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

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.

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

10.1101/2020.01.21.914507 ◽

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.

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High resolution single particle cryo-electron microscopy using beam-image shift

Journal of Structural Biology ◽

10.1016/j.jsb.2018.07.015 ◽

2018 ◽

Vol 204 (2) ◽

pp. 270-275 ◽

Cited By ~ 42

Author(s):

Anchi Cheng ◽

Edward T. Eng ◽

Lambertus Alink ◽

William J. Rice ◽

Kelsey D. Jordan ◽

...

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Single Particle ◽

Cryo Electron Microscopy ◽

Beam Image ◽

Resolution Single

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Optimized data acquisition workflow by sample thickness determination

10.1101/2020.12.01.392100 ◽

2020 ◽

Author(s):

Jan Rheinberger ◽

Gert Oostergetel ◽

Guenter P Resch ◽

Cristina Paulino

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Data Collection ◽

Data Acquisition ◽

Data Storage ◽

Single Particle ◽

Collection Efficiency ◽

Sample Thickness ◽

Cryo Electron Microscopy ◽

Key Parameter

AbstractSample thickness is a known key parameter in cryo-electron microscopy (cryo-EM) and can affect the amount of high-resolution information retained in the image. Yet, common data acquisition approaches in single particle cryo-EM do not take it into account. Here, we demonstrate how the sample thickness can be determined before data acquisition, allowing to identify optimal regions and restrict data collection to images with preserved high-resolution details. This quality over quantity approach, almost entirely eliminates the time- and storage-consuming collection of suboptimal images, which are discarded after a recorded session or during early image processing due to lack of high-resolution information. It maximizes data collection efficiency and lowers the electron microscopy time required per dataset. This strategy is especially useful, if the speed of data collection is restricted by the microscope hardware and software, or if data transfer, data storage and computational power are a bottleneck.SynopsisDetermining sample thickness, a key parameter in single particle cryo-electron microscopy, before data acquisition, and targeting only optimal areas, maximizes the data output from a single particle cryo-electron microscopy session. Scripts and optimized workflows for EPU and SerialEM are presented utilizing this concept.

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Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

10.1101/2021.06.15.448552 ◽

2021 ◽

Author(s):

Nicole Dimos ◽

Carl P.O. Helmer ◽

Andrea M. Chanique ◽

Markus C. Wahl ◽

Robert Kourist ◽

...

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Structural Biology ◽

Structure Analysis ◽

Cryo Electron Microscopy ◽

High Resolution Structure ◽

Fast Development ◽

Pharmaceutical Industries ◽

Exquisite Specificity ◽

Resolution Structure

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryo-EM) has revolutionized structural biology, its applicability to high-resolution structure analysis of comparatively small enzymes is so far largely unexplored. Here, we show that cryo-EM can reveal the structures of ~120 kDa plant borneol dehydrogenases at or below 2 Å resolution, paving the way for the fast development of new biocatalysts that provide access to bioactive terpenes and terpenoids.

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How Good Can Single-Particle Cryo-EM Become? What Remains Before It Approaches Its Physical Limits?

Annual Review of Biophysics ◽

10.1146/annurev-biophys-070317-032828 ◽

2019 ◽

Vol 48 (1) ◽

pp. 45-61 ◽

Cited By ~ 26

Author(s):

Robert M. Glaeser

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Single Particle ◽

Energy Use ◽

Optimal Choice ◽

Structural Basis ◽

Detective Quantum Efficiency ◽

Quantum Metrology ◽

Cryo Electron Microscopy ◽

Induced Motion

Impressive though the achievements of single-particle cryo–electron microscopy are today, a substantial gap still remains between what is currently accomplished and what is theoretically possible. As is reviewed here, twofold or more improvements are possible as regards ( a) the detective quantum efficiency of cameras at high resolution, ( b) converting phase modulations to intensity modulations in the image, and ( c) recovering the full amount of high-resolution signal in the presence of beam-induced motion of the specimen. In addition, potential for improvement is reviewed for other topics such as optimal choice of electron energy, use of aberration correctors, and quantum metrology. With the help of such improvements, it does not seem to be too much to imagine that determining the structural basis for every aspect of catalytic control, signaling, and regulation, in any type of cell of interest, could easily be accelerated fivefold or more.

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Real-time object locator for cryo-EM data collection --- You only navigate EM once ---

10.1101/2021.04.07.438905 ◽

2021 ◽

Author(s):

Koji Yonekura ◽

Saori Maki-Yonekura ◽

Hisashi Naitow ◽

Tasuku Hamaguchi ◽

Kiyofumi Takaba

Keyword(s):

Electron Microscopy ◽

Machine Learning ◽

Electron Diffraction ◽

Data Collection ◽

Real Time ◽

Single Particle ◽

Detection System ◽

Target Object ◽

Electron Crystallography ◽

Cryo Electron Microscopy

In cryo-electron microscopy (cryo-EM) data collection, locating a target object is the most error-prone. Here, we present a machine learning-based approach with a real-time object locator named yoneoLocr using YOLO, a well-known object detection system. Implementation showed its effectiveness in rapidly and precisely locating carbon holes in single particle cryo-EM and for locating crystals and evaluating electron diffraction (ED) patterns in automated cryo-electron crystallography (cryo-EX) data collection.

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