Locating Macromolecular Assemblies in Cells by 2D Template Matching with cisTEM
- Howard Hughes Medical Institute, United States
- University of Massachusetts Medical School, United States
- EMBL, Germany
- European Molecular Biology Laboratory, Germany
Abstract
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.
Data availability
All the code used for the 2D template matching has an open source license and is freely available from the cisTEM github repository, https://github.com/timothygrant80/cisTEM. The images of M. pneumoniae analyzed for this work, as well as the 70S reconstructions have been deposited in the EMPIAR and EMDB databases, https://www.ebi.ac.uk/pdbe/emdb/empiar/ and https://www.ebi.ac.uk/pdbe/emdb,
Article and author information
Author details
Nikolaus Grigorieff
Funding
European Research Council (760067)
- Julia Mahamid
Howard Hughes Medical Institute (N/A)
- Bronwyn A Lucas
- Benjamin A Himes
- Nikolaus Grigorieff
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, 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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Locating Macromolecular Assemblies in Cells by 2D Template Matching with cisTEM
eLife 10:e68946.
https://doi.org/10.7554/eLife.68946
Further reading
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- Cell Biology
- Structural Biology and Molecular Biophysics
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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- Structural Biology and Molecular Biophysics
Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor transcription factor that regulates gene expression programs in response to ligand binding. Endogenous and synthetic ligands, including covalent antagonist inhibitors GW9662 and T0070907, are thought to compete for the orthosteric pocket in the ligand-binding domain (LBD). However, we previously showed that synthetic PPARγ ligands can cooperatively cobind with and reposition a bound endogenous orthosteric ligand to an alternate site, synergistically regulating PPARγ structure and function (Shang et al., 2018). Here, we reveal the structural mechanism of cobinding between a synthetic covalent antagonist inhibitor with other synthetic ligands. Biochemical and NMR data show that covalent inhibitors weaken—but do not prevent—the binding of other ligands via an allosteric mechanism, rather than direct ligand clashing, by shifting the LBD ensemble toward a transcriptionally repressive conformation, which structurally clashes with orthosteric ligand binding. Crystal structures reveal different cobinding mechanisms including alternate site binding to unexpectedly adopting an orthosteric binding mode by altering the covalent inhibitor binding pose. Our findings highlight the significant flexibility of the PPARγ orthosteric pocket, its ability to accommodate multiple ligands, and demonstrate that GW9662 and T0070907 should not be used as chemical tools to inhibit ligand binding to PPARγ.
