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.

scholarly journals Coma-corrected rapid single-particle cryo-EM data collection on the CRYO ARM 300

2021 ◽  
Vol 77 (5) ◽  
pp. 555-564
Author(s):  
Rouslan G. Efremov ◽  
Annelore Stroobants
Keyword(s):  
High Resolution ◽  
Data Collection ◽  
Single Particle ◽  
Protein Complexes ◽  
Large Area ◽  
Beam Image ◽  
Resolution Electron ◽  
High Resolution Images ◽  
Electron Microscopes ◽  
Data Collection Procedure

Single-particle cryogenic electron microscopy has recently become a major method for determining the structures of proteins and protein complexes. This has markedly increased the demand for throughput of high-resolution electron microscopes, which are required to produce high-resolution images at high rates. An increase in data-collection throughput can be achieved by using large beam-image shifts combined with off-axis coma correction, enabling the acquisition of multiple images from a large area of the EM grid without moving the microscope stage. Here, the optical properties of the JEOL CRYO ARM 300 electron microscope equipped with a K3 camera were characterized under off-axis illumination conditions. It is shown that efficient coma correction can be achieved for beam-image shifts with an amplitude of at least 10 µm, enabling a routine throughput for data collection of between 6000 and 9000 images per day. Use of the benchmark for the rapid data-collection procedure (with beam-image shifts of up to 7 µm) on apoferritin resulted in a reconstruction at a resolution of 1.7 Å. This demonstrates that the rapid automated acquisition of high-resolution micrographs is possible using a CRYO ARM 300.

scholarly journals Practical Considerations of Using the K3 Camera in CDS Mode for High-resolution and High-throughput Single Particle Cryo-EM

2020 ◽  
Vol 26 (S2) ◽  
pp. 1320-1320
Author(s):  
Ming Sun ◽  
Caleigh Azumaya ◽  
Eric Tse ◽  
Daniel Southworth ◽  
Kliment Verba ◽  
...  
Keyword(s):  
High Resolution ◽  
High Throughput ◽  
Single Particle

A New ion Beam Based Etching/Coating System for Scanning Electron Microscopy (SEM) and Light Microscopy (LM)

1997 ◽  
Vol 3 (S2) ◽  
pp. 363-364
Author(s):  
R. Alani ◽  
R.J. Mitro ◽  
C.M. Tabatt ◽  
L. Malaszewski
Keyword(s):  
High Resolution ◽  
Ion Beam ◽  
Coating System ◽  
Etching Technique ◽  
Specimen Handling ◽  
Conductive Films ◽  
Resolution Electron ◽  
New Instrument ◽  
Electron Microscopes ◽  
And Performance

The design and performance of a new instrument, based on improved Penning ion guns [1] for etching and coating samples for SEM and LM in a single vacuum chamber, are described. The instrument is based on an existing high resolution ion beam coating system, which is capable of producing high quality ultra-thin and amorphous conductive films, required for present high resolution electron microscopes. [2]. The fact that in this system both etching and coating processes are combined in one chamber, the specimen handling and specimen contamination are minimized. Furthermore, the system eliminates the traditional multiple mounting /dismounting of samples to various holders for mechanical polishing, etching, coating and microscopy purposes. The specimen can stay with the same holder throughout the entire process, increasing the sample through-put. Moreover, the system offers an alternative method to the traditional “wet chemical etching,” technique with its well known problems.

scholarly journals cisTEM, user-friendly software for single-particle image processing

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Timothy Grant ◽  
Alexis Rohou ◽  
Nikolaus Grigorieff
Keyword(s):  
High Resolution ◽  
Single Particle ◽  
Imaging System ◽  
Computational Imaging ◽  
Computer Clusters ◽  
Large Computer ◽  
Transmission Electron ◽  
Resolution Electron ◽  
Map Generation ◽  
User Friendly

We have developed new open-source software called cisTEM (computational imaging system for transmission electron microscopy) for the processing of data for high-resolution electron cryo-microscopy and single-particle averaging. cisTEM features a graphical user interface that is used to submit jobs, monitor their progress, and display results. It implements a full processing pipeline including movie processing, image defocus determination, automatic particle picking, 2D classification, ab-initio 3D map generation from random parameters, 3D classification, and high-resolution refinement and reconstruction. Some of these steps implement newly-developed algorithms; others were adapted from previously published algorithms. The software is optimized to enable processing of typical datasets (2000 micrographs, 200 k – 300 k particles) on a high-end, CPU-based workstation in half a day or less, comparable to GPU-accelerated processing. Jobs can also be scheduled on large computer clusters using flexible run profiles that can be adapted for most computing environments. cisTEM is available for download from cistem.org.

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.

scholarly journals Energy optimization of a regular macromolecular crystallography beamline for ultra-high-resolution crystallography

2015 ◽  
Vol 22 (1) ◽  
pp. 172-174 ◽  
Author(s):  
Gerd Rosenbaum ◽  
Stephan L. Ginell ◽  
Julian C.-H. Chen
Keyword(s):  
High Resolution ◽  
Data Collection ◽  
Structural Biology ◽  
Practical Method ◽  
Accurate Data ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
Standard Operating ◽  
Photon Source ◽  
Synchrotron Beamlines

A practical method for operating existing undulator synchrotron beamlines at photon energies considerably higher than their standard operating range is described and applied at beamline 19-ID of the Structural Biology Center at the Advanced Photon Source enabling operation at 30 keV. Adjustments to the undulator spectrum were critical to enhance the 30 keV flux while reducing the lower- and higher-energy harmonic contamination. A Pd-coated mirror and Al attenuators acted as effective low- and high-bandpass filters. The resulting flux at 30 keV, although significantly lower than with X-ray optics designed and optimized for this energy, allowed for accurate data collection on crystals of the small protein crambin to 0.38 Å resolution.

scholarly journals New opportunities in structural biology with XFEL: from sources to structures

2014 ◽  
Vol 70 (a1) ◽  
pp. C19-C19
Author(s):  
Soichi Wakatsuki
Keyword(s):  
Data Collection ◽  
Radiation Damage ◽  
Structural Biology ◽  
Single Particle ◽  
De Novo ◽  
Reconstruction Algorithm ◽  
Simultaneous Recording ◽  
Atomic Resolution ◽  
X Ray ◽  
Biology Research

X-ray free electron lasers (XFEL) have shown the promise of providing new opportunities in structural biology research with their extremely high peak brilliance and short pulses. It is reaching the stage where biologically important questions can be tackled using XFEL based on the "diffract-before-destroy" concept. The first part of this presentation will focus on macromolecular crystallography using XFEL with results obtained at LCLS so far and future scope. R&D efforts being pursued at SLAC/LCLS include new beam modes, (two-color beam for de novo phasing, wider bandwidth for SAXS/WAXS and spectroscopy), beam multiplexing, a dedicated new station for in-air data collection, next generation detectors, data analysis incorporating pulse-by-pulse spectrometer measurements and post refinement. These projects are being pursued in collaboration with many groups locally and globally with a goal to provide integrated facilities for cutting edge structural biology research. For example, two-color self-seeded XFEL mode is being developed for simultaneous recording of diffraction data at two energies in order to optimize the dispersive difference between the two wavelengths for phasing. Another area of collaborative effort is a development of dedicated station for in-air data collection with a variety of sample delivery schemes. The second part will discuss a possible roadmap towards atomic resolution single particle imaging using XFEL. Here, key questions are ·Can XFEL single particle 3D structural analysis at atomic resolution be done? ·What is the pulse characteristics required? ·Can we overcome the radiation damage at soft X-ray regime? ·What is the highest resolution attainable in comparison with cryoEM? A workshop at LCLS is being organized to discuss these questions in 4 areas: radiation damage, image reconstruction algorithm, beam modes and instrumentation, sample delivery and heterogeneity. The outcome of the workshop and follow-up discussions will be presented.

High-throughput, high-resolution electron-beam lithography

1988 ◽  
Vol 32 (4) ◽  
pp. 494-501 ◽  
Author(s):  
H. C. Pfeiffer ◽  
T. R. Groves ◽  
T. H. Newman
Keyword(s):  
Electron Beam ◽  
High Resolution ◽  
High Throughput ◽  
Electron Beam Lithography ◽  
Resolution Electron
Sign in / Sign up

Export Citation Format

Share Document