Microscopy Conference 2023

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Visualizing electrons by single particle cryo-EM

Abstract text (incl. figure legends and references) Scattering of electrons by atoms in electron microscopy produces coulomb potential maps and hence EM maps reflect the charge of atoms in protein complexes. At resolution ranges between 5 Å and 10 Å, atomic scattering amplitudes are usually weaker the more negatively charged atoms are. Here, we compare the cryo EM structure of Rhodobacter capsulatus formate dehydrogenase (FDH) in the air oxidized, sodium azide inhibited state with an enzyme that is reduced by treatment with NADH. FDH catalyzes the reversible oxidation of formate to carbon dioxide. The electrons gained in this reaction at the active site molybdenum atom are transferred via an electron-transfer pathway formed by numerous FeS clusters and flavin mononucleotide (FMN) onto nicotinamide adenine dinucleotide (NAD+). At a resolution of about 3.25 Å, the difference map between the oxidized and NADH reduced states qualitatively visualizes negative charges on electron accepting atoms in the electron transfer chain of the reduced map.

IM 4: Development of cryo-EM instrumentation and techniques

LS 1: High-resolution cryo-EM

Poster

Aberration correctors for cryo electron microscopy

Abstract text (incl. figure legends and references) In cryo electron microscopy (cryo EM) the resolution of the TEM hasn&quot;t been the limiting factor for a long time. Recently, however, cryo EM has reached the single Ångstrom level [1] where the aberrations of the TEM start to play a significant role in the process of image formation. There are multiple aspects where aberration correctors might be very useful for cryo EM techniques: 1. Coma-free alignment: The present spherical aberration requires extremely high accuracy and stability of beam tilt alignment to prevent beam tilt induced axial coma. This condition has to be stable for a very long time to allow for long-term automated data acquisition. In a Cs-corrected TEM a beam tilt of several mrad is acceptable without noticeably introducing axial coma. 2. Beam-image-shifts: To avoid mechanical specimen shifts and improve throughput, it is common practice to use combined beam-image-shifts during image recording rather than physical specimen shifts. These virtual object shifts are affected by the unavoidable off-axial coma of the objective lens (Fig.1). Precise simultaneous adjustment of beam tilt during beam-image-shift allows to compensate for this effect [2]. Alternatively, a Cs-corrector can actively compensate the shift-induced coma below the object during image shift and in that case the beam tilt above the specimen does not need to change hence remains flexible, e.g. for dispersion correction. Cs-correctors allow for aberration-free beam-image-shifts of up to 10µm, which is extremely useful for efficient work-flows for single particle reconstructions. 3. Large cameras with a high number of pixels are important to increase throughput: The large field of view which is captured at once, however, is affected by the off-axial coma in that the coma increases linearly from the center of the image towards the outer areas. While this effect is negligible for 2k cameras, it can have a distinct impact on 4k detectors with a similar pixel size. Fortunately, this limitation can be overcome by the off-axial coma corrector (BCOR) which enables aplanatic imaging, i.e. freedom from spherical aberration and off-axial coma [3]. 4. Inelastically scattered electrons usually do not contribute to the high-resolution image contrast due to chromatic aberration. A Cc/Cs-corrector is expected to significantly improve contrast for larger sample thicknesses [4]. References: [1] K.M. Yip et al., Nature 587, 157–161 (2020). [2] Konings et al., Microsc. Microanal. 25 S2 (2019) 1012-1013. [3] H. Müller et al., Nucl. Instrum. Methods Phys. Res. A 645 (2011) 20-27. [4] J.L. Dickerson et al, Ultramicroscopy 237 (2022), 113510. [€] CEOS GmbH has received funding from the European Union&quot;s Horizon 2020 research and innovation program under grant agreement No. 823717 – ESTEEM3. Fig. 1 – (a) Simultaneous use of beam and image deflectors for virtual object shifts is always affected by off-axial coma (b, red). Uncorrected TEMs require an additional simultaneous beam tilt to counterbalance off-axial coma effects (magenta). In Cs-corrected TEMs (blue), the corrector elements compensate the off-axial coma contribution and the beam tilt remains flexible. For large cameras the off-axial coma even changes the image quality within the camera's field of view. To remove this effect as well, a corrector for off-axial coma (e.g. BCOR) has to be used (cyan, dashed).

Effect of multiple scattering and the ice thickness on the information transfer in cryo transmission electron microscopy

Abstract text (incl. figure legends and references) In the field of electron cryo-microscopy it remains a challenge to improve the resolution and to decrease the number of micrographs needed for challenging protein samples. To get a better understanding of the scattering inside a biological specimen and the surrounding glass-like ice, we performed multislice simulations of the tobacco mosaic virus (TMV) (PDBe: 6sag [1]) in ice. The molecular structure of ice was modelled by molecular dynamics (LAMMPS [2]) and the atomic model of TMV was added [3]. First, we varied the number of slices used for the multislice simulations. Secondly, we added additional ice above and below the TMV. The contrast transfer, as seen in Fig.1 by the Fourier ring correlations (FRC) of the exit waves decreases when the number of slices is reduced. The effect is strongest at high spatial frequencies. However, even for a simulation of a single slice that accounts for the propagation to the same optical plane as the multislice simulation, the FRC values are larger than 0.75 up to a resolution of 2.3 Å. So, multiple scattering has only a small effect up to this resolution, which suggests the principal applicability of dose-efficient scanning TEM (STEM) techniques that are based on single scattering, such as integrated differential phase contrast (iDPC) [4] and the single side band (SSB) ptychographic reconstruction. Compared to CTEM simulations, iDPC and the phase of SSB in Fig. 1 are easier to interpret because both contrast transfer functions do not show contrast inversions. To simulate the averaging of single particle reconstructions, we also calculated the average of 600 phase-corrected simulated CTEM images (defocus range: [-0.1,-1.0] μm, spherical aberration: 1.5 mm) for different ice thickness and compared the results to the ground truth, using the FRC curves (Fig. 2). The contrast transfer decreases with increasing ice thickness, which is reflected in a lower contrast in real space for TMVs and in lower FRC values. The decrease of the FRC values is stronger at higher spatial frequencies, with a plateau above a resolution of 3 Å (Fig. 2). The FRC decrease is most dominant when adding the first ice layers, whereas depositing further ice to form rather thick layer of up to 90nm affect the fidelity of the exit wave and the CTEM simulation as to the TMV less. Our results will be discussed in the context of experimental applicability of momentum-resolved STEM techniques to biological specimen under low-dose cryo-conditions, and to which extent such new acquisition schemes might improve the structural characterisations of proteins. Figure 1) (a): FRC curves of the exit waves for multislice simulations of TMV with different number of slices, the number is given in the legend. (b): iDPC reconstruction. (c): Angle of the SSB reconstruction. Figure 2) (a) and (b): Phase of the exit wave of simulations with minimal and maximal ice thickness (thickness in the label). (c) and (d): CTEM simulations of the exit waves in (a) and (b) with a defocus of -500 nm and a spherical aberration of 1.5 mm. (e) and (f): FRC curves of the exit wave and the average of 600 phase corrected CTEM simulations (defocus: -0.1 μm to -1.0 μm, spherical aberration: 1.5 mm). [1] Weis, et al. EMBO reports 20.11 (2019) [2] Thompson, et al. Computer Physics Communications 271 (2022) [3] Shang, et al. Journal of structural biology 180.1 (2012) [4] Lazic, et al. (accepted for publication).

IM 6: Phase-related techniques & 4D STEM

Design and deployment of a high-performance computing system for cryo-EM

Abstract text (incl. figure legends and references) The constant development of faster electron detectors and microscope software and the analysis of flexible and dynamic macromolecular complexes demand powerful computing resources. While the microscope operation and image processing are getting increasingly automated and require less manual input, the design and deployment of an HPC system, suitable to fulfill such demands, is still very tedious and can quickly become overwhelming, especially for not professional system administrators. Here we describe the design choices for our recent cryo-EM HPC system and explain how we automated its installation and deployment with a combination of Metal-As-A-Service (MAAS) and Ansible. The cluster incorporates multi-GPU nodes, highspeed Interconnection (InfiniBand HDR) and a parallel file system (BeeGFS). Our approach can be easily adapted to different storage and hardware systems and enables inexperienced personnel to set up an HPC with little time.

LS 4: Image analysis of large data sets

Functional and structural characterisation of DNA-protein interactions of DNA binding protein from Mycobacterium Smegmatis

Abstract text (incl. figure legends and references) Protein nanocages have been explored as potential carriers in biomedicine. These caged structure could be engineered by loading therapeutic and diagnostic molecules in the core, while their external surfaces can be used to enhance their biocompatibility and targeting abilities.These cage-like proteins have propensity to self-assemble from simple identical building blocks to highly ordered architecture. DNA binding proteins under starvation (Dps),are one of such cage forming protein which form dodecamer with pore diameter of 5nm. It helps in storing the iron to avoid the formation of free radical. It also helps in binding and condensing the DNA to protects the DNA from various oxidative stressesunder starvation condition. These properties of Dps protein could be used to develop DNA-protein nanocage for drug delivery system. One of such protein is M. smegmatis MsDps2, also showed DNA binding and iron storage capacity. MsDps2 binds the DNA without having any known DNA binding motifs. It is important to know the interaction of MsDps2 and DNA to explain the causes of bacterial resistance to antibiotics and to develop new antibacterial drug. Additionally, this DNA-protein interactions are important to know to construct the cage from both protein and DNA. However, different models were proposed for Dps and DNA interaction. But till date, there is a lag of structural information at atomic level for this Dps, including MsDps2 and DNA interaction To decipher the MsDps2 and DNA interaction, we performed EMSA, MST, CD and Fluorometry biochemical experiments.Furthermore, we employed single-molecule imaging (TIRF), negative staining TEM and cryo-EM structural analysis to characterize the MsDps2-DNA interactions. All the above mentioned experiments were performed using different length and concentrations of MsDps2 protein In this study, we reported that MsDps2 binds with DNA cooperatively and shows its compaction. EMSA studies suggested that the difference in the degree of compaction depends on DNA length and concentration of protein. We have used TIRF microscopy to further validate the compaction of DNA cooperatively on a real-time scale.Furthermore, our negative staining TEM imaging suggests that MsDps2 binds to DNA, which looks like pearls on a thread. To decipher the structure of the MsDps2-DNA complex, we have performed cryo-EM with 600bp of DNA with MsDps2.We resolved the structure of the MsDps2-DNA complex at a resolution of 7Å, whereas the core MsDps2 was solved at 2.4Åresolution.Based on this structural information, we have mutated four arginine residues (R4E,R5E,R104E,R114E) which were involved in DNA binding. Mutated constructs showed less affinity to DNA binding; however, there is no effect on MsDps2 oligomerisation. Our cryo-EM based structural studies might help us to employ bioengineer the MsDps2 constructs to develop new protein-DNA cargo.We plan to integrate our work with other experimental and computational methods like Molecular dynamics simulations, SAXS and computational modelling (AlphaFold and Haddock) to get more insightsinto protein-DNA interaction and characterize its flexibility.

LS 2: Cryo-tomography

Structural basis of TIRAP assembly revealed by cryo-EM

Abstract text (incl. figure legends and references) Toll-like receptors (TLRs) activate the innate immune response during pathogen infection. Upon ligand-induced receptor dimerization, intracellular adapter proteins, like TIRAP (TIR-domain containing adaptor protein), initiate the assembly of large signaling complexes. In resting TLR-positive cells, TIRAP multimerizes into filaments at the plasma membrane and is subsequently converted into dimers during TLR signaling. However, the exact process of TIRAP filament formation and interaction with TLR dimers remains unknown. Here, we utilize negative-stain and cryo-EM to investigate the mechanism of TIRAP multimerization. We demonstrate that the full-length human TIRAP forms filaments in a temperature-dependent and reversible manner in the absence of TLRs. Our 3.2 Å cryo-EM structure, together with the negative-stain analysis, indicates a sequential filament formation, starting from stable dimers that polymerize into thin filamentous intermediates and tubes with a diameter of about 30 nm. Together, our data support the presence of TIRAP filaments in resting TLR-positive cells. Furthermore, filamentous TIRAP may provide a pool of required adapter proteins to stimulate TLR-based signal transduction.

LS 7: Advances in sample preparation

Cryo-EM analyses of the conformational spectrum of an ABC transporter in various membrane mimics

Abstract text (incl. figure legends and references) Membrane proteins and especially ATP binding cassette (ABC) transporters play a major role in nature. ABC transporters are involved in numerous physiological processes and utilize ATP to translocate various compounds through the lipid bilayer. This process requires large-scale conformational changes and is highly dynamic. Therefore, structure determination is fundamental to understanding these transient proteins' function and mode of action. Cryo-electron microscopy enables structure determination of proteins in solution to high-resolution and is, therefore, ideally suited to investigate such structural dynamics. However, particles must be extracted from the membrane and stabilized in artificial environments to facilitate cryo-EM sample preparation. To mimic the native environment, components such as detergents or lipid-based systems that form nanodisc can be used. In this study, we investigated the impact of various hydrophobic environments on the archetypical ABC transporter MsbA which translocates the precursor of Lipopolysaccharide through the inner membrane of gram-negative bacteria. In addition, to different activity intensities, measured by phosphate release during ATP hydrolysis, we notice a strong bias of the observed conformational spectrum to the respective membrane mimic which complicates the accurate determination of the protein&quot;s behavior. We hope our experiments can serve as a blueprint to calibrate reconstituted membrane protein systems and are now progressing to an alternative test specimen for validation.

Towards structure of secretory immunoglobulin A with O-antigen substrate

Abstract text (incl. figure legends and references) Mucosal immune system is the largest component of immune system and mediates interactions with a diverse range of antigens from toxin molecules to pathogen organisms of dietary or environmental source. Moreover, it comprises physiologically diverse compartments such as ocular, oral, gastrointestinal, respiratory etc mucosae. Secretory Immunoglobulin A (SIgA) is the major antibody of mammalian mucosa. It is a polymeric antibody and interacts with a large and diverse set of antigens. Mode of action for SIgA seems to be mostly non-inflammatory and involves coating, clumping, cross-linking (Kathrin Moor, et al. 2017). Structure of SIgA was recently solved (Kumar Bharathkar, Parker, et al. 2020) and comprises two copies of IgA with one Joining-Chain (JC). The full molecular assembly furthermore comprises Secretory Component (SC). However, the Fab fragments were not resolved likely due to high flexibility. We characterized the SIgA using cryo electron microscopy (cryoEM) in presence of its substrate, the O-antigen. One SIgA and one O-antigen molecule comprise four Fabs and 50-60 repeating units, respectively. The molar ratio of SIgA/O-antigen is 2/1, which implies that two tetravalent SIgA molecules are bound to one molecule O-antigen comprising 50-60 antigen repeating units (corresponding to 1 Fab /6.25-7.5 antigen repeating units). Freshly glow discharged Quantifoil Cu R 2/2 grids were optimized for 0.5 mg/ml SIgA sample or its complex with O-antigen at a ratio of 2:1 using Vitrobot (3.5 µl, 22°C, 2-4 s blotting time). Data was collected using Titan Krios (Thermo Fisher Scientific) and GIF/K3 (Gatan) by applying 63 e/Å2 at 0.65 pix/Å. The collected 8060 movies were processed in cryoSPARC to solve the structure. Few conclusions can be drawn from a comparison of the published SIgA structure with our SIgA structure or the SIgA-O-antigen complex. The obtained structure of the SIgA shows no change in the overall structure upon binding of the O-antigen. However, addition of the O-antigen markedly improves the resolution of the complex (currently ca. 3.2 Å) possibly resulting from a higher stability of complex. Furthermore, the obtained structure is similar to the published structure referred to earlier, but differences are observed especially for parts of the SC where the polypeptide chain is better resolved, and a different atomic model can be built. Besides, we are working currently to resolve the Fabs and Fab-antigen interaction using cryoEM and additional techniques such X-ray crystallization. References: Kumar Bharathkar, Parker, et al. eLife 2020;9:e56098. DOI: https://doi.org/10.7554/eLife.56098 Kathrin Moor, et al. Nature 2017; DOI:10.1038/nature22058

PS 2
Poster session LS 1: High-resolution cryo-EM

Appointment

Session chairs

Programme

PS 2Poster session LS 1: High-resolution cryo-EM

Appointment

Session chairs

Programme

PS 2
Poster session LS 1: High-resolution cryo-EM