Figure 1. Detection of cytoplasmic mature 60S and mitochondrial ribosomes in 2D images of FIB-milled yeast lamella.

(A) Cryo-EM like density generated using the atomic coordinates of PDB: 6Q8Y that correspond with the mature 60S. (B) TEM image of the nuclear periphery from a FIB-milled yeast lamella. Yellow arrows indicate low-resolution features in the cytoplasm that may indicate the presence of ribosomes. Blue arrows indicate regions of similar size and contrast in the nucleoplasm. NE: nuclear envelope; NPC: nuclear pore complex. (C) Cryo-EM micrograph of yeast nuclear periphery from FIB-milled lamella with the results from a 2DTM search using the mature 60S template. Significant targets are indicated by mapping the template in the best matching locations and orientations (shown in yellow). The red box indicates the regions highlighted in (E) and (F). Scale bar = 50 nm. (D) 3D surface representation showing the pixel-wise 2DTM SNRs in the 50x50 pixel region of the normalized maximum intensity projection (MIP) indicated by the yellow box in (E). Each square represents 10x10 pixels. Colors represent the SNR value of each pixel as indicated by the scale bar below. (E) Normalized MIP showing the results of 2DTM using the template in (A) in the region of (C) indicated in red. (F) 3D slab indicating the locations and orientations of mature 60S-detected targets in the indicated region of (C). The red polypeptide indicates the location of the polypeptide exit tunnel on each 60S. (G) Bar chart indicating the number of mature 60S-detected targets identified in the indicated subcellular compartments in 28 images of the nuclear periphery. (H) Plot showing the density of mature 60S in the regions of the images corresponding to the nucleus (blue) or cytoplasm (red). Each dot represents a different image. The solid bar indicates the median. (I) 10 Å filtered 3D reconstruction calculated from 3991 60S subunits at the locations and orientations detected in 28 images, showing clear density for the 40S small subunit. The molecular model of the 60S used to generate the template in (A) is shown in yellow.

Figure 1—figure supplement 1. FIB-milled yeast cells and detection statistics relative to a Gaussian noise model.

(A) FIB-image of two yeast cells frozen on a cryo-EM grid. (B) FIB image of the lamella after milling the cells shown in (A). (C) Survival histogram showing the number of search locations with 2DTM SNR values above a given threshold from a 2DTM search using the mature 60S template in Figure 1A.

Figure 1—figure supplement 2. Background 2DTM SNRs in the nucleus and vacuole relative to the cytoplasm.

(A) Cryo-EM image from Figure 1B indicating the image subregions from the normalized MIP that were used to calculate the histograms in B-E. (B) Histogram showing the distribution of pixel-wise 2DTM SNRs in the normalized MIP resulting from 2DTM using the template in Figure 1A, corresponding to the cytoplasmic (red) or nuclear (blue) subregions of the image in (A). Only values below the threshold allowing a single false positive per image (2DTM SNR = 7.85) are shown. (C) As in (B), showing the indicated subset of the histogram. (D) As in (B) showing the 2DTM SNRs in the cytoplasmic (red) and vacuole (dark blue). (E) As in (D), showing the indicated subset of the histogram.

Figure 1—figure supplement 3. Reconstruction using 60S coordinates shows features consisitent with detection of 80S ribosomes.

(A) FSC from two half-maps using a 175 Å mask obtained for the 3D reconstruction shown in Figure 1I calculated using the mature 60S-detected targets identified by 2DTM. (B) Reconstruction from 3991 in situ mature 60S-detected targets from yeast lamellae. Colors show the estimated local resolution. The 10Å-filtered reconstruction in Figure 1I is shown in transparent grey. (C) As in (B), showing the comparison with the mature 60S template, filtered to 10 Å. The cut-away shows clear density for the 40S, which is outside of the template and a tRNA with an estimated local resolution of 4–7 Å.