Close

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

Posted on: 21 May 2019

Preprint posted on 9 May 2019

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.

 

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

Read preprint (No Ratings Yet)

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.

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

Also in the biochemistry category:

Triglyceride metabolism controls inflammation and APOE4-associated disease states in microglia

Roxan A. Stephenson, Kory R. Johnson, Linling Cheng, et al.

Selected by 22 August 2024

Gustavo Stelzer, Marcus Oliveira

Biochemistry

Impaired 26S proteasome causes learning and memory deficiency and induces neuroinflammation mediated by NF-κB in mice

Christa C. Huber, Eduardo Callegari, Maria Paez, et al.

Selected by 22 August 2024

Gustavo Stelzer, Marcus Oliveira

Biochemistry

Notch3 is a genetic modifier of NODAL signalling for patterning asymmetry during mouse heart looping

Tobias Holm Bønnelykke, Marie-Amandine Chabry, Emeline Perthame, et al.

Selected by 06 June 2024

Bhaval Parmar

Developmental Biology

Also in the biophysics category:

Global coordination of protrusive forces in migrating immune cells

Patricia Reis-Rodrigues, Nikola Canigova, Mario J. Avellaneda, et al.

Selected by 10 October 2024

yohalie kalukula

Biophysics

Engineered Nanotopographies Induce Transient Openings in the Nuclear Membrane

Einollah Sarikhani, Vrund Patel, Zhi Li, et al.

Selected by 23 September 2024

Sristilekha Nath

Bioengineering

Spiral-eyes: A soft active matter model of in vivo corneal epithelial cell migration

Kaja Kostanjevec, Rastko Sknepnek, Jon Martin Collinson, et al.

Selected by 03 September 2024

Prasanna Padmanaban

Biophysics

Also in the molecular biology category:

Non-disruptive inducible labeling of ER-membrane contact sites using the Lamin B Receptor

Laura Downie, Nuria Ferrandiz, Megan Jones, et al.

Selected by 15 October 2024

Jonathan Townson

Cell Biology

HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos

Estefania Sanchez-Vasquez, Marianne E. Bronner, Magdalena Zernicka-Goetz

Selected by 11 October 2024

Anchel De Jaime Soguero

Developmental Biology

The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites

Joelle Lo, Katherine F. Vaeth, Gurprit Bhardwaj, et al.

Selected by 24 September 2024

Felipe Del Valle Batalla

Neuroscience

preLists in the biochemistry category:

BSCB-Biochemical Society 2024 Cell Migration meeting

This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.

 



List by Reinier Prosee

Peer Review in Biomedical Sciences

Communication of scientific knowledge has changed dramatically in recent decades and the public perception of scientific discoveries depends on the peer review process of articles published in scientific journals. Preprints are key vehicles for the dissemination of scientific discoveries, but they are still not properly recognized by the scientific community since peer review is very limited. On the other hand, peer review is very heterogeneous and a fundamental aspect to improve it is to train young scientists on how to think critically and how to evaluate scientific knowledge in a professional way. Thus, this course aims to: i) train students on how to perform peer review of scientific manuscripts in a professional manner; ii) develop students' critical thinking; iii) contribute to the appreciation of preprints as important vehicles for the dissemination of scientific knowledge without restrictions; iv) contribute to the development of students' curricula, as their opinions will be published and indexed on the preLights platform. The evaluations will be based on qualitative analyses of the oral presentations of preprints in the field of biomedical sciences deposited in the bioRxiv server, of the critical reports written by the students, as well as of the participation of the students during the preprints discussions.

 



List by Marcus Oliveira et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

20th “Genetics Workshops in Hungary”, Szeged (25th, September)

In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf

 



List by Nándor Lipták

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Also in the molecular biology category:

2024 Hypothalamus GRC

This 2024 Hypothalamus GRC (Gordon Research Conference) preList offers an overview of cutting-edge research focused on the hypothalamus, a critical brain region involved in regulating homeostasis, behavior, and neuroendocrine functions. The studies included cover a range of topics, including neural circuits, molecular mechanisms, and the role of the hypothalamus in health and disease. This collection highlights some of the latest advances in understanding hypothalamic function, with potential implications for treating disorders such as obesity, stress, and metabolic diseases.

 



List by Nathalie Krauth

BSCB-Biochemical Society 2024 Cell Migration meeting

This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.

 



List by Reinier Prosee

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra
Close