Enabling a Paradigm Shift in CryoEM Sample Preparation with Chameleon
WHERE AND WHEN
DATE: Wednesday, October 27, 2021
TIME: 11 a.m. – 12 p.m. EDT
LOCATION: Via Zoom: mit.zoom.us/j/94586100937
SPEAKER: Michele C. Darrow, SPT Labtech LTD., Melbourn Science Park, Hertfordshire, UK
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DETAILS
In the cryo electron microscopy (cryoEM) structure determination workflow, improvements in microscope stability, direct detectors and image processing have shifted the bottleneck to sample preparation. The process of obtaining a film of vitreous ice of an appropriate thickness, with evenly distributed particles is not straightforward. Furthermore, conventional sample preparation methods have now been shown to suffer from the ubiquitous effects of the air-water interface (AWI), leading to negative sample outcomes and a reduction in achievable resolution [1].
There are a variety of experimental methods which have been developed over the past 40 years that attempt to overcome conventional specimen preparation failures. A recent survey of optimization methods found that there are not yet any workflows specific to all samples, or even specific sample types [2]. Only a consensus that optimization is required for most samples and that approximately 10 different methods will be attempted in combination before improvement is seen and a high-resolution dataset can be achieved [2]. Additionally, none of the currently available methods can be determined to work in advance of sample preparation; although in experts’ hands, experience can lead to a few common-sense first steps [2]. This iterative cycle of optimization and screening is time and resource expensive and when it fails, the brute force option of collecting ever larger datasets is even more so. Recent advances in automation of data collection strategies have reduced this barrier, but they do not address the root-cause of the issue – poor quality samples.
The chameleon system is a blot-free, pico-litre dispense instrument for quickly and robustly freezing samples for use in cryoEM [3]. The chameleon system was developed from Spotiton [4,5] and uses self-wicking copper nanowire grids to form the thin sample film [6]. This process occurs ‘on-the-fly’ as the grid passes in front of the dispenser on its way to the cryogen bowl, resulting in a stripe of sample across the frozen grid.
Chameleon provides many benefits:
- Blot-free high-speed plunging
- Automated grid handling
- Grid screening based on film thickness
- Intuitive automated workflows
- Sample tracking and recording
- Cryogen feedback and control
Importantly, by varying the plunge time during grid preparation, the chameleon system can be used to understand sample specific behaviours and overcome sample specific AWI effects. Together, with walk-up usability, the chameleon democratizes opportunities for cutting-edge research despite poorly behaved cryoEM samples. Observations and updates on the chameleon system and associated grids will be discussed.
References:
[1] A Noble, et al. Nature Methods 15 (2018), p. 793-795
[2] B. Carragher, et al. J. Microsc 276 (2019), p. 39-45.
[3] MC Darrow, et al. Microscopy and Microanalysis, 25 (2019), p. 994-995
[4] I Razinkov, et al. Journal of Structural Biology 195 (2016), p. 190-198
[5] V Dandey, et al. Journal of Structural Biology 202 (2018), p. 161-169
[6] H Wei, et al. Journal of Structural Biology 202 (2018), p. 170-174