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. 2019 Apr 1;8:e42747. doi: 10.7554/eLife.42747

Figure 5. Sub-tomogram averaging of FAS vitrified on hydrophilized graphene.

(A) Zero degree view of tomographic tilt series. (B) Slab of vitrified buffer delimited by carbon and ice contaminants, indicating adsorption of FAS complexes to the graphene-water interface. (C) Subtomogram averaging confirms the structural integrity of FAS. (D) Three-dimensional impression (not drawn to scale) indicating the relative position of Quantifoil carbon film (dark grey) and hydrophilized graphene (mid-grey) on the copper support grid (dark red). The solution containing FAS particles (light grey) was applied from the uncoated side of the grid.

Figure 5.

Figure 5—figure supplement 1. Chemical doping of graphene-coated Quantifoil grids.

Figure 5—figure supplement 1.

(A) Quantifoil R0.6/1 grid coated with graphene. (B) Electron diffraction pattern of the area shown in (A). (C) Same grid with a droplet of water. (D) Same grid as in (A) after chemical doping with 1-pyrCA. (E) Electron diffraction pattern of area imaged in (D). (F) Doped grid with a droplet of water.
Figure 5—figure supplement 2. Slices through tomographic volume of FAS on hydrophilized graphene.

Figure 5—figure supplement 2.

Four-pixel slices through NAD-filtered tomograms at various heights show atmospheric ice contamination (slices #1 and #4). FAS particles are evenly spread in one layer (slice #2) on the graphene film. A layer of contaminants on the lower surface identifies the position of the electron-transparent graphene sheet (slice #3).
Figure 5—figure supplement 3. Angular distribution plot after subtomogram averaging of the particle dataset on hydrophilized graphene support (Figure 5C).

Figure 5—figure supplement 3.

Figure 5—figure supplement 4. Multi-reference alignment of FAS on hydrophilized graphene.

Figure 5—figure supplement 4.

Classification by MRA (A) indicates that all particles are intact. The map of the best class (class 1) does not show any sign of denaturation (B). Published cryo-EM map of FAS (Gipson et al., 2010) shown for comparison on the right.
Figure 5—figure supplement 5. Resolution estimate of subtomogram averages.

Figure 5—figure supplement 5.

FSC of the two unfiltered half-maps of FAS before (blue) and after (cyan) masking. FSC performed on the half-maps with phases randomized beyond 60 Å (grey). FSC curves before and after masking indicate resolutions of 24.6 Å and 17.1 Å, respectively.
Figure 5—video1. Three-dimensional rendering of FAS on hydrophilized graphene.
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DOI: 10.7554/eLife.42747.017
Tomographic reconstruction of sample in Figure 5. The distribution of FAS particles is visible in the sequence of xy and xz slices and volume segmentation. Holey carbon film (light gray), FAS (orange), graphene contamination (dark grey), atmospheric ice contamination (dark blue) and vitreous buffer (cyan). Density map and orientations from subtomogram averaging were used to place copies of FAS into the tomographic reconstruction. Vertical section: single layer of particles adsorbed to the graphene-water interface.