Rapid tilt-series acquisition for electron cryotomography

Chreifi, Georges; Chen, Songye; Metskas, Lauren Ann; Kaplan, Mohammed; Jensen, Grant J.

doi:10.1016/j.jsb.2018.12.008

Cited by 93 publications

(51 citation statements)

References 27 publications

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“…In our implementation of hStA only the first "high-dose" image is used for the final reconstruction which relaxes the quality requirements for the remaining projections in the tilt series. Furthermore, hybrid tomographic acquisition and processing may be naturally combined with collecting fast tilt series (Chreifi et al, 2019, Eisenstein et al, 2019 to increase throughput. If combined with conservatively increasing the angular step and/or reducing the angular coverage of the tilt series, throughput could further increased to one tomogram every few minutes, improving the current throughput by 10-fold.…”

Section: Discussionmentioning

confidence: 99%

Subnanometer-resolution structure determinationin situby a hybrid subtomogram averaging – single particle cryoEM – workflow

Sánchez

Zhang

Chen

et al. 2020

Preprint

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Cryo electron tomography (cryo-ET) combined with subtomogram averaging (StA) enables structural determination of macromolecules in their native context. A few structures were reported by StA at resolution higher than 4.5 Å, however all of these are from viral structural proteins or vesicle coats. Reaching high resolution for a broader range of samples is uncommon due to beam-induced sample drift, poor signal-to-noise ratio (SNR) of images, challenges in CTF correction, limited number of particles. Here we propose a strategy to address these issues, which consists of a tomographic data collection scheme and a processing workflow. Tilt series are collected with higher electron dose at zero-degree tilt in order to increase SNR. Next, after performing StA conventionally, we extract 2D projections of the particles of interest from the higher SNR images and use the single particle analysis tools to refine the particle alignment and generate a reconstruction. We benchmarked our proposed hybrid StA (hStA) workflow and improved the resolution for tobacco mosaic virus from 7.2 to 5.2 Å and the resolution for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. We demonstrate that hStA can improve the resolution obtained by conventional StA and promises to be a useful tool for StA projects aiming at subnanometer resolution or higher.

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Section: Discussionmentioning

confidence: 99%

Subnanometer-resolution structure determinationin situby a hybrid subtomogram averaging – single particle cryoEM – workflow

Sánchez

Zhang

Chen

et al. 2020

Preprint

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“…Recently the development of electron cryotomography has allowed the visualization and understanding of complex structure of microorganisms. Also this scientific tool permits to visualization of intact cells and another biological structures close to naïve state in a molecular level 52,53 .…”

Section: -Discussionmentioning

confidence: 99%

Antimicrobial effects of Photodynamic Therapy to gram negative bacteria envelope revealed by Cryo-electron tomography

Garcez

Kaplan

Núñez

et al. 2019

17th International Photodynamic Association World Congress

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Cryo-electron tomography (cryo-ET) is an emerging technology that enables thin samples, including small intact prokaryotic cells, to be imaged in three dimensions in a near-native 'frozenhydrated' state to a resolution sufficient to recognize very large macromolecular complexes in situ. This methodology has been fundamental to provide information about cellular ultrastructure. This study used cryo-ET to evaluate the photodynamic effect on the viability and envelope architecture of a Gram-negative bacteria. Bacterial suspension of E. coli minicells were submitted to photodynamic treatment with methylene blue solution (100µM) and a 100mW low power diode laser emitting at 660nm with 6 and 18J of energy. As a control group, a suspension of minicells were submitted to 462 IU/mL penicillin G for 60 min at 30 °C, to compare the damage in cell wall structure. After treatment bacteria were immediately plunge-frozen across EM grids and standard cryo-ET tilt series were collected, 3D images reconstructions were calculated and recorded. The imagens showed detachment of the bacterial cell walls and mesosome-like structures. In addition, some sites showed interrupted stretches in both inner and outer membranes and cell wall degradation, indicating bacterial envelope damage. Cryo-electron tomography revealed that the effects of photodynamic therapy on Gram negative bacteria was based on damage to the outer membrane, cell wall and inner membrane and occurs in an energy-dependent manner.

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“…Accordingly, this opens up the possibility to prepare cryo-lamellae with a 24 h continuous 231 operation of the microscope and therefore lead to ~80 or more lamellae/day. Considering the latest 232 developments in regards to fast cryo-ET acquisition it will be possible to match this throughput on the 233 TEM (Chreifi et al, 2019). Even in the situation where the above-described throughput is not required, 234 a protocol providing up to 40 lamellae during a daytime session will shorten the user queue around 235 cryo-FIBs, therefore, allowing more projects to be conducted in facilities where the availability of 236 instrumentation is limited in relation to the number of users.…”

Section: Cryo-temmentioning

confidence: 99%

Automated cryo-lamella preparation for high-throughput in-situ structural biology

Buckley

Gervinskas

Taveneau

et al. 2019

Preprint

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17Cryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) 18 milling enables structural biology studies to be performed directly within the cellular environment. 19Cryo-preserved cells are milled and a lamella with a thickness of 200-300 nm provides an electron 20 transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a 21 tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we 22 introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the 23 amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae 24 and demonstrate that this method allows an increased throughput, achieving a rate of 5 lamellae/hour 25 without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is 26 consistent throughout the preparation and their compatibility with cryo-ET analyses. 27 28

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Rapid tilt-series acquisition for electron cryotomography

Cited by 93 publications

References 27 publications

Subnanometer-resolution structure determinationin situby a hybrid subtomogram averaging – single particle cryoEM – workflow

Subnanometer-resolution structure determinationin situby a hybrid subtomogram averaging – single particle cryoEM – workflow

Antimicrobial effects of Photodynamic Therapy to gram negative bacteria envelope revealed by Cryo-electron tomography

Automated cryo-lamella preparation for high-throughput in-situ structural biology

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