scholarly journals Epoxidized graphene grid for high-throughput high-resolution cryoEM structural analysis

2021 ◽  
Author(s):  
Junso Fujita ◽  
Fumiaki Makino ◽  
Haruyasu Asahara ◽  
Maiko Moriguchi ◽  
Shota Kumano ◽  
...  
Keyword(s):  
Structural Analysis ◽  
High Resolution ◽  
Data Collection ◽  
High Throughput ◽  
Particle Density ◽  
Orientation Distribution ◽  
Biological Macromolecules ◽  
Density Maps ◽  
V1 Atpase ◽  
Density Map

Many specimens suffer from low particle density and/or preferred orientation in cryoEM specimen grid preparation, making data collection and structure determination time consuming. We developed an epoxidized graphene grid (EG-grid) that effectively immobilizes protein particles by applying an oxidation reaction using photoactivated ClO2· and further chemical modification. The particle density and orientation distribution are both dramatically improved, having enabled us to reconstruct the density map of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), at 1.99 and 2.16 Å resolution from only 504 and 241 micrographs, respectively. A low concentration sample solution of 0.1 mg ml–1 was sufficient to reconstruct a 3.10 Å resolution density map of SARS-CoV-2 spike protein from 1,163 micrographs. The density maps of V1-ATPase, β-galactosidase, and apoferritin were also reconstructed at 3.03, 1.81, and 1.29 Å resolution, respectively. These results indicate that the EG-grid will be a powerful tool for high-throughput cryoEM data collection to accelerate high-resolution structural analysis of biological macromolecules.

High Resolution Microscopy of Multi-Layered Biological Crystals

1982 ◽  
Vol 40 ◽  
pp. 80-83
Author(s):  
H.A. Cohen ◽  
T.W. Jeng ◽  
W. Chiu
Keyword(s):  
High Resolution ◽  
Low Dose ◽  
Three Dimensional ◽  
Data Interpretation ◽  
Two Dimensional ◽  
Biological Objects ◽  
Density Maps ◽  
Density Map ◽  
Complex Crystal ◽  
High Resolution Microscopy

This tutorial will discuss the methodology of low dose electron diffraction and imaging of crystalline biological objects, the problems of data interpretation for two-dimensional projected density maps of glucose embedded protein crystals, the factors to be considered in combining tilt data from three-dimensional crystals, and finally, the prospects of achieving a high resolution three-dimensional density map of a biological crystal. This methodology will be illustrated using two proteins under investigation in our laboratory, the T4 DNA helix destabilizing protein gp32*I and the crotoxin complex crystal.

scholarly journals In-depth structural analysis of glycans in the gas phase

2019 ◽  
Vol 10 (5) ◽  
pp. 1272-1284 ◽  
Author(s):  
Eike Mucha ◽  
Alexandra Stuckmann ◽  
Mateusz Marianski ◽  
Weston B. Struwe ◽  
Gerard Meijer ◽  
...  
Keyword(s):  
Structural Analysis ◽  
High Resolution ◽  
Gas Phase ◽  
High Throughput ◽  
Glycan Analysis ◽  
The Past

Although there have been substantial improvements in glycan analysis over the past decade, the lack of both high-resolution and high-throughput methods hampers progress in glycomics.

Simplified transfer of frozen-hydrated specimens into a commercially available cooling holder

1984 ◽  
Vol 42 ◽  
pp. 274-275
Author(s):  
B. D. Athey ◽  
J.P. Langmore
Keyword(s):  
Electron Microscope ◽  
Structural Analysis ◽  
High Resolution ◽  
Liquid Nitrogen ◽  
Manual Dexterity ◽  
New Method ◽  
Biological Macromolecules ◽  
Cooling Stage

The examination of frozen-hydrated specimens maintained at liquid nitrogen (LN2) temperatures within the electron microscope is now established as an important technique to perform high resolution structural analysis of biological macromolecules. In some commercially available side-entry cold stages, it is often impossible to take advantage of this new method because hydration of the frozen specimen during grid placement and subsequent transfer of the stage into the EM makes the sample unusable. Recently, modifications to allow for frost-free transfer under LN2 have been reported for the JEOL EMSCH cooling stage, available for the JEOL JEM 100CX. Detailed below is a further change to this holder which greatly improves the reproducability of specimen transfer, reducing the time and manual dexterity needed for this process.

Structure Refinement Against Synchrotron Laue Data: Strategies for Data Collection and Reduction

1998 ◽  
Vol 54 (3) ◽  
pp. 367-377 ◽  
Author(s):  
Xiaojing Yang ◽  
Zhong Ren ◽  
Keith Moffat
Keyword(s):  
High Resolution ◽  
Data Collection ◽  
Data Reduction ◽  
Structure Refinement ◽  
Detailed Comparison ◽  
Density Maps ◽  
Reduction Strategies ◽  
High Resolution Structure ◽  
Collection Strategies ◽  
Resolution Structure

The synchrotron Laue technique has been applied to high-resolution structure refinement of the ribotoxin, restrictocin [Yang & Moffat (1996). Structure, 4, 837–852]. By employing carefully designed data-collection strategies and the data-reduction algorithms incorporated in the software system LaueView [Ren & Moffat (1995a). J. Appl. Cryst. 28, 461–481; Ren & Moffat (1995b). J. Appl. Cryst. 28, 482–493], a set of high-resolution Laue data with a completeness and accuracy comparable to excellent monochromatic data was obtained. Through detailed comparison with the monochromatic data and electron-density maps derived from the Laue data, optimum data-collection and reduction strategies were identified and the application of Laue diffraction techniques to conventional crystallographic refinement was demonstrated.

scholarly journals Assessing the JEOL CRYO ARM 300 for high-throughput automated single-particle cryo-EM in a multiuser environment

IUCrJ ◽  
2020 ◽  
Vol 7 (4) ◽  
pp. 707-718
Author(s):  
Marcus Fislage ◽  
Alexander V. Shkumatov ◽  
Annelore Stroobants ◽  
Rouslan G. Efremov
Keyword(s):  
High Resolution ◽  
Data Collection ◽  
Structural Biology ◽  
High Throughput ◽  
Single Particle ◽  
Illumination System ◽  
Resolution Electron ◽  
Access Mode ◽  
User Access ◽  
And Performance

Single-particle cryo-EM has become an indispensable structural biology method. It requires regular access to high-resolution electron cryogenic microscopes. To fully utilize the capacity of the expensive high-resolution instruments, the time used for data acquisition and the rate of data collection have to be maximized. This in turn requires high stability and high uptime of the instrument. One of the first 300 kV JEOL CRYO ARM 300 microscopes has been installed at the cryo-EM facility BECM at VIB-VUB, Brussels, where the microscope is used for continuous data collection on multiple projects. Here, the suitability and performance of the microscope is assessed for high-throughput single-particle data collection. In particular, the properties of the illumination system, the stage stability and ice contamination rates are reported. The microscope was benchmarked using mouse heavy-chain apoferritin which was reconstructed to a resolution of 1.9 Å. Finally, uptime and throughput statistics of the instrument accumulated during the first six months of the facility operation in user access mode are reported.

Microfabricated mounts for high-throughput macromolecular cryocrystallography

2003 ◽  
Vol 36 (6) ◽  
pp. 1455-1460 ◽  
Author(s):  
R. E. Thorne ◽  
Z. Stum ◽  
J. Kmetko ◽  
K. O'Neill ◽  
R. Gillilan
Keyword(s):  
Data Collection ◽  
High Throughput ◽  
Thermal Mass ◽  
Biological Macromolecules ◽  
X Ray ◽  
Small Crystals ◽  
Patterned Structures ◽  
X Ray Background ◽  
Design Customization ◽  
Viscous Solutions

A new approach is described for mounting microcrystals of biological macromolecules for cryocrystallography. The sample mounts are prepared by patterning thin polyimide films by standard microfabrication techniques. The patterned structures contain a small hole for the crystal connected to a larger holeviaa drainage channel, allowing removal of excess liquid and easier manipulation in viscous solutions. These polyimide structures are wrapped around small metal rods. The resulting curvature increases their rigidity and allows a convenient scoop-like action in retrieving crystals. The polyimide contributes minimally to X-ray background and absorption, and can be treated to obtain desired hydrophobicity or hydrophilicity. The new mounts are fully compatible with existing automated sample-handling hardware for cryocrystallography. Their potential advantages include completely reproducible sample hole sizes to below 10 µm; accurate and reproducible sample positioning and good sample-to-mount contrast, simplifying alignment; more convenient manipulation of small crystals; easier removal of excess liquid and reduced background scatter; reduced thermal mass and more rapid flash-cooling; and easy design customization and mass production. They are especially well suited to data collection from the smaller crystals produced in high-throughput crystallization trials, and are suitable for automated crystal retrieval. They should be more generally useful for X-ray data collection from small organic and inorganic crystals of all types.

scholarly journals Practical considerations for using K3 cameras in CDS mode for high-resolution and high-throughput single particle cryo-EM

2020 ◽  
Author(s):  
Ming Sun ◽  
Caleigh Azumaya ◽  
Eric Tse ◽  
Adam Frost ◽  
Daniel Southworth ◽  
...  
Keyword(s):  
High Resolution ◽  
Data Collection ◽  
High Throughput ◽  
First Generation ◽  
Direct Detection ◽  
Protein Complexes ◽  
Large Data ◽  
Optimal Dose ◽  
Data Sets ◽  
Dose Rates

AbstractDetector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed nonCDS mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 Å with a 1.346 Å physical pixel). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834Å physical pixel size) we achieved a resolution of 1.65 Å within 9 hours of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 Å resolution with 5 minutes of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community.

scholarly journals Non-uniform refinement: Adaptive regularization improves single particle cryo-EM reconstruction

2019 ◽  
Author(s):  
Ali Punjani ◽  
Haowei Zhang ◽  
David J. Fleet
Keyword(s):  
Membrane Proteins ◽  
Single Particle ◽  
Iterative Refinement ◽  
Biological Macromolecules ◽  
Disordered Structures ◽  
Spatial Uniformity ◽  
Density Maps ◽  
Density Map ◽  
Particle Images ◽  
Lipid Nanodiscs

AbstractSingle particle cryo-EM is a powerful method for studying proteins and other biological macromolecules. Many of these molecules comprise regions with varying structural properties including disorder, flexibility, and partial occupancy. These traits make computational 3D reconstruction from 2D images challenging. Detergent micelles and lipid nanodiscs, used to keep membrane proteins in solution, are common examples of locally disordered structures that can negatively affect existing iterative refinement algorithms which assume rigidity (or spatial uniformity). We introduce a cross-validation approach to derive non-uniform refinement, an algorithm that automatically regularizes 3D density maps during iterative refinement to account for spatial variability, yielding dramatically improved resolution and 3D map quality. We find that in common iterative refinement methods, regularization using spatially uniform filtering operations can simultaneously over- and under-regularize local regions of a 3D map. In contrast, non-uniform refinement removes noise in disordered regions while retaining signal useful for aligning particle images. Our results include state-of-the-art resolution 3D reconstructions of multiple membrane proteins with molecular weight as low as 90kDa. These results demonstrate that higher resolutions and improved 3D density map quality can be achieved even for small membrane proteins, an important use case for single particle cryo-EM, both in structural biology and drug discovery. Non-uniform refinement is implemented in the cryoSPARC software package and has already been used successfully in several notable structural studies.

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