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Cited by 53Advanced Electron Microscopy Techniques and ApplicationsRNA and protein synthesis mechanismsEnzyme Structure and Function

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

Open access
Lucas, Bronwyn A, Zhang, Kexin, Loerch, Sarah, Grigorieff, Nikolaus·2022·eLife
DOI 10.7554/elife.79272PMC PMC9444246

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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.

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Metadata

Authors
Lucas, B. A., Zhang, K., Loerch, S., & Grigorieff, N.
Affiliations
Howard Hughes Medical Institute (US)
Corresponding author
  • Lucas, Bronwyn A
  • Grigorieff, Nikolaus
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2022-08-25
Volume
11
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.79272
PMC
PMC9444246
OpenAlex
W4293102039
Cited by
53
References
1
Citations by year
2026: 7 · 2025: 13 · 2024: 13 · 2023: 16 · 2022: 4
FWCI
5.72
Type
journal article
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Chan Zuckerberg Initiative (2021-234617)
  • Howard Hughes Medical Institute
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Enzyme Structure and Function
Keywords
template matching, signal-to-noise ratio, maximum likelihood classification, ribosome localization, Saccharomyces cerevisiae, Mycoplasma pneumoniae, subcellular localization, focused ion beam milling
MeSH terms
Models, Molecular, Ribosomes, Saccharomyces cerevisiae, Likelihood Functions, Cryoelectron Microscopy, Saccharomyces cerevisiae Proteins

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