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

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.

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

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.

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

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.

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

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.

scholarly journals Advances in cryo-EM and ED with a cold-field emission beam and energy filtration —Refinements of the CRYO ARM 300 system in RIKEN SPring-8 center—

Microscopy ◽  
2020 ◽  
Author(s):  
Saori Maki-Yonekura ◽  
Tasuku Hamaguchi ◽  
Hisashi Naitow ◽  
Kiyofumi Takaba ◽  
Koji Yonekura
Keyword(s):  
Field Emission ◽  
Direct Detection ◽  
Quality Data ◽  
Particle Analysis ◽  
High Quality Data ◽  
Camera Control ◽  
Cryo Electron Microscopy ◽  
Control Computer ◽  
Gpu Clusters ◽  
Resolution Single

Abstract We have designed and evaluated a cryo-electron microscopy (cryo-EM) system for higher-resolution single particle analysis and high-precision electron 3D crystallography. The system comprises a JEOL CRYO ARM 300 electron microscope—the first machine of this model—and a direct detection device camera, a scintillator-coupled camera, GPU clusters connected with a camera control computer and software for automated-data collection and efficient and accurate operation. The microscope provides parallel illumination of a highly coherent 300-kV electron beam to a sample from a cold-field emission gun and filters out energy-loss electrons through the sample with an in-column energy filter. The gun and filter are highly effective in improving imaging and diffraction, respectively, and have provided high quality data since July 2018. We here report on the characteristics of the cryo-EM system, updates, our progress and future plan for running such cryo-EM machines in RIKEN SPring-8 Center.

An overview of the recent advances in cryo-electron microscopy for life sciences

2021 ◽  
Author(s):  
Anshul Assaiya ◽  
Ananth Prasad Burada ◽  
Surbhi Dhingra ◽  
Janesh Kumar
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structure Determination ◽  
Electron Tomography ◽  
Protein Structures ◽  
Particle Analysis ◽  
X Ray Crystallography ◽  
Cryo Electron Microscopy ◽  
Recent Advances ◽  
Cellular Environment

Cryo-electron microscopy (CryoEM) has superseded X-ray crystallography and NMR to emerge as a popular and effective tool for structure determination in recent times. It has become indispensable for the characterization of large macromolecular assemblies, membrane proteins, or samples that are limited, conformationally heterogeneous, and recalcitrant to crystallization. Besides, it is the only tool capable of elucidating high-resolution structures of macromolecules and biological assemblies in situ. A state-of-the-art electron microscope operable at cryo-temperature helps preserve high-resolution details of the biological sample. The structures can be determined, either in isolation via single-particle analysis (SPA) or helical reconstruction, electron diffraction (ED) or within the cellular environment via cryo-electron tomography (cryoET). All the three streams of SPA, ED, and cryoET (along with subtomogram averaging) have undergone significant advancements in recent times. This has resulted in breaking the boundaries with respect to both the size of the macromolecules/assemblies whose structures could be determined along with the visualization of atomic details at resolutions unprecedented for cryoEM. In addition, the collection of larger datasets combined with the ability to sort and process multiple conformational states from the same sample are providing the much-needed link between the protein structures and their functions. In overview, these developments are helping scientists decipher the molecular mechanism of critical cellular processes, solve structures of macromolecules that were challenging targets for structure determination until now, propelling forward the fields of biology and biomedicine. Here, we summarize recent advances and key contributions of the three cryo-electron microscopy streams of SPA, ED, and cryoET.

scholarly journals User Manual for Tomography-Guided 3D Reconstruction of Subcellular Structures (TYGRESS)

2019 ◽  
Author(s):  
Zhiguo Shang ◽  
Kangkang Song ◽  
Xiaofeng Fu ◽  
Xiaochu Lou ◽  
Nikolaus Grigorieff ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
3D Reconstruction ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Intact Cells ◽  
Cryo Electron Microscopy ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging ◽  
Crowded Environment

Abstract Recent advances in cryo-electron microscopy (cryo-EM) are paving the way to determining isolated three-dimensional (3D) macromolecular structures at near-atomic resolution using single-particle cryo-electron microscopy (SP-cryo-EM). However, determining the subcellular structures in intact cells and organelles using cryo-electron tomography (cryo-ET) and subtomogram averaging, another cryo-EM technique, with comparable resolution remains a challenge. Current methodologies can only reach a resolution of several nanometers in most samples studied. Here, we introduce a new hybrid method, called Tomography-Guided 3D Reconstruction of Subcellular Structures (TYGRESS) that is able to achieve structural determination of subcellular structures within their natural crowded environment with nanometer-resolution by combining the advantages of cryo-ET and SP-cryo-EM.

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

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S7-S7
Author(s):  
Roman Kamyshinsky ◽  
Yury Chesnokov ◽  
Liubov Dadinova ◽  
Andrey Mozhaev ◽  
Alexander Vasiliev ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Particle Analysis ◽  
Phase Plate ◽  
Dna Fragments ◽  
Linear Dna ◽  
Thermofisher Scientific ◽  
X Ray Scattering ◽  
Subtomogram Averaging ◽  
Dps Protein ◽  
Dna Complex

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.

scholarly journals Cryo-Electron microscopy for the study of self-assembled poly(ionic liquid) nanoparticles and protein supramolecular structures

2020 ◽  
Vol 298 (7) ◽  
pp. 707-717
Author(s):  
Zdravko Kochovski ◽  
Guosong Chen ◽  
Jiayin Yuan ◽  
Yan Lu
Keyword(s):  
Electron Microscopy ◽  
Ionic Liquid ◽  
Sample Preparation ◽  
Self Assembly ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Particle Analysis ◽  
Cryo Electron Microscopy ◽  
Self Assembled ◽  
Poly Ionic Liquid

Abstract Cryo-electron microscopy (cryo-EM) is a powerful structure determination technique that is well-suited to the study of protein and polymer self-assembly in solution. In contrast to conventional transmission electron microscopy (TEM) sample preparation, which often times involves drying and staining, the frozen-hydrated sample preparation allows the specimens to be kept and imaged in a state closest to their native one. Here, we give a short overview of the basic principles of Cryo-EM and review our results on applying it to the study of different protein and polymer self-assembled nanostructures. More specifically, we show how we have applied cryo-electron tomography (cryo-ET) to visualize the internal morphology of self-assembled poly(ionic liquid) nanoparticles and cryo-EM single particle analysis (SPA) to determine the three-dimensional (3D) structures of artificial protein microtubules.

scholarly journals Orientating the Future Bio-Macromolecular Electron Microscopy

2018 ◽  
Author(s):  
Fei Sun
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Electron Tomography ◽  
Particle Analysis ◽  
Electron Radiation ◽  
Conformational Heterogeneity ◽  
Single Particle Analysis ◽  
Cryo Electron Microscopy ◽  
Air Water Interface ◽  
The Future

With forty years of developments, bio-macromolecule cryo-electron microscopy has met its revolution of resolution and is playing a very important role in structural biology study. According to different specimen states, cryo-electron microscopy (cryo-EM) involves three specific techniques, single particle analysis (SPA), electron tomography and sub-tomogram averaging, and electron diffraction. All these three techniques have not realized their full potentials of solving structures of bio-macromolecules and therefore need to be developed in the future. In this review, the current existing bottlenecks of cryo-EM SPA are discussed with theoretical analysis, which includes air-water interface during specimen cryo-vitrification, bio-macromolecular conformational heterogeneity, focus gradient within thick specimen, and electron radiation damage. Besides, potential solutions of these bottlenecks are proposed and discussed, which are worthy of further investigations in the future.

scholarly journals Orientating the Future Bio-Macromolecular Electron Microscopy

2018 ◽  
Author(s):  
Fei Sun
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Electron Tomography ◽  
Particle Analysis ◽  
Electron Radiation ◽  
Conformational Heterogeneity ◽  
Single Particle Analysis ◽  
Cryo Electron Microscopy ◽  
Air Water Interface ◽  
The Future

With forty years of developments, bio-macromolecule cryo-electron microscopy has met its revolution of resolution and is playing a very important role in structural biology study. According to different specimen states, cryo-electron microscopy (cryo-EM) involves three specific techniques, single particle analysis (SPA), electron tomography and sub-tomogram averaging, and electron diffraction. All these three techniques have not realized their full potentials of solving structures of bio-macromolecules and therefore need to be developed in the future. In this review, the current existing bottlenecks of cryo-EM SPA are discussed with theoretical analysis, which includes air-water interface during specimen cryo-vitrification, bio-macromolecular conformational heterogeneity, focus gradient within thick specimen, and electron radiation damage. Besides, potential solutions of these bottlenecks are proposed and discussed, which are worthy of further investigations in the future.

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