Optimized data acquisition workflow by sample thickness determination

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.

Download Full-text

Optimized cryo-EM data-acquisition workflow by sample-thickness determination

Acta Crystallographica Section D Structural Biology ◽

10.1107/s205979832100334x ◽

2021 ◽

Vol 77 (5) ◽

pp. 565-571

Author(s):

Jan Rheinberger ◽

Gert Oostergetel ◽

Guenter P. Resch ◽

Cristina Paulino

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Data Collection ◽

Data Acquisition ◽

Data Storage ◽

Collection Efficiency ◽

Sample Thickness ◽

Access Time ◽

Time Data ◽

Data Set

Sample 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, it is demonstrated how the sample thickness can be determined before data acquisition, allowing the identification of optimal regions and the restriction of automated 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 a lack of high-resolution information. It maximizes the data-collection efficiency and lowers the electron-microscopy time required per data set. This strategy is especially useful if the speed of data collection is restricted by the microscope hardware and software, or if microscope access time, data transfer, data storage and computational power are a bottleneck.

Download Full-text

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.

Download Full-text

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

IUCrJ ◽

10.1107/s2052252520013482 ◽

2020 ◽

Vol 7 (6) ◽

pp. 1179-1187 ◽

Cited By ~ 1

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Amino and PEG-amino graphene oxide grids enrich and protect samples for high-resolution single particle cryo-electron microscopy

Journal of Structural Biology ◽

10.1016/j.jsb.2019.107437 ◽

2020 ◽

Vol 209 (2) ◽

pp. 107437 ◽

Cited By ~ 7

Author(s):

Feng Wang ◽

Zanlin Yu ◽

Miguel Betegon ◽

Melody G. Campbell ◽

Tural Aksel ◽

...

Keyword(s):

Electron Microscopy ◽

Graphene Oxide ◽

High Resolution ◽

Single Particle ◽

Cryo Electron Microscopy ◽

Resolution Single

Download Full-text

High-Resolution Insights into Nascent-Chain Conformation by Single-Particle Cryo-Electron Microscopy

Biophysical Journal ◽

10.1016/j.bpj.2020.11.1895 ◽

2021 ◽

Vol 120 (3) ◽

pp. 296a

Author(s):

Meranda Masse ◽

Christopher Morgan ◽

Wanting Wei ◽

Edward W. Yu ◽

Silvia Cavagnero

Keyword(s):

Electron Microscopy ◽

High Resolution ◽

Single Particle ◽

Chain Conformation ◽

Cryo Electron Microscopy ◽

Nascent Chain

Download Full-text

Amino and PEG-Amino Graphene Oxide Grids Enrich and Protect Samples for High-resolution Single Particle Cryo-electron Microscopy

10.1101/813972 ◽

2019 ◽

Cited By ~ 2

Author(s):

Feng Wang ◽

Zanlin Yu ◽

Miguel Betegon ◽

Melody Campbell ◽

Tural Aksel ◽

...

Keyword(s):

Electron Microscopy ◽

Graphene Oxide ◽

High Resolution ◽

Single Particle ◽

Particle Distribution ◽

Water Interface ◽

Amino Groups ◽

Cryo Electron Microscopy ◽

Air Water Interface ◽

Resolution Single

AbstractCryo-EM samples prepared using the traditional methods often suffer from too few particles, poor particle distribution, or strongly biased orientation, or damage from the air-water interface. Here we report that functionalization of graphene oxide (GO) coated grids with amino groups concentrates samples on the grid with improved distribution and orientation. By introducing a PEG spacer, particles are kept away from both the GO surface and the air-water interface, protecting them from potential denaturation.

Download Full-text

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

10.1101/861740 ◽

2019 ◽

Cited By ~ 1

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.

Download Full-text