Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms

Abstract

With faithful sample preservation and direct imaging of fully hydrated biological material, cryoelectron tomography (cryo-ET) provides an accurate representation of molecular architecture of cells. However, detection and precise localization of macromolecular complexes within cellular environments is aggravated by the presence of many molecular species and molecular crowding. We developed a template-free image processing procedure for accurate tracing of complex networks of densities in cryo-electron tomograms, a comprehensive and automated detection of heterogeneous membranebound complexes and an unsupervised classification. Applying this procedure to tomograms of intact cells and isolated endoplasmic reticulum (ER), we detected and classified small protein complexes like the ER protein translocons, which were not detected by other methods before. This classification provided sufficiently homogeneous particle sets and initial references to allow subsequent de novo subtomogram averaging. Therefore the procedure presented allows a comprehensive detection and a structural analysis of complexes in their native state. In addition, we present structural evidence that different ribosome-free translocon species are present at the ER membrane, determine their 3D structure, and show that they have different localization patterns forming nanodomains.

Deep Learning Improves Macromolecule Identification in 3D Cellular Cryo-Electron Tomograms

“…The combination of algorithms with different designs, paves the way towards successful identification of macromolecules in cryo-ET. Having an accurate picture of the actual number of macromolecules in situ has paramount importance to analyze their spatial distribution 8 .…”

Revealing the polarity of actin filaments by cryo-electron tomography

“…The more distal the position, the more uniform the polarity distribution, while mixed polarity can be observed mainly in the proximal regions ( Technical developments in sample preparation allowed cryo-ET to provide fundamental insights into cellular assemblies and processes in-situ (Mahamid et al, 2016;Marko et al, 2007;Schaffer et al, 2019). However, it also requires the development of novel tools to analyse the data (Chen et al, 2017;Himes and Zhang, 2018;Martinez-Sanchez et al, 2020;Song et al, 2019). APT complements these tools and allows to analyse actin filaments in physiological relevant processes, which would provide a better understanding of actin networks, such as the actin cortex (Chugh et al, 2017), stereocilia organization (Metlagel et al, 2019) or remodelling of actin during phagocytosis (Gerisch, 2011).…”

Trans-synaptic assemblies link synaptic vesicles and neuroreceptors

“…Cryo-ET density was traced in 3D using the discrete Morse theory segmentation and the topological persistence simplification [46,40]. The resulting network of greyscale minima, saddle points and arcs provided an accurate representation of the proteins and lipids present at the synapse (Figure 1a, b, Supplemental video 1).…”

“…The number and abundance of structurally similar types of complexes present at the synapse is not known a priori. We used the affinity propagation (AP) clustering in order to classify the particle sets according to their morphological features and remove false positive complexes, because AP is better suited for this task than the other commonly used methods [48,40]. As rotational averages around vectors normal to the plasma membrane were subjected to AP clustering, the results were sensitive to the particle orientation with respect to the membrane, but not to the rotation around the membrane normal.…”

“…To overcome problems arising from the presence of many molecular species and molecular crowding, here we extended the procedure we recently developed for automated, template-free detection and unsupervised classification of heterogeneous membrane-bound complexes [40] ( Figure S1). This allowed us to localize different synaptic complexes at a single nanometer precision, obtain their native average structures, explore their relation to synaptic vesicles (SVs) and synaptic plasma membranes, and resolve the trans-synaptic assemblies that they form.…”