In situ single particle classification reveals distinct 60S maturation intermediates in cells
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.
Micrographs, templates and scaled maximum intensity projections (MIPs) in this study have been deposited to EMPIAR and are accessible with the following public access code: EMPIAR-10998
Article and author information
Chan Zuckerberg Initiative (2021-234617)
- Bronwyn A Lucas
- Nikolaus Grigorieff
Howard Hughes Medical Institute
- Nikolaus Grigorieff
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Sjors HW Scheres, MRC Laboratory of Molecular Biology, United Kingdom
- Received: April 5, 2022
- Preprint posted: April 10, 2022 (view preprint)
- Accepted: August 24, 2022
- Accepted Manuscript published: August 25, 2022 (version 1)
- Version of Record published: September 5, 2022 (version 2)
© 2022, Lucas et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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