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:10.1107/s2052252519011345

Cited by 90 publications

(54 citation statements)

References 31 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 Leeds, one of Thompson's favorite parts of her lab is the corner scattered with pond misters, tubing and other hardware, where they're building a time-resolved cryo-EM device, or TED. 10 "It looks likein the best possible way -somebody's shed, " she says.…”

Section: Build Your Ownmentioning

confidence: 99%

Molecular motion on ice

Dance¹

2020

Nat Methods

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“…For example, neuronal networkbased particle pickers [58,65,66] allow to minimize false positives and, moreover, implement micrograph denoising algorithms to enable detection of low-signal particles. Gentler cryo-EM specimen preparation methods [67][68][69][70][71][72] are expected to reduce the number of damaged particles. In combination, this should result in a cleaner and healthier pool of particleswhether this is really the case, still needs to be investigated.…”

Section: Challenges In Statistical Analysismentioning

confidence: 99%

Cryo‐EM of ABC transporters: an ice‐cold solution to everything?

Januliene

Moeller

2020

FEBS Letters

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High-resolution cryo-EM has revolutionized how we look at ABC transporters and membrane proteins in general. An ever-increasing number of software tools and faster processing now allow dissecting the molecular details of nanomachines at atomic precision. Considering the further benefits of significantly reduced sample demands and increased speed, cryo-EM will dominate the structure determination of membrane proteins in the near future without compromising on data quality or detail. Moreover, improved and new algorithms make it now possible to resolve the conformational spectrum of macromolecular machines under turnover conditions and to analyze heterogeneous samples at high-resolution. The future of cryo-EM is, therefore, bright, and the growing number of imaging facilities and groups active in this field will amplify this trend even further. Nevertheless, expectations have to be managed, as cryo-EM alone cannot provide an ultimate answer to all scientific questions. In this review, we discuss the capabilities and limitations of cryo-EM together with possible solutions for studies of ABC transporters.

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A cryo-EM grid preparation device for time-resolved structural studies

Abstract: Time-resolved structural studies are becoming an important tool in understanding biological function. Here we describe a cryo-EM grid freezing device capable of rapidly mixing and plunge freezing grids within 10 ms.

Cited by 90 publications

References 31 publications

Through-grid wicking enables high-speed cryoEM specimen preparation

Through-grid wicking enables high-speed cryoEM specimen preparation

Molecular motion on ice

Cryo‐EM of ABC transporters: an ice‐cold solution to everything?

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