VitroJet: new features and case studies

Henderikx, Rene J. M.; Mann, Daniel; Domanska, Aušra; Dong, Jing; Shahzad, Saba; Lak, Behnam; Filopoulou, Aikaterini; Ludig, Damian; Grininger, Martin; Momoh, Jeffrey; Laanto, Elina; Oksanen, Hanna M.; Bisikalo, Kyrylo; Williams, Pamela A.; Butcher, Sarah J.; Peters, Peter J.; Beulen, Bart W. A. M. M.

doi:10.1107/s2059798324001852

Cited by 2 publications

References 70 publications

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CryoEM grid preparation: a closer look at advancements and impact of preparation mode and new approaches

Hirst,

Thomas,

Davies

et al. 2024

Biochemical Society Transactions

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Sample preparation can present a significant hurdle within single particle cryo-electron microscopy (cryoEM), resulting in issues with reproducibility, data quality or an inability to visualise the sample. There are several factors which can influence this, including sample or buffer composition, grid type, route of sample preparation and interactions with the air–water interface (AWI). Here, we review some of the current routes for sample preparation and the associated challenges. We discuss a range of approaches for overcoming these challenges, such as minimising the grid preparation time, surfactants, grid type and biochemical approaches such as nanomagnetic beads. Finally, we discuss how a set of commercially available protein samples may serve as a benchmark suite for future technologies. This provides a route to compare techniques’ abilities not just to generate high-resolution structures but also to overcome the challenges traditionally associated with cryoEM. As the field continues to produce new approaches to sample preparation and we start to better understand the underlying principles behind the behaviour of proteins within a thin film and in response to different environments, especially grid composition, it is hoped that more universal solutions can be provided that make the intractable systems tractable, improve resolution and, importantly, speed up data collection and reduce the currently required dataset sizes.

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CryoEM grid preparation: a closer look at advancements and impact of preparation mode and new approaches

Hirst,

Thomas,

Davies

et al. 2024

Biochemical Society Transactions

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Ice thickness control and measurement in the VitroJet for time-efficient single particle structure determination

Henderikx,

Schotman,

Shahzad

et al. 2023

Preprint

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In cryo-electron microscopy structure determination, embedding biomolecules in vitreous ice of optimal thickness is critical. Ice thickness assessment and selection of suitable holes for data collection are currently part of time-consuming preparatory routines performed on expensive electron microscopes. For this reason, we developed a method to determine ice thickness during sample preparation using the optical camera integrated in the VitroJet. As a result, the ice thickness of buffer-suspended holes on an EM grid can be determined faithfully in the working range relevant for single particle cryo-EM. Dependent on the thickness, the error is below ± 20 nm in the range between 0 - 70 nm down to ± 10 nm in the thinnest ice regions (10 - 40 nm). Single particle structures of apoferritin were determined at two distinct thicknesses of 30 nm and 70 nm to demonstrate the suitability of optical layer thickness measurements for time-efficient cryo-EM structure determination.

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VitroJet: new features and case studies

Cited by 2 publications

References 70 publications

CryoEM grid preparation: a closer look at advancements and impact of preparation mode and new approaches

CryoEM grid preparation: a closer look at advancements and impact of preparation mode and new approaches

Ice thickness control and measurement in the VitroJet for time-efficient single particle structure determination

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