scholarly journals Size matters: optimal mask diameter and box size for single-particle cryogenic electron microscopy

2020 ◽  
Author(s):  
Toshio Moriya ◽  
Naruhiko Adachi ◽  
Masato Kawasaki ◽  
Yusuke Yamada ◽  
Akira Shinoda ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Protein Structures ◽  
Atomic Resolution ◽  
Phase Plate ◽  
Contrast Transfer Function ◽  
Protein Size ◽  
Typical Parameter ◽  
Acceleration Voltage ◽  
Processing Steps

AbstractRecently it has been demonstrated that single-particle cryogenic electron microscopy (cryo-EM) at 200 keV is capable of determining protein structures, including those smaller than 100 kDa, at sub-3.0 Å resolutions, without using significant defocus or a phase plate. However, the majority of near-atomic resolution cryo-EM structures has been determined using 300 keV. Consequently, many typical parameter settings for the cryo-EM computational image processing steps, especially those associated with the contrast transfer function, are based on the accumulated experience of 300 kV cryo-EM. We have therefore revised these parameters, established theoretical bases for criteria to find an optimal mask diameter and box size for a given dataset irrespective of acceleration voltage or protein size, and proposed a protocol. Considering the defocus distributions of the datasets, merely optimizing the mask diameters and box sizes yielded meaningful resolution improvements for the reconstruction of < 200 kDa proteins using 200 kV cryo-EM.

A3Near-atomic Resolution Single Particle Analysis with the Volta Phase Plate

Microscopy ◽  
2015 ◽  
Vol 64 (suppl 1) ◽  
pp. i9.2-i9
Author(s):  
Radostin Danev ◽  
Maryam Khoshouei ◽  
Wolfgang Baumeister
Keyword(s):  
Single Particle ◽  
Atomic Resolution ◽  
Particle Analysis ◽  
Phase Plate ◽  
Single Particle Analysis ◽  
Resolution Single

scholarly journals Using the Volta phase plate with defocus for cryo-EM single particle analysis

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Radostin Danev ◽  
Dimitry Tegunov ◽  
Wolfgang Baumeister
Keyword(s):  
Data Acquisition ◽  
Single Particle ◽  
Hybrid Approach ◽  
Particle Analysis ◽  
Phase Plate ◽  
Single Particle Analysis ◽  
Resolution Limit ◽  
Contrast Transfer Function ◽  
Acquisition Speed ◽  
Acquisition Method

Previously, we reported an in-focus data acquisition method for cryo-EM single-particle analysis with the Volta phase plate (Danev and Baumeister, 2016). Here, we extend the technique to include a small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also removes the resolution limit inherent to the in-focus method thus allowing 3D reconstructions with resolutions better than 3 Å.

scholarly journals Contrast Transfer Function-Based Exit-Wave Reconstruction and Denoising of Atomic-Resolution Transmission Electron Microscopy Images of Graphene and Cu Single Atom Substitutions by Deep Learning Framework

Nanomaterials ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1977
Author(s):  
Jongyeong Lee ◽  
Yeongdong Lee ◽  
Jaemin Kim ◽  
Zonghoon Lee
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Transfer Function ◽  
Signal To Noise Ratio ◽  
Atomic Resolution ◽  
Single Atom ◽  
Monolayer Graphene ◽  
Signal To Noise ◽  
Contrast Transfer Function ◽  
Transmission Electron

The exit wave is the state of a uniform plane incident electron wave exiting immediately after passing through a specimen and before the atomic-resolution transmission electron microscopy (ARTEM) image is modified by the aberration of the optical system and the incoherence effect of the electron. Although exit-wave reconstruction has been developed to prevent the misinterpretation of ARTEM images, there have been limitations in the use of conventional exit-wave reconstruction in ARTEM studies of the structure and dynamics of two-dimensional materials. In this study, we propose a framework that consists of the convolutional dual-decoder autoencoder to reconstruct the exit wave and denoise ARTEM images. We calculated the contrast transfer function (CTF) for real ARTEM and assigned the output of each decoder to the CTF as the amplitude and phase of the exit wave. We present exit-wave reconstruction experiments with ARTEM images of monolayer graphene and compare the findings with those of a simulated exit wave. Cu single atom substitution in monolayer graphene was, for the first time, directly identified through exit-wave reconstruction experiments. Our exit-wave reconstruction experiments show that the performance of the denoising task is improved when compared to the Wiener filter in terms of the signal-to-noise ratio, peak signal-to-noise ratio, and structural similarity index map metrics.

Instrumentation at the National Center for Electron Microscopy: The atomic resolution microscope

1983 ◽  
Vol 41 ◽  
pp. 310-311 ◽  
Author(s):  
R. Gronsky ◽  
G. Thomas
Keyword(s):  
Electron Microscopy ◽  
Atomic Resolution ◽  
Objective Lens ◽  
Contrast Transfer Function ◽  
Voltage Electron ◽  
Variable Voltage ◽  
New Instrument ◽  
Performance Specifications ◽  
Electron Microscopes ◽  
Point To Point

The Atomic Resolution Microscope (ARM) is one of two unique high voltage electron microscopes at the Lawrence Berkeley Laboratory's National Center for Electron Microscopy (NCEM). This paper reports on the latest results from this new instrument which was manufactured by JEOL, Ltd. to the performance specifications of the NCEM, delivered in January of 1983, and soon to be open to access by the entire microscopy community. Details of its history and development are given in reference 1; its performance specifications are reviewed below.Adopting as a design definition for resolution the first zero crossover of th% phase contrast transfer function at Scherzer defocus, the ARM (Fig. 1) maintains 1.7Å point-to-point resolution over its 400kV to 1000kV operating range. Consequently the microscope can be tuned to a voltage which is below the threshold for knock-on damage in a specimen and used to directly image its contiguous-atom structure. The key to this variable-voltage, high-resolution performance is a top-entry objective stage, which, in addition to ± 40° biaxial tilting, incorporates a height (Z)-control to alter specimen position within the objective lens.

scholarly journals 2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Melody G Campbell ◽  
David Veesler ◽  
Anchi Cheng ◽  
Clinton S Potter ◽  
Bridget Carragher
Keyword(s):  
Electron Microscopy ◽  
Drug Design ◽  
Single Particle ◽  
Protein Structures ◽  
Rational Drug Design ◽  
20S Proteasome ◽  
Thermoplasma Acidophilum ◽  
Structure Based Drug Design ◽  
Cryo Electron Microscopy ◽  
Pharmaceutical Industries

Recent developments in detector hardware and image-processing software have revolutionized single particle cryo-electron microscopy (cryoEM) and led to a wave of near-atomic resolution (typically ∼3.3 Å) reconstructions. Reaching resolutions higher than 3 Å is a prerequisite for structure-based drug design and for cryoEM to become widely interesting to pharmaceutical industries. We report here the structure of the 700 kDa Thermoplasma acidophilum 20S proteasome (T20S), determined at 2.8 Å resolution by single-particle cryoEM. The quality of the reconstruction enables identifying the rotameric conformation adopted by some amino-acid side chains (rotamers) and resolving ordered water molecules, in agreement with the expectations for crystal structures at similar resolutions. The results described in this manuscript demonstrate that single particle cryoEM is capable of competing with X-ray crystallography for determination of protein structures of suitable quality for rational drug design.

scholarly journals Structure Determination by Single-Particle Cryo-Electron Microscopy: Only the Sky (and Intrinsic Disorder) is the Limit

2019 ◽  
Vol 20 (17) ◽  
pp. 4186 ◽  
Author(s):  
Emeka Nwanochie ◽  
Vladimir N. Uversky
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Structure Determination ◽  
Single Particle ◽  
Protein Structures ◽  
X Ray ◽  
X Ray Crystallography ◽  
Intrinsically Disordered ◽  
Small Proteins ◽  
Cryo Electron Microscopy

Traditionally, X-ray crystallography and NMR spectroscopy represent major workhorses of structural biologists, with the lion share of protein structures reported in protein data bank (PDB) being generated by these powerful techniques. Despite their wide utilization in protein structure determination, these two techniques have logical limitations, with X-ray crystallography being unsuitable for the analysis of highly dynamic structures and with NMR spectroscopy being restricted to the analysis of relatively small proteins. In recent years, we have witnessed an explosive development of the techniques based on Cryo-electron microscopy (Cryo-EM) for structural characterization of biological molecules. In fact, single-particle Cryo-EM is a special niche as it is a technique of choice for the structural analysis of large, structurally heterogeneous, and dynamic complexes. Here, sub-nanometer atomic resolution can be achieved (i.e., resolution below 10 Å) via single-particle imaging of non-crystalline specimens, with accurate 3D reconstruction being generated based on the computational averaging of multiple 2D projection images of the same particle that was frozen rapidly in solution. We provide here a brief overview of single-particle Cryo-EM and show how Cryo-EM has revolutionized structural investigations of membrane proteins. We also show that the presence of intrinsically disordered or flexible regions in a target protein represents one of the major limitations of this promising technique.

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