TY - JOUR AB - The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) has been well documented. Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4–6 Å resolution range based on Fourier-Shell correlation (FSC). In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Å. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes. AU - Obr, Martin AU - Hagen, Wim J.H. AU - Dick, Robert A. AU - Yu, Lingbo AU - Kotecha, Abhay AU - Schur, Florian KM ID - 11155 IS - 2 JF - Journal of Structural Biology KW - Structural Biology SN - 1047-8477 TI - Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs VL - 214 ER - TY - JOUR AB - A precise quantitative description of the ultrastructural characteristics underlying biological mechanisms is often key to their understanding. This is particularly true for dynamic extra- and intracellular filamentous assemblies, playing a role in cell motility, cell integrity, cytokinesis, tissue formation and maintenance. For example, genetic manipulation or modulation of actin regulatory proteins frequently manifests in changes of the morphology, dynamics, and ultrastructural architecture of actin filament-rich cell peripheral structures, such as lamellipodia or filopodia. However, the observed ultrastructural effects often remain subtle and require sufficiently large datasets for appropriate quantitative analysis. The acquisition of such large datasets has been enabled by recent advances in high-throughput cryo-electron tomography (cryo-ET) methods. This also necessitates the development of complementary approaches to maximize the extraction of relevant biological information. We have developed a computational toolbox for the semi-automatic quantification of segmented and vectorized filamentous networks from pre-processed cryo-electron tomograms, facilitating the analysis and cross-comparison of multiple experimental conditions. GUI-based components simplify the processing of data and allow users to obtain a large number of ultrastructural parameters describing filamentous assemblies. We demonstrate the feasibility of this workflow by analyzing cryo-ET data of untreated and chemically perturbed branched actin filament networks and that of parallel actin filament arrays. In principle, the computational toolbox presented here is applicable for data analysis comprising any type of filaments in regular (i.e. parallel) or random arrangement. We show that it can ease the identification of key differences between experimental groups and facilitate the in-depth analysis of ultrastructural data in a time-efficient manner. AU - Dimchev, Georgi A AU - Amiri, Behnam AU - Fäßler, Florian AU - Falcke, Martin AU - Schur, Florian KM ID - 10290 IS - 4 JF - Journal of Structural Biology KW - Structural Biology SN - 1047-8477 TI - Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data VL - 213 ER - TY - JOUR AB - Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications. AU - Fäßler, Florian AU - Zens, Bettina AU - Hauschild, Robert AU - Schur, Florian KM ID - 8586 IS - 3 JF - Journal of Structural Biology KW - electron microscopy KW - cryo-EM KW - EM sample preparation KW - 3D printing KW - cell culture SN - 1047-8477 TI - 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy VL - 212 ER - TY - JOUR AB - The bacterial cell wall is composed of the peptidoglycan (PG), a large polymer that maintains the integrity of the bacterial cell. Due to its multi-gigadalton size, heterogeneity, and dynamics, atomic-resolution studies are inherently complex. Solid-state NMR is an important technique to gain insight into its structure, dynamics and interactions. Here, we explore the possibilities to study the PG with ultra-fast (100 kHz) magic-angle spinning NMR. We demonstrate that highly resolved spectra can be obtained, and show strategies to obtain site-specific resonance assignments and distance information. We also explore the use of proton-proton correlation experiments, thus opening the way for NMR studies of intact cell walls without the need for isotope labeling. AU - Bougault, Catherine AU - Ayala, Isabel AU - Vollmer, Waldemar AU - Simorre, Jean-Pierre AU - Schanda, Paul ID - 8409 IS - 1 JF - Journal of Structural Biology KW - Structural Biology SN - 1047-8477 TI - Studying intact bacterial peptidoglycan by proton-detected NMR spectroscopy at 100 kHz MAS frequency VL - 206 ER - TY - JOUR AB - Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25 nm in the x–y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells. AU - Perkovic, Mario AU - Kunz, Michael AU - Endesfelder, Ulrike AU - Bunse, Stefanie AU - Wigge, Christoph AU - Yu, Zhou AU - Hodirnau, Victor-Valentin AU - Scheffer, Margot P. AU - Seybert, Anja AU - Malkusch, Sebastian AU - Schuman, Erin M. AU - Heilemann, Mike AU - Frangakis, Achilleas S. ID - 9655 IS - 2 JF - Journal of Structural Biology SN - 1047-8477 TI - Correlative light- and electron microscopy with chemical tags VL - 186 ER -