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Shake-it-off: A simple ultrasonic cryo-EM specimen preparation device

John L. Rubinstein, Hui Guo, Zev A. Ripstein, Ali Haydaroglu, Aaron Au, Christopher M. Yip, Justin M. Di Trani, Samir Benlekbir, Timothy Kwok

Preprint posted on 9 May 2019 https://www.biorxiv.org/content/10.1101/632125v1.abstract?%3Fcollection=

Article now published in Acta Crystallographica Section D Structural Biology at http://dx.doi.org/10.1107/S2059798319014372

Self-wicking grids allow millisecond sample preparation of microlitre samples for CryoEM

Selected by David Wright

Background

Recent developments in cryogenic electron microscopy (CryoEM) have led to the emergence of the technique for high resolution structure determination of proteins (1). In many cases, this technique is more suitable than X-ray crystallography, as samples are not crystallised and can be imaged in near-native conditions. One important non-native aspect of CryoEM preparation is the introduction of an air-water interface, which can lead to sample degradation/unfolding or preferential orientation, and can hinder structure determination (2). An emerging method to lessen the damaging effect of the air-water interface is to reduce the length of time that a sample is exposed to it. Typical cryoEM grid preparation takes several seconds; however, several methods have been developed to reduce this well into the millisecond scale. So far this has mainly been achieved with rapid spraying techniques (3, 4); however, self-wicking grids are another good alternative, which may be easier for non-specialist groups to achieve. In this preprint the authors describe the use of treated grids, novel sample application and rapid plunging to achieve freezing times of approximately 90 ms, which may reduce sample deterioration at the air-water interface.

Results

A typical cryoEM protocol is as follows: cryoEM grids are prepared, a sample is applied and then blotted with filter paper, then the grid is frozen by plunging into liquid ethane. The set-up in this preprint prepares the grids and applies the sample in such a way that blotting is not required, speeding up the process of cryoEM grid preparation by several orders of magnitude. This article and its references contain protocols that allow the production of self-wicking grids from commercially-available components. In addition, the ultrasonic sprayer is adapted from a humidifier and the plunger components can be made using widely-available 3D-printers. The system is controlled using an inexpensive Raspberry pi system using open source software, meaning that the whole system could be replicated in multiple research institutions.

As a proof-of-principle the authors obtain the structure of horse spleen apoferritin at 2.6 Å resolution, which is on par with previous articles. This protein is routinely used in method development in the cryoEM field, as it is available commercially, is very stable and results in high resolution data, so is a very good test case.

Interestingly, on the grid shown in figure 4 the ice is not uniform, which is similar to blotted grids. This is not an issue for data collection, as an experienced user is able to select optimum ice thickness. Part of the appeal of this protocol is the use of such a small volume of purified protein: the authors state that this 1 μl sample could potentially be used to make several grids.

One caveat is that the 90 ms timeframe that is estimated may not quite be short enough to prevent deterioration at the air-water interface. At this time, if shorter timescales are required, then sprayers should be the method of choice. On the other hand, these sprayers generally require at least an order of magnitude more purified protein sample, which may render them less suitable for widespread use. The widespread availability of this equipment would also allow time-resolved studies of molecular machines, rather than the usual static snapshots obtained in standard cryoEM sample preparation.

In summary, the shake-it-off system appears to be a useful method to determine the high resolution structures of proteins with low sample requirements and the potential reduction in air-water interface mediated damage.

Comments and questions

  1. Apoferritin is an extremely stable protein sample, has this set-up been tested with air-sensitive samples?
  2. It would be very interesting to see if there really were fewer particles at the air-water interface, are there any plans to measure this?
  3. Denaturation at the air-water interface may occur quicker than 90 ms, are there any ways to speed up this system?

Why I chose this article

I have an interest in the structure determination of delicate membrane protein samples by cryoEM, so I am always interested in reading about interesting method developments. There were several things that appealed to me about this preprint: firstly, its open source nature, allowing others to try this with their own samples; secondly, how the preprint brings together several smaller technological improvements resulting in a large step; and finally, how this technique will allow further time-resolved studies in a great number of labs.

References

  1. Kühlbrandt W. The Resolution Revolution. Science. 2014;343(6178):1443.
  2. Chen J, Noble AJ, Kang JY, Darst SA. Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: Bacterial RNA polymerase and CHAPSO. Journal of Structural Biology: X. 2019 2019/01/01/;1:100005.
  3. Kontziampasis D, Klebl DP, Iadanza MG, Scarff CA, Kopf F, Sobott F, et al. A Cryo-EM Grid Preparation Device for Time-Resolved Structural Studies. bioRxiv. 2019:563254.
  4. Kaledhonkar S, Fu Z, White H, Frank J. Time-Resolved Cryo-electron Microscopy Using a Microfluidic Chip. Methods in molecular biology (Clifton, NJ). 2018;1764:59-71. PubMed PMID: 29605908. Epub 2018/04/02. eng.

 

Posted on: 21 May 2019

doi: https://doi.org/10.1242/prelights.10774

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Author's response

John Rubinstein shared

  1. Apoferritin is an extremely stable protein sample, has this set-up been tested with air-sensitive samples?

We are just starting to test the device with specimens that we think are sensitive to the air-water interface. However, there is some evidence that it does indeed reduce air-water interface interactions. The air-water interface isn’t all bad. It can cause denaturation of proteins and induce preferred orientations. But it can also concentrate proteins and let you work with samples at reduced concentration. Zev Ripstein in our group works with several proteins that show preferred orientations. He can often avoid this problem by adding detergents to his final buffer because detergents can also reduce air-water interface interactions for proteins. With detergents he needs a higher-concentration of protein than in detergent-free conditions. When he tried SIO with one of these proteins without detergents, he found that he got rid of the preferred orientations usually seen in detergent-free conditions, but he saw a low density of particles and would need to use concentrations comparable to when detergents are included. That result is exactly what you would expect if the speed of the device reduces air-water interface interactions.

  1. It would be very interesting to see if there really were fewer particles at the air-water interface, are there any plans to measure this?

Yes, it is in the plans. Alex Noble in Bridget Carragher and Clint Potters group in New York has shown this nicely with the Spotiton device using tomography. We’re using the same sort of self-wicking grids that the New York group developed and freezing them with a speed comparable to Spotiton. It would be surprising and interesting if one device reduced the number of particles at the air-water interface while the other didn’t

  1. Denaturation at the air-water interface may occur quicker than 90 ms, are there any ways to speed up this system?

Video 2 from the preprint shows there are a couple of things that can be sped up. First, there appears to be a lag after spraying and before plunging starts. This lag can be removed by adjusting the timing so that (at least from the Raspberry Pi’s perspective) the plunging process is initiated before spraying is completed – or maybe even before spraying is started. Second, the grid travels 30 mm from the sprayer to the cryogen. This distance can be easily decreased. However, we don’t know how fast self-wicking grids can work. The Spotiton approach takes a similar amount of time and it’s not clear how much that can be decreased with us still obtaining good ice.

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