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. 2018 May;202(2):161-169.
doi: 10.1016/j.jsb.2018.01.002.

Spotiton: New features and applications

Venkata P Dandey  1 , Hui Wei  1 , Zhening Zhang  1 , Yong Zi Tan  2 , Priyamvada Acharya  3 , Edward T Eng  1 , William J Rice  1 , Peter A Kahn  4 , Clinton S Potter  2 , Bridget Carragher  5
Affiliations

Spotiton: New features and applications

Venkata P Dandey et al. J Struct Biol. 2018 May.

Abstract

We present an update describing new features and applications of Spotiton, a novel instrument for vitrifying samples for cryoEM. We have used Spotiton to prepare several test specimens that can be reconstructed using routine single particle analysis to ∼3 Å resolution, indicating that the process has no apparent deleterious effect on the sample integrity. The system is now in routine and continuous use in our lab and has been used to successfully vitrify a wide variety of samples.

Keywords: cryoEM vitrification; nanowire grids; piezo dispensing.

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Figures

Figure 1
Figure 1
(a) Overall layout of the Spotiton instrument. (b) Grid positioned in front of the piezo tip (magnified view is inset) with upper grid camera viewing dispensing process from behind.
Figure 2
Figure 2
Camera inspection of drop dispensing using cosine mode: (a) satisfactory drop dispensing, (b) unsatisfactory drop dispensing.
Figure 3
Figure 3
Snapshots of the grid flying past the (a) upper grid camera and (b) lower grid camera after sample deposition.
Figure 4
Figure 4
20S proteasome test sample. (a, b) Overall atlas of the grid showing the stripe of vitrified sample shaded blue. F (c–d) Sequentially higher magnification images of the vitrified sample. (e–f) High magnification images taken from the hole shown in (d).
Figure 5
Figure 5
Apoferritin test sample. (a, b) Overall atlas of the grid showing the stripe of vitrified sample shaded blue. (c–d) Sequentially higher magnification images of the vitrified sample. (e–f) High magnification images taken from the hole shown in (d).
Figure 6
Figure 6
3D reconstruction of 20S proteasome test sample. (a) 3D map rendered with Chimera. (b) Representative 2D class averages. (c) Euler angle distribution. (d) Histogram of directional FSC (blue), global FSC (red) and ± 1 S.D from mean of the directional FSC. FSC curve indicates a resolution of 3.0Å.
Figure 7
Figure 7
3D reconstruction of Apoferritin test sample. (a) 3D map rendered with Chimera. (b) Representative 2D class averages. (c) Euler angle distribution. (d) Histogram of directional FSC (blue), global FSC (red) and ± 1 S.D from mean of the directional FSC. FSC curve indicates a resolution of 2.8Å.
Figure 8
Figure 8
(a–c) Sequentially higher magnification images of a vitrified sample of an integral membrane protein in a nanodisc. (d–f) Sequentially higher magnification images of a vitrified sample of an HIV trimer prepared in the presence of detergent.
See this image and copyright information in PMC

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