine hemagglutinin, insulin receptor complex, and apoferritin.