Abstract
With faithful sample preservation and direct imaging of fully hydrated biological material, cryo-electron 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 membrane-bound 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.
Footnotes
- Added an application of our procedure to in situ data. - Direct comparison shows superiority of the methods used in our procedure over those currently used in the field.