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.
Highlights
A major goal in structural biology is to understand the molecular mechanisms of biological processes that occur inside cells by studying the underlying proteins and their assemblies, collectively referred to here as ‘complexes.’ To realize this goal, X-ray crystallography and electron cryo-microscopy have been used to generate high-resolution density maps of purified complexes that could be interpreted by atomic models (Berman et al, 2002)
We described a 2D template matching (2DTM) technique that may overcome some of the limitations of 3D template matching (3DTM) (Rickgauer et al, 2017). 2DTM matches projections of 3D templates to features found in single-exposure (2D) images of nominally untilted specimens, at 4 Aor higher resolution depending on the sample and targets to be localized (Rickgauer et al, 2017)
We provide strategies to overcome some of the major limitations of molecular localization in cells using 2DTM
Summary
A major goal in structural biology is to understand the molecular mechanisms of biological processes that occur inside cells by studying the underlying proteins and their assemblies, collectively referred to here as ‘complexes.’ To realize this goal, X-ray crystallography and electron cryo-microscopy (cryo-EM) have been used to generate high-resolution density maps of purified complexes that could be interpreted by atomic models (Berman et al, 2002). Avoiding multiple exposures and high specimen tilt angles helps preserve the high-resolution signal in these 2D images (Brilot et al, 2012), and 2DTM can utilize this signal to detect complexes with high precision, as well as high angular and positional in-plane accuracy (x,y coordinates) In crowded environments such as the cell, this added signal comes at the expense of increased background (structural noise) in the images due to overlapping density from other molecules, and a relatively large error in localizing the depth of the targets within the sample (z coordinate) (Rickgauer et al, 2017). We show that 2DTM is a versatile method, with comparable sensitivity and improved precision relative to 3DTM, pointing to potentially broad applications for both in situ visual proteomics and in situ structure determination, including de novo structure determination
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