1. Cell Biology
  2. Structural Biology and Molecular Biophysics

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

  1. Bronwyn A Lucas  Is a corresponding author
  2. Kexin Zhang
  3. Sarah Loerch
  4. Nikolaus Grigorieff  Is a corresponding author
  1. University of Massachusetts Medical School, United States
  2. University of California, Santa Cruz, United States
https://doi.org/10.7554/eLife.79272
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  1. Bronwyn A Lucas
  2. Kexin Zhang
  3. Sarah Loerch
  4. Nikolaus Grigorieff
(2022)
In situ single particle classification reveals distinct 60S maturation intermediates in cells
eLife 11:e79272.
https://doi.org/10.7554/eLife.79272
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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.

Data availability

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

The following data sets were generated

Article and author information

Author details

  1. Bronwyn A Lucas

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    bronwyn.lucas@umassmed.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9162-0421

  2. Kexin Zhang

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    Competing interests
    No competing interests declared.

  3. Sarah Loerch

    Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1731-516X

  4. Nikolaus Grigorieff

    RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
    For correspondence
    niko@grigorieff.org
    Competing interests
    Nikolaus Grigorieff, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1506-909X

Funding

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.

Copyright

© 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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  1. Bronwyn A Lucas
  2. Kexin Zhang
  3. Sarah Loerch
  4. Nikolaus Grigorieff
(2022)
In situ single particle classification reveals distinct 60S maturation intermediates in cells
eLife 11:e79272.
https://doi.org/10.7554/eLife.79272

Share this article

https://doi.org/10.7554/eLife.79272

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Further reading

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics

    Baited reconstruction with 2D template matching for high-resolution structure determination in vitro and in vivo without template bias

    Bronwyn A Lucas, Benjamin A Himes, Nikolaus Grigorieff
    Research Advance

    Previously we showed that 2D template matching (2DTM) can be used to localize macromolecular complexes in images recorded by cryogenic electron microscopy (cryo-EM) with high precision, even in the presence of noise and cellular background (Lucas et al., 2021; Lucas et al., 2022). Here, we show that once localized, these particles may be averaged together to generate high-resolution 3D reconstructions. However, regions included in the template may suffer from template bias, leading to inflated resolution estimates and making the interpretation of high-resolution features unreliable. We evaluate conditions that minimize template bias while retaining the benefits of high-precision localization, and we show that molecular features not present in the template can be reconstructed at high resolution from targets found by 2DTM, extending prior work at low-resolution. Moreover, we present a quantitative metric for template bias to aid the interpretation of 3D reconstructions calculated with particles localized using high-resolution templates and fine angular sampling.

    1. Structural Biology and Molecular Biophysics

    Locating macromolecular assemblies in cells by 2D template matching with cisTEM

    Bronwyn A Lucas, Benjamin A Himes ... Nikolaus Grigorieff
    Tools and Resources Updated

    For a more complete understanding of molecular mechanisms, it is important to study macromolecules and their assemblies in the broader context of the cell. This context can be visualized at nanometer resolution in three dimensions (3D) using electron cryo-tomography, which requires tilt series to be recorded and computationally aligned, currently limiting throughput. Additionally, the high-resolution signal preserved in the raw tomograms is currently limited by a number of technical difficulties, leading to an increased false-positive detection rate when using 3D template matching to find molecular complexes in tomograms. We have recently described a 2D template matching approach that addresses these issues by including high-resolution signal preserved in single-tilt images. A current limitation of this approach is the high computational cost that limits throughput. We describe here a GPU-accelerated implementation of 2D template matching in the image processing software cisTEM that allows for easy scaling and improves the accessibility of this approach. We apply 2D template matching to identify ribosomes in images of frozen-hydrated Mycoplasma pneumoniae cells with high precision and sensitivity, demonstrating that this is a versatile tool for in situ visual proteomics and in situ structure determination. We benchmark the results with 3D template matching of tomograms acquired on identical sample locations and identify strengths and weaknesses of both techniques, which offer complementary information about target localization and identity.