Time-resolved cryoEM using Spotiton

Dandey, Venkata P.; Wei, Hui; Bobe, Daija; Maruthi, Kashyap; Kopylov, Mykhailo; Eng, Edward T.; Kahn, Peter; Hinshaw, Jenny E.; Kundu, Nidhi; Nimigean, Crina M.; Chen, Fan; Sukomon, Nattakan; Darst, Seth A.; Saecker, Ruth M.; Chen, James; Malone, Brandon; Potter, C. S.; Carragher, Bridget

doi:10.1101/2020.04.20.050518

Cited by 6 publications

(8 citation statements)

References 49 publications

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“…It has the disadvantage of using relatively large volumes for each grid, such as hundreds of nanolitres up to one microlitre, although it may be possible to reduce this volume further. Alternative sample-application methods include the inkjet approach implemented in the Spotiton device (Jain et al, 2012;Dandey et al, 2018) and the microfluidic sprayers used with several time-resolved specimen-preparation devices (Feng et al, 2017;Kaledhonkar et al, 2018;Kontziampasis et al, 2019;Lu et al, 2014;Dandey et al, 2020). Through-grid wicking could also prove useful for preparing optimal ice when applying nanolitre samples to a grid with a microcapillary (Arnold et al, 2016(Arnold et al, , 2017 or from a solid surface (Ravelli et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

Through-grid wicking enables high-speed cryoEM specimen preparation

Tan

Rubinstein

2020

Acta Crystallogr D Struct Biol

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Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air–water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass-fiber filter held against the opposite side, often called the `back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic specimen application and through-grid wicking were combined in a high-speed specimen-preparation device that was named `Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations.

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Section: Discussionmentioning

confidence: 99%

Through-grid wicking enables high-speed cryoEM specimen preparation

Tan

Rubinstein

2020

Acta Crystallogr D Struct Biol

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“…In addition to trapping transient intermediates, rapid sample preparation has found to have other benefits. By preparing the sample in a few milliseconds, we and others found that the sample did not strongly concentrate at the airwater interfaces (Ma ¨eots et al, 2020;Dandey et al, 2020), although Klebl, Gravett et al (2020) provide conflicting observations. This is a well known effect that can hinder many sample-preparation methods (D'Imprima et al, 2019;Klebl, Gravett et al, 2020;Glaeser, 2018).…”

Section: Topical Reviews 5 Benefits Of Rapid Sample Preparationmentioning

confidence: 85%

Structural dynamics: review of time-resolved cryo-EM

Mäeots¹,

Enchev²

2022

Acta Crystallogr D Struct Biol

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The structural determination of biological macromolecules has been transformative for understanding biochemical mechanisms and developing therapeutics. However, the ultimate goal of characterizing how structural dynamics underpin biochemical processes has been difficult. This is largely due to significant technical challenges that hinder data collection and analysis on the native timescales of macromolecular dynamics. Single-particle cryo-EM provides a powerful platform to approach this challenge, since samples can be frozen faster than the single-turnover timescales of most biochemical reactions. In order to enable time-resolved analysis, significant innovations in the handling and preparation of cryo-EM samples have been implemented, bringing us closer to the goal of the direct observation of protein dynamics in the milliseconds to seconds range. Here, the current state of time-resolved cryo-EM is reviewed and the most promising future research directions are discussed.

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“…In this study, we present a major step in the development of time-resolved structural studies, by developing a reliable and robust methodology for the initiation, synchronisation, and freeze-trapping of biochemical reactions onto cryo-EM sample support grids for subsequent analysis. There have been other developments of time-resolved cryo-EM methods 22,23,26,[53][54][55] , including a very recently reported adaptation of existing technology, Spotiton, for time-resolved studies 56 . Some devices already enabled outstanding contributions to resolving unknown biochemistry 26,54,55 , however important steps remain to be improved for the general usability of trEM.…”

Section: Discussionmentioning

confidence: 99%

Modular microfluidics enables kinetic insight from time-resolved cryo-EM

et al. 2020

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Mechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this information in a broadly applicable manner and thus structural studies of static macromolecules are often complemented by biophysical analysis. Moreover, the common strategy of utilizing mutants or crosslinking probes to stabilize intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. Here we report a time-resolved sample preparation method for cryo-electron microscopy (trEM) using a modular microfluidic device, featuring a 3D-mixing unit and variable delay lines that enables automated, fast, and blot-free sample vitrification. This approach not only preserves high-resolution structural detail but also substantially improves sample integrity and protein distribution across the vitreous ice. We validate the method by visualising reaction intermediates of early RecA filament growth across three orders of magnitude on sub-second timescales. The trEM method reported here is versatile, reproducible, and readily adaptable to a broad spectrum of fundamental questions in biology.

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Time-resolved cryoEM using Spotiton

Cited by 6 publications

References 49 publications

Through-grid wicking enables high-speed cryoEM specimen preparation

Through-grid wicking enables high-speed cryoEM specimen preparation

Structural dynamics: review of time-resolved cryo-EM

Modular microfluidics enables kinetic insight from time-resolved cryo-EM

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