Routine determination of ice thickness for cryo-EM grids - PubMed (original) (raw)

Routine determination of ice thickness for cryo-EM grids

William J Rice et al. J Struct Biol. 2018 Oct.

Abstract

Recent advances in instrumentation and automation have made cryo-EM a popular method for producing near-atomic resolution structures of a variety of proteins and complexes. Sample preparation is still a limiting factor in collecting high quality data. Thickness of the vitreous ice in which the particles are embedded is one of the many variables that need to be optimized for collection of the highest quality data. Here we present two methods, using either an energy filter or scattering outside the objective aperture, to measure ice thickness for potentially every image collected. Unlike geometrical or tomographic methods, these can be implemented directly in the single particle collection workflow without interrupting or significantly slowing down data collection. We describe the methods as implemented into the Leginon/Appion data collection workflow, along with some examples from test cases. Routine monitoring of ice thickness should prove helpful for optimizing sample preparation, data collection, and data processing.

Keywords: Cryo-EM; Energy filter; Ice thickness; Inelastic scattering; Mean free path.

Copyright © 2018 Elsevier Inc. All rights reserved.

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Figures

Figure 1.

Figure 1.

(A) Representative grid atlas collected in Leginon, showing an evident gradient in ice thickness. (B) A “square” level image from this same grid shows evidence of varying thickness, including occasional empty holes.

Figure 2.

Figure 2.

(A) Determination of mean free path for inelastic scattering by electron tomography. Images were collected for both aldolase and proteasome samples with and without a 15 eV energy slit. Tomograms were collected at the same areas as thickness was measured. Thickness versus Log (Itot/IZLP) is plotted. The line of best fit (green) had a slope of 395 nm. (B) Thickness determined using the energy filter for untilted and 45 degree tilted images. Inset: an overview of 4 holes are shown where the measurements were made, untilted (left) and tilted (right). For each hole, thickness was measured as described for both tilted and untilted images, and plotted as tilted thickness versus untilted thickness (purple crosses). The green line is a plot of the equation y=1.41x, which is where the points should ideally lie. (C) Determination of the ALS coefficient using thickness as measured by the energy filter. Thickness was measured over many images using the energy filter and eq. (1). Thickness plotted versus log I0/I as described in the text. For this experiment, the slope was 332 nm.

Figure 3.

Figure 3.

(A) Histogram of thickness values as measured on a rabbit muscle aldolase sample frozen on gold Ultrafoil grids. (B) Histogram of thickness values as measured on a T20S proteasome test sample frozen on C-flat carbon grids. (C-J) Plots of mean radial intensity of Fourier transforms of image averages versus resolution for various ice thicknesses. Blue bar: 3.9 Å−1 resolution. (C): 0–25 nm thickness. (D): 25–50 nm thickness. (E): 50–75 nm thickness. (F) 75–100 nm thickness. (G): 100–125 nm thickness. (H): 125–150 nm thickness. (I): 150–175 nm thickness. (J): 175–200 nm thickness.

Figure 4:

Figure 4:

Plots of Thon ring extent, as measured by CTFFIND4 (Å−1), versus ice thickness for several samples. (A): glutamate dehydrogenase. (B, C): rabbit muscle aldolase. (D): T20S proteasome. Thon ring extent goes to very low resolution where the ice is too thin or absent, is optimal at thin ice, and generally rises as ice thickens.

Figure 5:

Figure 5:

Integration of ice thickness determination into Leginon. (A) The control panel for the node is shown. The user provides various parameters for thickness determination and chooses how often the thickness is measured. (B) Thickness plots for an experiment as shown in the Appion summary pages. (C) Thickness information is displayed when viewing the image in the Leginon web interface.

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