Abstract OR-2: The Formation of Dps-DNA Complexes under Different Conditions According to Cryo-EM and SAXS

Background: The effect of Dps-DNA co-crystals formation, which occurs in stressed Escherichia coli cells exposed to extreme conditions, is well described in the literature. However, the exact mechanisms of co-crystals formation are yet to be postulated remaining largely unknown. Here we summarize the results obtained by our group over the last few years using cryo-Electron Microscopy (cryo-EM) and Small Angle X-ray Scattering (SAXS). Methods: Samples for cryo-EM were plunge frozen in liquid ethane with Vitrobot Mark IV and studied with Titan Krios (ThermoFisher Scientific, US) cryo-EM, equipped with Falcon 2 direct electron detector, Image corrector (CEOS, Germany), and Volta phase plate. Single Particle Analysis (SPA) and cryo-Electron Tomography (cryo-ET) studies were conducted with 300 kV accelerating voltage in low dose mode using EPU and Tomography software (ThermoFisher Scientific, US). Cryo-EM data processing was conducted using Warp, CryoSPARC, IMOD, EMAN, and Relion software packages. SAXS measurements were performed at the EMBL on the P12 BioSAXS beam line at the PETRAIII storage ring (DESY, Hamburg). Results: In this work, Dps-DNA complex formation is thoroughly studied using complementary cryo-EM (including SPA, cryo-ET, and subtomogram averaging) and SAXS methods. The formation of individual complexes of Dps with small linear DNA fragments and the Dps-Dps interaction was visualized using cryo-EM. It was found that Dps-DNA complex remains stable under various conditions and while the addition of different ions leads to the disruption of co-crystals, the process is completely or partially reversible. Conclusion: Recent studies conducted by our group showed that Dps-DNA co-crystals adopt triclinic or cubic crystal lattice (FEBS Lett., 2019; Biomolecules, 2020). Here we present the results on the studies of Dps interaction with small linear DNA fragments, demonstrate the effects of MgCl2, FeSO4, and EDTA on the Dps-DNA complex and individual Dps protein structure, discuss the influence of the temperature and time on the co-crystals.

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Structural Rearrangement of Dps-DNA Complex Caused by Divalent Mg and Fe Cations

International Journal of Molecular Sciences ◽

10.3390/ijms22116056 ◽

2021 ◽

Vol 22 (11) ◽

pp. 6056

Author(s):

Liubov Dadinova ◽

Roman Kamyshinsky ◽

Yury Chesnokov ◽

Andrey Mozhaev ◽

Vladimir Matveev ◽

...

Keyword(s):

Chelating Agent ◽

Structural Rearrangement ◽

Particle Analysis ◽

X Ray Scattering ◽

Cryo Electron Microscopy ◽

Complete Restoration ◽

Hydrophobic Pore ◽

Dps Protein ◽

Dna Complex ◽

Free Metal

Two independent, complementary methods of structural analysis were used to elucidate the effect of divalent magnesium and iron cations on the structure of the protective Dps-DNA complex. Small-angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) demonstrate that Mg2+ ions block the N-terminals of the Dps protein preventing its interaction with DNA. Non-interacting macromolecules of Dps and DNA remain in the solution in this case. The subsequent addition of the chelating agent (EDTA) leads to a complete restoration of the structure of the complex. Different effect was observed when Fe cations were added to the Dps-DNA complex; the presence of Fe2+ in solution leads to the total complex destruction and aggregation without possibility of the complex restoration with the chelating agent. Here, we discuss these different responses of the Dps-DNA complex on the presence of additional free metal cations, investigating the structure of the Dps protein with and without cations using SAXS and cryo-EM. Additionally, the single particle analysis of Dps with accumulated iron performed by cryo-EM shows localization of iron nanoparticles inside the Dps cavity next to the acidic (hydrophobic) pore, near three glutamate residues.

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Polymorphic Protective Dps–DNA Co-Crystals by Cryo Electron Tomography and Small Angle X-Ray Scattering

Biomolecules ◽

10.3390/biom10010039 ◽

2019 ◽

Vol 10 (1) ◽

pp. 39 ◽

Cited By ~ 2

Author(s):

Roman Kamyshinsky ◽

Yury Chesnokov ◽

Liubov Dadinova ◽

Andrey Mozhaev ◽

Ivan Orlov ◽

...

Keyword(s):

Small Angle ◽

Electron Tomography ◽

Actual Structure ◽

X Ray ◽

Cell Parameters ◽

X Ray Scattering ◽

Cryo Electron Tomography ◽

Dps Protein ◽

Ray Scattering

Rapid increase of intracellular synthesis of specific histone-like Dps protein that binds DNA to protect the genome against deleterious factors leads to in cellulo crystallization—one of the most curious processes in the area of life science at the moment. However, the actual structure of the Dps–DNA co-crystals remained uncertain in the details for more than two decades. Cryo-electron tomography and small-angle X-ray scattering revealed polymorphous modifications of the co-crystals depending on the buffer parameters. Two different types of the Dps–DNA co-crystals are formed in vitro: triclinic and cubic. Three-dimensional reconstruction revealed DNA and Dps molecules in cubic co-crystals, and the unit cell parameters of cubic lattice were determined consistently by both methods.

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Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs

10.1101/2022.01.10.475481 ◽

2022 ◽

Author(s):

Martin Obr ◽

Wim JH Hagen ◽

Robert A Dick ◽

Lingbo Yu ◽

Abhay Kotecha ◽

...

Keyword(s):

Field Emission ◽

Electron Tomography ◽

Particle Analysis ◽

Energy Filtering ◽

Cryo Electron Microscopy ◽

Electron Detection ◽

Thermo Fisher Scientific ◽

Subtomogram Averaging ◽

New Generation ◽

Fisher Scientific

The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo- EM) image processing has been well documented for single particle analysis (SPA). 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 Angstrom resolution range. In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Angstrom. 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.

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Phase Contrast Cryo-Electron Tomography and Single Particle Analysis with a New Phase Plate

Microscopy and Microanalysis ◽

10.1017/s1431927614002888 ◽

2014 ◽

Vol 20 (S3) ◽

pp. 232-233

Author(s):

Maryam Khoshouei ◽

Radostin Danev ◽

Günther Gerisch ◽

Maria Ecke ◽

Juergen Plitzko ◽

...

Keyword(s):

Single Particle ◽

Phase Contrast ◽

Electron Tomography ◽

Particle Analysis ◽

Phase Plate ◽

Single Particle Analysis ◽

Cryo Electron Tomography ◽

New Phase

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Subnanometer-resolution structure determination in situ by a hybrid subtomogram averaging – single particle cryoEM – workflow

10.1101/2020.04.24.057299 ◽

2020 ◽

Author(s):

Ricardo Sanchez ◽

Yingyi Zhang ◽

Wenbo Chen ◽

Lea Dietrich ◽

Misha Kudryashev

Keyword(s):

Single Particle ◽

Electron Tomography ◽

Signal To Noise Ratio ◽

Particle Analysis ◽

Electron Dose ◽

Particle Alignment ◽

Zero Degree ◽

Subtomogram Averaging

ABSTRACTCryo electron tomography (cryo-ET) combined with subtomogram averaging (StA) enables structural determination of macromolecules in their native context. A few structures were reported by StA at resolution higher than 4.5 Å, however all of these are from viral structural proteins or vesicle coats. Reaching high resolution for a broader range of samples is uncommon due to beam-induced sample drift, poor signal-to-noise ratio (SNR) of images, challenges in CTF correction, limited number of particles. Here we propose a strategy to address these issues, which consists of a tomographic data collection scheme and a processing workflow. Tilt series are collected with higher electron dose at zero-degree tilt in order to increase SNR. Next, after performing StA conventionally, we extract 2D projections of the particles of interest from the higher SNR images and use the single particle analysis tools to refine the particle alignment and generate a reconstruction. We benchmarked our proposed hybrid StA (hStA) workflow and improved the resolution for tobacco mosaic virus from 7.2 to 5.2 Å and the resolution for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. We demonstrate that hStA can improve the resolution obtained by conventional StA and promises to be a useful tool for StA projects aiming at subnanometer resolution or higher.

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In situ cryo-electron tomography reveals gradient organization of ribosome biogenesis in intact nucleoli

Nature Communications ◽

10.1038/s41467-021-25413-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Philipp S. Erdmann ◽

Zhen Hou ◽

Sven Klumpe ◽

Sagar Khavnekar ◽

Florian Beck ◽

...

Keyword(s):

Ribosome Biogenesis ◽

Electron Tomography ◽

Molecular Data ◽

Small Subunit ◽

Particle Analysis ◽

Cryo Electron Tomography ◽

Cellular Context ◽

New Perspective ◽

Subtomogram Averaging

AbstractRibosomes comprise a large (LSU) and a small subunit (SSU) which are synthesized independently in the nucleolus before being exported into the cytoplasm, where they assemble into functional ribosomes. Individual maturation steps have been analyzed in detail using biochemical methods, light microscopy and conventional electron microscopy (EM). In recent years, single particle analysis (SPA) has yielded molecular resolution structures of several pre-ribosomal intermediates. It falls short, however, of revealing the spatiotemporal sequence of ribosome biogenesis in the cellular context. Here, we present our study on native nucleoli in Chlamydomonas reinhardtii, in which we follow the formation of LSU and SSU precursors by in situ cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). By combining both positional and molecular data, we reveal gradients of ribosome maturation within the granular component (GC), offering a new perspective on how the liquid-liquid-phase separation of the nucleolus supports ribosome biogenesis.

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Combining high throughput and high quality for cryo-electron microscopy data collection

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320008347 ◽

2020 ◽

Vol 76 (8) ◽

pp. 724-728

Author(s):

Felix Weis ◽

Wim J. H. Hagen

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Data Collection ◽

Electron Tomography ◽

3D Structure ◽

Particle Analysis ◽

High Resolution Data ◽

Cryo Electron Microscopy ◽

Microscopy Data ◽

Subtomogram Averaging

Cryo-electron microscopy (cryo-EM) can be used to elucidate the 3D structure of macromolecular complexes. Driven by technological breakthroughs in electron-microscope and electron-detector development, coupled with improved image-processing procedures, it is now possible to reach high resolution both in single-particle analysis and in cryo-electron tomography and subtomogram-averaging approaches. As a consequence, the way in which cryo-EM data are collected has changed and new challenges have arisen in terms of microscope alignment, aberration correction and imaging parameters. This review describes how high-end data collection is performed at the EMBL Heidelberg cryo-EM platform, presenting recent microscope implementations that allow an increase in throughput while maintaining aberration-free imaging and the optimization of acquisition parameters to collect high-resolution data.

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Benchmarking tomographic acquisition schemes for high-resolution structural biology

10.1101/742254 ◽

2019 ◽

Cited By ~ 1

Author(s):

Beata Turoňová ◽

Wim J. H. Hagen ◽

Martin Obr ◽

Hans-Georg Kräusslich ◽

Martin Beck

Keyword(s):

Structural Biology ◽

Electron Tomography ◽

Atomic Resolution ◽

Phase Plate ◽

Macromolecular Complexes ◽

Cryo Electron Tomography ◽

Experimental Parameters ◽

Native Context ◽

Subtomogram Averaging ◽

Hiv 1

AbstractCryo electron tomography with subsequent subtomogram averaging is a powerful technique to structurally analyze macromolecular complexes in their native context. Although close to atomic resolution, in principle, can be obtained, it is not clear how individual experimental parameters contribute to the attainable resolution. Here, we have used immature HIV-1 lattice as a benchmarking sample to optimize the attainable resolution for subtomogram averaging. We systematically tested various experimental parameters such as the order of projections, different angular increments and the use of the Volta phase plate. We find that although any of the prominently used acquisition schemes is sufficient to obtain subnanometer resolution, dose-symmetric acquisition provides considerably better outcome. We discuss our findings in order to provide guidance for data acquisition. Our data is publicly available at EMPIAR-10277 as well as EMD-10207 and might be used to further develop processing routines.

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Protective Dps–DNA co‐crystallization in stressed cells: an in vitro structural study by small‐angle X‐ray scattering and cryo‐electron tomography

FEBS Letters ◽

10.1002/1873-3468.13439 ◽

2019 ◽

Vol 593 (12) ◽

pp. 1360-1371 ◽

Cited By ~ 7

Author(s):

Liubov A. Dadinova ◽

Yurii M. Chesnokov ◽

Roman A. Kamyshinsky ◽

Ivan A. Orlov ◽

Maxim V. Petoukhov ◽

...

Keyword(s):

Structural Study ◽

Small Angle ◽

Electron Tomography ◽

X Ray ◽

X Ray Scattering ◽

Cryo Electron Tomography ◽

Stressed Cells ◽

Ray Scattering

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A guided approach for subtomogram averaging of challenging macromolecular assemblies

10.1101/2020.02.01.930297 ◽

2020 ◽

Author(s):

Danielle Grotjahn ◽

Saikat Chowdhury ◽

Gabriel C. Lander

Keyword(s):

Electron Tomography ◽

A Priori ◽

Three Dimensional ◽

Structural Heterogeneity ◽

Dimensional Structure ◽

Diverse Range ◽

Macromolecular Assemblies ◽

Cryo Electron Tomography ◽

Subtomogram Averaging

AbstractCryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

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