A Cryo-EM Grid Preparation Device for Time-Resolved Structural Studies

Kontziampasis, Dimitrios; Klebl, David P.; Iadanza, M.G.; Scarff, Charlotte A.; Kopf, Florian; Sobott, Frank; Monteiro, Diana C. F.; Trebbin, Martin; Muench, Stephen P.; White, Howard D.

doi:10.1101/563254

Cited by 31 publications

(47 citation statements)

References 25 publications

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“…The major advances reported here as well as by others 21,26 prompt further meaningful improvements to trEM. Currently, the amount of sample consumed per experiment is large in comparison to other sample preparation approaches 33,60,61 .…”

Section: Discussionsupporting

confidence: 59%

“…To overcome these limitations and develop a general time-resolved sample preparation method for cryo-EM (trEM) requires building a miniaturized mixer and bioreactor able to rapidly initiate and synchronize biochemical reactions, followed by spreading the incubated sample onto a cryo-EM grid without the need for manual operation or blotting, collectively faster than the lifetime of the structures of interest 19 . Previous work has shown that combining reactants in microfluidic devices followed by rapid application of sample by gasassisted spraying is in principle possible [20][21][22][23][24][25] , and has yielded fascinating new insights into biology 26 . However, substantial technical challenges remain unaddressed.…”

Section: Introductionmentioning

confidence: 99%

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Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

Mäeots

Lee

Nans

et al. 2020

Preprint

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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 otherwise shortlived reaction intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. To overcome these limitations and allow visualisation of biochemical processes at near-atomic spatial resolution and millisecond time scales, we developed a time-resolved sample preparation method for cryo-electron microscopy (trEM). We integrated a modular microfluidic device, featuring a 3D-mixing unit and a delay line of variable length, with a gas-assisted nozzle and motorised plunge-freeze set-up that enables automated, fast, and blot-free sample vitrification. This sample preparation not only preserves high-resolution structural detail but also substantially improves protein distribution across the vitreous ice. We validated the method by examining the formation of RecA filaments on single-stranded DNA. We could reliably visualise reaction intermediates of early filament growth across three orders of magnitude on sub-second timescales. Quantification of the trEM data allowed us to characterize the kinetics of RecA filament growth. The trEM method reported here is versatile, easy to reproduce and thus readily adaptable to a broad spectrum of fundamental questions in biology.

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Section: Discussionsupporting

confidence: 59%

Section: Introductionmentioning

confidence: 99%

Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

Mäeots

Lee

Nans

et al. 2020

Preprint

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“…To improve sample deposition on EM grids, several developments have been presented, including droplet-based methods [20][21][22][23][24] , e.g. on nanowire grids 25 , and using capillaries followed by sample thinning by controlled water evaporation 26 .…”

Section: Pin Printingmentioning

confidence: 99%

Automated cryo-EM sample preparation by pin-printing and jet vitrification

Ravelli

Nijpels

Henderikx

et al. 2019

Preprint

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The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide controlled and efficient sample preparation for cryo-EM studies.Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device's performance was validated by resolving 4 standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.

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“…The TED was primarily designed to perform time-resolved experiments by rapidly mixing constituents before vitrification on the millisecond timescale. However, in this study we only make use of its ability to deposit a single sample and vitrify it on a very fast timescale (R6 ms) (Kontziampasis et al, 2019). A conventional EM grid is placed on a plunging arm, which has an adjustable speed within a high-humidity chamber at room temperature.…”

Section: Introductionmentioning

confidence: 99%