Through-grid wicking enables high-speed cryoEM specimen preparation
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Abstract
Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air–water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass-fiber filter held against the opposite side, often called the `back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic specimen application and through-grid wicking were combined in a high-speed specimen-preparation device that was named `Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations.
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###Reviewer #3:
The method described, Back-it-up (BIU), builds upon the recently published Shake-it-off (SIO) system for EM grid preparation by eliminating the requirement for self-wicking, nano-wire grids (along with their inherent limitations including grid-to-grid variability and limited wicking capacity) by back-blotting standard copper-faced EM grids with highly absorbent glass fiber filter paper. Additional modifications to the SIO unit are reported that enable grid preparation (sample application-to-vitrification) times on the order of ~100ms. Although the achievement of this time constant has been reported for the Spotiton and chameleon automated grid preparation robots, these systems are technically complex and expensive to build or buy. As reported here, BIU represents for labs of modest financial resources a robust, …
###Reviewer #3:
The method described, Back-it-up (BIU), builds upon the recently published Shake-it-off (SIO) system for EM grid preparation by eliminating the requirement for self-wicking, nano-wire grids (along with their inherent limitations including grid-to-grid variability and limited wicking capacity) by back-blotting standard copper-faced EM grids with highly absorbent glass fiber filter paper. Additional modifications to the SIO unit are reported that enable grid preparation (sample application-to-vitrification) times on the order of ~100ms. Although the achievement of this time constant has been reported for the Spotiton and chameleon automated grid preparation robots, these systems are technically complex and expensive to build or buy. As reported here, BIU represents for labs of modest financial resources a robust, reproducible high speed cryo-EM grid preparation device for around $1000 that uses a fraction of the sample volume required by typical automatic plunge freezer and can achieve sub-second plunge times that reduce the negative effects (denaturation, preferred orientation) of the air-water interface on the protein sample.
This study is well organized. First, it clearly demonstrates and provides visual supporting evidence of the absorptive capacity of the glass fiber filters. Next they validate the filters on a commonly used grid prep device using back-blotting. Finally, the authors use multiple samples and plunge speeds to demonstrate the utility and effectiveness of combining the glass fiber filters and a modified SIO device to prepare grids that yielded high resolution EM structural data.
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###Reviewer #2:
General Assessment:
The paper is well crafted and a clever improvement on current methods by combining the shake-it-off system with a Leica GP3 back blotter switching out the filter paper for a glass fiber pad. This improvement has likewise shown impressive results, and this information should be disseminated to help the field move forward. However there are a couple of issues, with borderline tangential material, that must be dealt with.
Substantive Concerns:
There are two major substantive concerns. The first revolves around the use of the influenza A hemagglutinin trimer in a direct apples to apples comparison with the work of Noble et.al. In their paper using spotiton they showed that dropping from 500ms to 100ms not only reduced the preferred orientation dramatically, but it also changed the thickness distribution …
###Reviewer #2:
General Assessment:
The paper is well crafted and a clever improvement on current methods by combining the shake-it-off system with a Leica GP3 back blotter switching out the filter paper for a glass fiber pad. This improvement has likewise shown impressive results, and this information should be disseminated to help the field move forward. However there are a couple of issues, with borderline tangential material, that must be dealt with.
Substantive Concerns:
There are two major substantive concerns. The first revolves around the use of the influenza A hemagglutinin trimer in a direct apples to apples comparison with the work of Noble et.al. In their paper using spotiton they showed that dropping from 500ms to 100ms not only reduced the preferred orientation dramatically, but it also changed the thickness distribution of the ice in the holes. Thus the paper left the reader with a bit of an open question about whether it was a thickness effect or a temporal effect that resulted in the reduction of the preferred orientation problem. This is especially pertinent given their tomography work showing that the influenza A hemagglutinin trimer displays extreme sensitivity to the thickness of ice. For example, when the ice is too thin the trimer is completely excluded, then when the ice is just barely thick enough there is a region where only the top view orientation is possible, and finally only in the thicker ice (100-150nm) are side views possible. Thus, when attempting to compare the results from the BIU to the results from Noble et. al. the ice thickness becomes a confounding factor to the assignment of the improved distribution due to reduced time between blotting and vitrification. It is quite likely that the BIU's enhanced results are not a product of the reduced time between deposition and vitrification but rather due to the BIU producing a thicker ice in the middle of the holes due to the different thinning method, thus allowing for more side views as shown in Noble et. al.. Therefore the lines 265-271 seem, to this reviewer, to be much too strong of a conclusion; however, given the importance of the observation this reviewer suggests that the authors simply remove lines 269-271 and leave the important observation as an important observation.
The sentence starting on line 169 should be removed. A biosafety cabinet alone is insufficient to allow this invention to be compatible with BSL3/4 safety protocols, as the aerosol generated not only contaminates everything in the biosafety cabinet, but also will stay in the air for quite some time afterwards, long enough that a researcher might accidentally make the mistake of releasing whatever pathogen they are working with.
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###Reviewer #1:
I found no faults with this study and believe it is a timely contribution to the subfield of cryoEM sample preparation. Given the lower costs associated with this technology than the alternatives, it is possible that through-grid wicking with glass fiber will be widely adopted.
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript. Adam Frost (University of California) served as the Reviewing Editor.
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