Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales

Abstract

Summary A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic “silver bullet” for improving the quality of every cryoEM sample preparation.

Cryo-EM structure of human mitochondrial HSPD1

“…We used novel fast grid preparation techniques as an alternative, with a home-built device designed for time-resolved EM or a chameleon grid-making device (SPT Labtech). This could not eliminate binding to the air-water interface but gave modest improvements in angular distribution, but, in the case of the home-built device, it also increased ice thickness ( Klebl et al, 2020 ).…”

Current limitations to high-resolution structure determination by single-particle cryoEM

“…For aerosols and inkjet printing, this includes the time of flight through air. In practice, the shortest times from bulk liquid sample to vitrification reported to date are around 6–30 ms (Klebl et al ., 2020 a ) – two orders of magnitude slower than protein diffusion. It follows that current cryoEM specimen preparation technology cannot outrun the physics of protein diffusion in aqueous solution, and thus does not eliminate the interaction of proteins with the air-water interface.…”

“…A systematic study (Klebl et al ., 2020 a ) evaluates and compares the merits of inkjet printing and microfluidic spraying to standard blotting of three different, well-characterized samples: apoferritin, Escherichia coli ribosomes and the mitochondrial heat shock protein HSPD1. The three techniques produced grids suitable for high-resolution cryoEM data collection of all three samples, but even at the shortest preparation time of 6 ms, achieved with a microfluidics device, a significant degree of preferential orientation on the air-water interface was evident (Fig.…”

Structural insight intoPichia pastorisfatty acid synthase

“…Particle orientation distribution was visualized using an adapted version 44 of a script from Naydenova & Russo (2017) 45 .…”