Microscopy Conference 2023

Prof. Dr.

Session chairs

In 2020, cryo-EM breached another barrierer and it has now been shown that it can visualise individual atoms within a macromolecular complex. Significant improvements in software and mircoscope design were mandatory for this achievement. However, what is possible for ideal samples or test-specimen must not necessarily be true for research-based biological applications, highlighting the prerequisite of sample quality and optimization which continues to be the major challenge en route to atomic-resolution cryo-EM structures. The session will showcase how to improve the sample handling, microscope setup and image processing to obtain high-resolution structures using cryo-EM.

Optimizing cryo-EM data acquisition

Abstract text (incl. figure legends and references) Cryo-electron microscopy (cryo-EM) allows for the visualization of biomolecules at near-atomic resolution and has become an increasingly important tool for structural biologists. The presentation will discuss several strategies for optimizing cryo-EM data acquisition in order to improve the efficiency and throughput of experiments. Innovative approaches that have been developed over the years will be covered, such as aberration-free image shift (AFIS), and fringe-free imaging (FFI), which have been shown to improve the throughput of cryo-EM data collection. Additionally, the use of &quot;Virtual Maps&quot; for screening samples with problematic particle distribution will be discussed, a strategy that helps to prioritize areas of the sample for imaging and reduce the overall time and resources required for the experiment. Lastly, screening cryo-EM samples using an energy filter&quot;s plasmon regime will be presented. Through the use of these optimization strategies, researchers can obtain high-quality data from cryo-EM experiments, leading to informative structural models, in a shorter amount of data acquisition time.

LS 1: High-resolution cryo-EM

LSLB: Late breaking abstracts

Invited talk

Understanding redox protein mechanism through structure

Abstract text (incl. figure legends and references) Redox proteins carry out sophisticated reactions that shape our bodies, our planets and our climate. Understanding the mechanism of redox proteins depends on an atomic-level understanding not only of the protein structure, but of structural changes associated with reduction and oxidation processes. Because structural studies by single-particle cryo-EM have only recently achieved the resolutions that allow for atomic-level insight into mechanism, the tools for preparation of samples under defined redox conditions are lacking. As our group has worked toward understanding the structure and function of redox proteins, we have brought together a set of tools for better assessing redox-dependent structural changes. Applying these tools to proteins involved in hydrogen production and methanogenesis have allowed us to gain new insight into these processes.

LS 7: Advances in sample preparation

Structure of the HOPS tethering complex, a lysosomal membrane fusion machinery

Abstract text (incl. figure legends and references) Lysosomes are essential for cellular recycling, nutrient signaling, autophagy and the invasion of pathogenic bacteria and viruses. Lysosomal fusion is fundamental to cell survival and requires HOPS, a conserved heterohexameric tethering complex. On the membranes to be fused, HOPS binds small membrane-associated GTPases and assembles SNAREs for fusion, but how the complex fulfils its function remained speculative. Here, we used cryo-electron microscopy to reveal the structure of HOPS. Unlike previously reported, HOPS is surprisingly rigid and extensive flexibility is confined to its extremities, where GTPase binding occurs. The SNARE-binding module is rigidly attached to the core, therefore, ideally positioned between the membranes to catalyze fusion. Our results explain HOPS dual functionality and unravel why tethering complexes are an essential part of the membrane fusion machinery. Figure 1. Composition and architecture of the yeast HOPS complex. A, Composite cryo-EM map from local refinement maps. B, Atomic model of the HOPS complex. C, Schematic representation of the HOPS complex. References: [1] Shvarev, Schoppe, König et al. (2022) bioRxiv, doi.org/10.1101/2022.05.05.490745.

Abstract talk

Cryo-EM structures of C. glutamicum pre- and posttranslocational 70S ribosome in complex with Kasugamycin

Abstract text (incl. figure legends and references) The Actinobacteria Corynebacterium glutamicum is the workhorse in modern biotechnology, e.g. in industrial biosynthesis of amino acids and heterologous protein production. The detailed architecture of the C. glutamicum translation machinery, the 70S ribosome, is hitherto unknown, impeding its structure-function analysis, and limiting drug optimization. The majority of aminoglycoside antibiotic producers are Actinobacteria, still C. glutamicum is sensitive to the antibiotic Kasugamycin (KSG). Our study aims to understand the impact KSG has on the 70S ribosome of C. glutamicum (Cg70S) and how the simple mutations ΔbS22 and ΔKsgA contribute to resistance. Therefore, we present the three-dimensional structures of the wildtype, ΔbS22 and ΔKsgA ribosome solved to high resolution (&lt;3 Å) with cryo-electron microscopy. By exploiting the potential of single particle analysis in the structural analysis of dynamic processes, particles were classified in 3D heterogeneous refinement according to their t-RNA position in the ribosome. We captured Cg70S ribosomes in the pre- and posttranslocational state in absence and presence of KSG. For both states, we were able to localize the KSG-binding pocket within the mRNA path and in close proximity to the P-tRNA binding site. In direct comparison of the molecular models derived from the WT, ΔbS22 and ΔKsgA EM density maps, it became clear that the deletions do not significantly alter the molecular structure of the KSG binding pocket. Interestingly however, depending on the absence or presence of KSG in wildtype- or ΔbS22-Cg70S the particle ratio between the two distinct states differs significantly, suggesting a role of bS22 in the stabilization of the pretranslocational state. ΔKsgA results in undermethylation of the 16s rRNA, but the structure of the KSG binding pocket is preserved. Here, the alteration of the protein content of the small ribosomal subunit, visible in the EM density maps, seems to be the key to resistance by influencing the presence of bS22 and other small ribosomal subunit proteins and thereby modifying ribosomal dynamics. The here presented cryo-EM structures of the C. glutamicum ribosome provide the structural framework for understanding antibiotic KSG resistance.

LS 4: Image analysis of large data sets

Dipl.-Ing. Dr.

Visualizing cellulose chains with cryo scanning transmission electron microscopy

Abstract text (incl. figure legends and references) Cellulose is the most abundant biopolymer on earth and is found in the cell walls of most plants and some algae, but also occurs in bacteria, fungi and even some sea animals. Plant cellulose, in particular, has a wide range of applications as a renewable, biodegradable and non-toxic material in papers, textiles, packaging and medical products, to name a few. Cellulose in plants is organized in supramolecular structures where the basic structural element the cellulose microfibril (CMF). The supramolecular cellulose comprises of ordered periodic crystalline regions that can be liberated by acid hydrolysis into nanoparticles, namely the cellulose nanocrystals (CNC), with dimensions of 100-200 nm in length and a few nanometers in width. Despite its abundance in nature and its technological relevance, the structural details of cellulose still remain elusive and various structural models have been proposed in recent years [1]. Transmission electron microscopy (TEM) under cryogenic conditions has proven to be a highly valuable technique for the structural analysis of such biomolecules. However, imaging cellulose at sufficiently high resolution has been challenging due to its very high susceptibility to electron beam damage, combined with the low contrast provided by its light constituents. These problems have been addressed in the past by applying contrast agents, staining with uranyl acetate or low voltage imaging [2]. While, by this, great progress has been made regarding the visualization of nanoscale cellulose features, atomic scale visualization still remains problematic [3]. Here, we report on the visualization of sulfated cellulose chains by low-dose cryo high-resolution scanning TEM (STEM). To this end we exploit the high contrast provided by indvidual heavy ions in annular dark field (ADF) imaging for visualization of cellulose chains. For imaging a FEI Titan G2 STEM, operated at 300 kV, has been used. Samples were prepared by drop casting the CNCs, dispersed in water, on a TEM grid, which is covered by a 2-3 nm thick amorphous carbon film. During imaging the sample is kept at liquid nitrogen temperature. In the obtained ADF images (Figure 1) the individual atoms providing contrast can clearly be discerned and exhibit a regular, linear arrangement along the long axis of the CNCs. By comparing the micrographs with multislice simulations based on atomistic structural models, we obtain information about possible arrangements of the sulfate groups, linked to the position of carbon 6 sites in the glucose unit within single CNC chains. Exemplary, a possible structural configuration on the amorphous carbon substrate is depicted in Figure 1c with the corresponding ADF multislice simulation shown in (f). References [1] Habibi, Youssef et al.; Chemical reviews 110 (6), pp. 3479–3500. DOI: 10.1021/cr900339w. [2] Sugiyama, J. et al.; International Journal of Biological Macromolecules 9 (3), pp. 122–130. DOI: 10.1016/0141-8130(87)90039-0. [2] Ogawa, Yu et al.; Cellulose 26 (1), pp. 5–15. DOI: 10.1007/s10570-018-2076-9. Acknowledgements The authors acknowledge financial support by the European Union"s Horizon 2020 research and innovation program under Grant 823717-ESTEEM3.

IM 4: Development of cryo-EM instrumentation and techniques

Common structural features of ATTRwt and ATTRV30M amyloid fibrils from different patients

Abstract text (incl. figure legends and references) Introduction: Wild type transthyretin-derived amyloid (ATTRwt) is the major component of non-hereditary transthyretin amyloidosis. Its accumulation in the heart of elderly patients is life threatening. A variety of genetic variants of transthyretin can lead to hereditary transthyretin amyloidosis, which shows different clinical symptoms, like age of onset and pattern of organ involvement. However, in the case of non-hereditary and in some cases of hereditary ATTR amyloidosis fibril deposits are located primarily in heart tissue. Objectives: The goal of this study was the analysis of ATTRwt amyloid fibrils structure from the heart of a patient with non-hereditary transthyretin amyloidosis and comparison with previous published structures. Materials &amp; methods: Fibrils were extracted from the amyloidotic tissue of an explanted heart of a male ATTRwt patient. Cryo electron microscopy with subsequent structure modelling and mass spectrometry was used to study these fibrils. Results: A density map of ex vivo ATTRwt fibrils with a resolution of 2.78 Å was reconstructed. The structure is formed by stacked N- and C-terminal fragments of transthyretin forming an amyloid fibril which was confirmed with mass spectrometry. They form a spearhead like shape in their cross section. The N-terminal fragment has an extensive hydrophobic core. The C-terminal fragment on the other hand contains a big cavity surrounded by polar and charged amino acids and is probably filled with water. Conclusion: We compare the structure of ATTRwt fibrils with previously published V30M variants of ATTR fibrils extracted from heart and eye of different patients. All so far published structures show a remarkably similar cross-sectional shape of the N- and C-terminal fragments with only some minor differences. This demonstrates common features for ATTR fibrils despite differences in mutations and patients.

LS 1
High resolution cryo-EM

Appointment

Session chairs

Description

Programme

LS 1High resolution cryo-EM

Appointment

Session chairs

Description

Programme

LS 1
High resolution cryo-EM