In situ single particle classification reveals distinct 60S maturation intermediates in cells

Abstract

Previously we showed that high-resolution template matching can localize ribosomes in two-dimensional electron cryo-microscopy (cryo-EM) images of untilted Mycoplasma pneumoniae cells with high precision (Lucas et al., 2021). Here we show that comparing the signal-to-noise ratio (SNR) observed with 2DTM using different templates relative to the same cellular target can correct for local variation in noise and differentiate related complexes in focused ion beam (FIB)-milled cell sections. We use a maximum likelihood approach to define the probability of each particle belonging to each class, thereby establishing a statistic to describe the confidence of our classification. We apply this method in two contexts to locate and classify related intermediate states of 60S ribosome biogenesis in the Saccharomyces cerevisiae cell nucleus. In the first, we separate the nuclear pre-60S population from the cytoplasmic mature 60S population, using the subcellular localization to validate assignment. In the second, we show that relative 2DTM SNRs can be used to separate mixed populations of nuclear pre-60S that are not visually separable. 2DTM can distinguish related molecular populations without the need to generate 3D reconstructions from the data to be classified, permitting classification even when only a few target particles exist in a cell.

Quantification of gallium cryo-FIB milling damage in biological lamella

“…The maximum 2DTM SNR decreased with increasing lamella thickness (SI Appendix, Fig. S1B) as observed previously (15)(16)(17)(18). However, we observed a different 2DTM SNR profile as a function of z-coordinate in regions of different thicknesses.…”

“…Any damage introduced during FIB-milling will therefore decrease the correlation with the undamaged template, leading to a lower 2DTM SNR. Ribosomes are present at high density and relatively evenly distributed in the cytoplasm of the yeast Sacchromyces cerevisiae (18), and therefore present an ideal 2DTM target to quantify differences in target integrity. We prepared FIB-milled lamellae of S. cerevisiae cells of thickness varying from 120 nm to 260 nm ( Fig.…”

“…1 E and F ), indicating that this is unlikely to be the result of defocus estimation error. We used the tilt axis and angle estimated from the contrast transfer function (CTF) fit (18, 19) to adjust the coordinate frame to reflect the position of each LSU relative to the lamella center ( Fig. 1 G and H ).…”

“…Changes to the 2DTM SNR provide a read-out of the relative similarity of a target molecule to a given template. We have previously shown that relative 2DTM SNRs discriminate between molecular states and can reveal target identity (17, 18). In this study we show that changes in 2DTM SNRs can also reflect damage introduced during FIB-milling and radiation damage introduced during cryo-EM imaging.…”

“…3A-D, we applied a series of sharp low-pass filters in steps of 0.01 Å -1 to the template using the e2proc3d.py function in EMAN2 (37). We used the locations and orientations from the refined 2DTM search with the full-length template to recalculate the 2DTM SNR with each modified template using the program refine_template (18) by keeping the positions and orientations fixed. The normalized cross-correlation was determined by dividing the SNR calculated with each low-pass filtered template to the SNR of the full-length template for each target.…”