Abstract
Phase plates in cryo-electron tomography (cryoET) improve contrast, increasing the ability to discern separate molecules and molecular complexes in dense biomolecular environments. Here, we applied this new technology to the activation of the human complement system. Binding of C1 to antigen-antibody complexes initiates a cascade of proteolytic events that deposits molecules onto adjacent surfaces and terminates with the formation of membrane-attack-complex (MAC) pores in the targeted membranes. We imaged steps in this process using a Volta phase plate mounted on a Titan Krios equipped with a Falcon-II direct electron detector. The data show patches of single-layer antibodies on the surface and C1 bound to antibody platforms, with ca. ∼4% of instances where C1r and C1s proteases have dissociated from C1, and potentially instances of C1 transiently interacting with its substrate C4 or product C4b. Next, extensive deposition of C4b and C3b molecules is apparent, although individual molecules cannot always be properly distinguished with the current methods. Observations of MAC pores include formation of both single and composite pores, and instances of potential soluble-MAC dissociation upon failure of membrane insertion. Overall, application of the Volta phase plate cryoET markedly improved the contrast in the tomograms, which allowed for individual components to be more readily interpreted. However, variability in the phase shift induced by the phase-plate during the course of an experiment, together with incomplete sampling during tomogram acquisition, limited the interpretability of the resulting tomograms. Our studies exemplify the potential in studying molecular processes with complex spatial topologies by phase-plate cryoET.
Original language | English |
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Pages (from-to) | 155-162 |
Number of pages | 8 |
Journal | Journal of Structural Biology |
Volume | 197 |
Issue number | 2 |
DOIs | |
Publication status | Published - 1 Feb 2017 |
Funding
This work was supported by the Council for Chemical Sciences (CW) of the Netherlands Organization for Scientific Research (NWO grant 700.57.010), the Netherlands Centre for Electron Nanoscopy (NeCEN), Leiden (NWO grant 175.010.2009.001) and by the Institute for Chemical Immunology, an NWO Gravitation project funded by the Ministry of Education, Culture and Science of the government of the Netherlands. F.G.A. Faas was supported by NanoNextNL of the Government of the Netherlands and 130 partners. We thank David Mastronarde for advice with IMOD and Suzan Rooijakkers for critical reading of the manuscript. Appendix A
Keywords
- C1
- Complement
- Electron tomography
- Membrane attack complex
- Phase plate