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.

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Cryo-electron microscopy (CryoEM) has superseded X-ray crystallography and NMR to emerge as a popular and effective tool for structure determination in recent times.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 Approaches to altering particle distributions in cryo-electron microscopy sample preparation

2018 ◽  
Vol 74 (6) ◽  
pp. 560-571 ◽  
Author(s):  
Ieva Drulyte ◽  
Rachel M. Johnson ◽  
Emma L. Hesketh ◽  
Daniel L. Hurdiss ◽  
Charlotte A. Scarff ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Sample Preparation ◽  
Single Particle ◽  
Structural Information ◽  
Particle Analysis ◽  
Diverse Range ◽  
Sample Distribution ◽  
High Resolution Data ◽  
Cryo Electron Microscopy

Cryo-electron microscopy (cryo-EM) can now be used to determine high-resolution structural information on a diverse range of biological specimens. Recent advances have been driven primarily by developments in microscopes and detectors, and through advances in image-processing software. However, for many single-particle cryo-EM projects, major bottlenecks currently remain at the sample-preparation stage; obtaining cryo-EM grids of sufficient quality for high-resolution single-particle analysis can require the careful optimization of many variables. Common hurdles to overcome include problems associated with the sample itself (buffer components, labile complexes), sample distribution (obtaining the correct concentration, affinity for the support film), preferred orientation, and poor reproducibility of the grid-making process within and between batches. This review outlines a number of methodologies used within the electron-microscopy community to address these challenges, providing a range of approaches which may aid in obtaining optimal grids for high-resolution data collection.

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 High-resolution cryo-EM using beam-image shift at 200 keV

2020 ◽  
Author(s):  
Jennifer N. Cash ◽  
Sarah Kearns ◽  
Yilai Li ◽  
Michael A. Cianfrocco
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Data Collection ◽  
Data Quality ◽  
Single Particle ◽  
Aberration Correction ◽  
Cryo Electron Microscopy ◽  
Beam Image ◽  
Recent Advances ◽  
Shift Data

ABSTRACTRecent advances in single-particle cryo-electron microscopy (cryo-EM) data collection utilizes beam-image shift to improve throughput. Despite implementation on 300 keV cryo-EM instruments, it remains unknown how well beam-image shift data collection affects data quality on 200 keV instruments and how much aberrations can be computationally corrected. To test this, we collected and analyzed a cryo-EM dataset of aldolase at 200 keV using beam-image shift. This analysis shows that beam tilt on the instrument initially limited the resolution of aldolase to 4.9Å. After iterative rounds of aberration correction and particle polishing in RELION, we were able to obtain a 2.8Å structure. This analysis demonstrates that software correction of microscope aberrations can provide a significant improvement in resolution at 200 keV.

scholarly journals Live analysis and reconstruction of single-particle cryo-electron microscopy data with CryoFLARE

2019 ◽  
Author(s):  
Andreas D. Schenk ◽  
Simone Cavadini ◽  
Nicolas H. Thomä ◽  
Christel Genoud
Keyword(s):  
Electron Microscopy ◽  
Data Collection ◽  
Single Particle ◽  
Integrated Reporting ◽  
Seamless Integration ◽  
High Resolution Data ◽  
User Input ◽  
Cryo Electron Microscopy ◽  
New Processing ◽  
Set Up

AbstractEfficient, reproducible and accountable single-particle cryo-electron microscopy structure determination is tedious and often impeded by lack of a standardized procedure for data analysis and processing. To address this issue, we have developed the FMI Live Analysis and Reconstruction Engine (CryoFLARE). CryoFLARE is a modular open-source platform offering easy integration of new processing algorithms developed by the cryo-EM community. It provides a user-friendly interface that allows fast setup of standardized workflows, serving the need of pharmaceutical industry and academia alike who need to optimize throughput of their microscope. To consistently document how data is processed, CryoFLARE contains an integrated reporting facility to create reports.Live analysis and processing parallel to data acquisition are used to monitor and optimize data quality. Problems at the level of the sample preparation (heterogeneity, ice thickness, sparse particles, areas selected for acquisition, etc.) or misalignments of the microscope optics can quickly be detected and rectified before data collection is continued. Interfacing with automated data collection software for retrieval of acquisition metadata reduces user input needed for analysis, and with it minimizes potential sources of errors and workflow inconsistencies. Local and remote export support in Relion-compatible job and data format allows seamless integration into the refinement process. The support for non-linear workflows and fine-grained scheduling for mixed workflows with separate CPU and GPU based calculation steps ensures optimal use of processing hardware. CryoFLARE’s flexibility allows it to be used for all types of image acquisitions, ranging from sample screening to high-resolution data collection, and offers a new alternative for setting up image processing workflows. It can be used without modifications of the hardware/software delivered by the microscope supplier. As it is running on a server in parallel to the hardware used for acquisition, it can easily be set up for remote display connections and fast control of the acquisition status.

scholarly journals Autologous Antibody Responses to an HIV Envelope Glycan Hole Are Not Easily Broadened in Rabbits

2020 ◽  
Vol 94 (7) ◽  
Author(s):  
Yuhe R. Yang ◽  
Laura E. McCoy ◽  
Marit J. van Gils ◽  
Raiees Andrabi ◽  
Hannah L. Turner ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Neutralizing Antibodies ◽  
Cross Reactivity ◽  
High Resolution Data ◽  
Subtype B ◽  
Cryo Electron Microscopy ◽  
Antibody Complex ◽  
Subtype A ◽  
Hiv Envelope

ABSTRACT Extensive studies with subtype A BG505-derived HIV envelope glycoprotein (Env) immunogens have revealed that the dominant autologous neutralizing epitope in rabbits is located in an exposed region of the heavily glycosylated trimer that lacks potential N-linked glycosylation sites at positions 230, 241, and 289. The Env derived from B41, a subtype B virus, shares a glycan hole centered on positions 230 and 289. To test whether broader neutralization to the common glycan hole can be achieved, we immunized rabbits with B41 SOSIP (gp120-gp41 disulfide [SOS] with an isoleucine-to-proline mutation [IP] in gp41) alone, as well as B41 and BG505 coimmunization. We isolated autologous neutralizing antibodies (nAbs) and described their structure in complex with the B41 Env. Our data suggest that distinct autologous nAb lineages are induced by BG505 and B41 immunogens, even when both were administered together. In contrast to previously described BG505 glycan hole antibodies, the B41-specific nAbs accommodate the >97% conserved N241 glycan, which is present in B41. Single-particle cryo-electron microscopy studies confirmed that B41- and BG505-specific nAbs bind to overlapping glycan hole epitopes. We then used our high-resolution data to guide mutations in the BG505 glycan hole epitope in an attempt to broaden the reactivity of a B41-specific nAb, but we recovered only partial binding. Our data demonstrate that the lack of cross-reactivity in glycan hole antibodies is due to amino acid differences within the epitope, and our attempts to rationally design cross-reactive trimers resulted in only limited success. Thus, even for the immunodominant glycan hole shared between BG505 and B41, the prospect of designing prime-boost immunogens remains difficult. IMPORTANCE A glycan hole is one of the most dominant autologous neutralizing epitopes targeted on BG505 and B41 SOSIP trimer-immunized rabbits. Our high-resolution cryo-electron microscopy (cryoEM) studies of B41 in complex with a B41-specific antibody complex elucidate the molecular basis of this strain-specific glycan hole response. We conclude that even for the immunodominant glycan hole shared between BG505 and B41, the prospect of designing prime-boost immunogens remains difficult.

scholarly journals High-resolution cryo-EM using beam-image shift at 200 keV

IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 1179-1187 ◽  
Author(s):  
Jennifer N. Cash ◽  
Sarah Kearns ◽  
Yilai Li ◽  
Michael A. Cianfrocco
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Data Collection ◽  
Single Particle ◽  
Particle Motion ◽  
Data Set ◽  
Cryo Electron Microscopy ◽  
Beam Image ◽  
Shift Data ◽  
Resolution Structure

Recent advances in single-particle cryo-electron microscopy (cryo-EM) data collection utilize beam-image shift to improve throughput. Despite implementation on 300 keV cryo-EM instruments, it remains unknown how well beam-image-shift data collection affects data quality on 200 keV instruments and the extent to which aberrations can be computationally corrected. To test this, a cryo-EM data set for aldolase was collected at 200 keV using beam-image shift and analyzed. This analysis shows that the instrument beam tilt and particle motion initially limited the resolution to 4.9 Å. After particle polishing and iterative rounds of aberration correction in RELION, a 2.8 Å resolution structure could be obtained. This analysis demonstrates that software correction of microscope aberrations can provide a significant improvement in resolution at 200 keV.

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 Low cost, high performance processing of single particle cryo-electron microscopy data in the cloud

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Michael A Cianfrocco ◽  
Andres E Leschziner
Keyword(s):  
Electron Microscopy ◽  
Cloud Computing ◽  
High Resolution ◽  
Single Particle ◽  
High Performance ◽  
Low Cost ◽  
Computing Environment ◽  
Cryo Electron Microscopy ◽  
Electron Microscopes ◽  
Microscopy Data

The advent of a new generation of electron microscopes and direct electron detectors has realized the potential of single particle cryo-electron microscopy (cryo-EM) as a technique to generate high-resolution structures. Calculating these structures requires high performance computing clusters, a resource that may be limiting to many likely cryo-EM users. To address this limitation and facilitate the spread of cryo-EM, we developed a publicly available ‘off-the-shelf’ computing environment on Amazon's elastic cloud computing infrastructure. This environment provides users with single particle cryo-EM software packages and the ability to create computing clusters with 16–480+ CPUs. We tested our computing environment using a publicly available 80S yeast ribosome dataset and estimate that laboratories could determine high-resolution cryo-EM structures for $50 to $1500 per structure within a timeframe comparable to local clusters. Our analysis shows that Amazon's cloud computing environment may offer a viable computing environment for cryo-EM.

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.

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