High-confidence 3D template matching for cryo-electron tomography

Abstract

Visual proteomics attempts to build atlases of the molecular content of cells but the automated annotation of cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures of macromolecules. However, their applicability remains limited in terms of both the abundance and size of the molecular targets. Here we show that the performance of TM is greatly improved by using template-specific search parameter optimization and by including higher-resolution information. We establish a TM pipeline with systematically tuned parameters for the automated, objective and comprehensive identification of structures with confidence 10 to 100-fold above the noise level. We demonstrate high-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, fatty acid synthases, lipid membranes and microtubules, and individual subunits inside crowded eukaryotic cells. We provide software tools for the generic implementation of our method that is broadly applicable towards realizing visual proteomics.

Passage of the HIV capsid cracks the nuclear pore

“…Moreover, by analyzing the NPC architecture through subtomogram averaging, differences across a small set of individual particles could not be readily detected. Here, by using template matching of NPC subunits 42,43 we could reliably detect damage of individual NPCs and, by greatly expanding the statistics, connect NPC cracking to HIV capsid passage.…”

“…The obtained bin4 STA averages for CR, IR and NR were masked to only include a single subunit and then used as search templates (lowpass filtered to 30 Å) to perform template matching in full 3D-CTF-corrected bin4 tomograms with visible NPCs using GAPSTOP™ [https://gitlab.mpcdf.mpg.de/bturo/gapstop_tm] 42 based on the STOPGAP software package 73 . The angular search range was set to a 6-degree sampling which has been shown to increase TM performance 42 . The constrained cross-correlation (CCC) peaks which had values 7x above the mean of the CCC-volume were extracted with their corresponding orientations and then manually cleaned in the ArtiaX plugin 70 for ChimeraX 72 to obtain only plausible candidates that are oriented correctly in the nuclear envelope.…”

“…https://gitlab.mpcdf.mpg.de/bturo/gapstop_tm]42 . The angular search range was set to 5degree angular sampling.…”

“…https://gitlab.mpcdf.mpg.de/bturo/gapstop_tm]42 based on the STOPGAP software package73 . The angular search range was set to a 6-degree sampling which has been shown to increase TM performance42 .…”

“…https://gitlab.mpcdf.mpg.de/bturo/gapstop_tm]42 based on the STOPGAP software package73 . The angular search range was set to a 6-degree sampling which has been shown to increase TM performance42 . The constrained cross-correlation (CCC) peaks which had values 7x above the mean of the CCC-volume were extracted with their corresponding orientations and then manually cleaned in the ArtiaX plugin 70 for ChimeraX 72 to obtain only plausible candidates that are oriented correctly in the nuclear envelope.…”