CryoEM-enabled visual proteomics reveals de novo structures of oligomeric protein complexes from Azotobacter vinelandii
-
1.
California Institute of Technology
Abstract
Single particle cryoelectron microscopy (cryoEM) and cryoelectron tomography (cryoET) are powerful methods for unveiling unique and functionally relevant structural states. Aided by mass spectrometry and machine learning, they promise to facilitate the visual exploration of proteomes. Leveraging visual proteomics, we interrogate structures isolated from a complex cellular milieu by cryoEM to identify and classify molecular structures and complexes de novo. That approach determines the identity of six distinct oligomeric protein complexes from partially purified extracts of Azotobacter vinelandii using both anaerobic and aerobic cryoEM. Identification of the first unknown species, phosphoglucoisomerase (Pgi1), is achieved by comparing three automated model building programs: CryoID, DeepTracer, and ModelAngelo with or without a priori proteomics data. All three programs identify the Pgi1 protein, revealed to be in a new decameric state, as well as additional globular structures identified as glutamine synthetase (GlnA) and bacterioferritin (Bfr). Large filamentous assemblies are observed in tomograms reconstructed from cryoFIB milled lamellae of nitrogen-fixing A. vinelandii. Enrichment of these species from the cells by centrifugation allows for structure determination of three distinct filament types by helical reconstruction methods: the Type 6 Secretion System non-contractile sheath tube (TssC), a novel filamentous form of the soluble pyridine transhydrogenase (SthA), and the flagellar filament (FliC). The multimeric states of Pgi1 and SthA stand out in contrast to known crystallographic structures and offer a new structural framework from which to evaluate their activities. Overall, by allowing the study of near-native oligomeric protein states, cryoEM-enabled visual proteomics reveals novel structures that correspond to relevant species observed in situ.
Copyright and License
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
Acknowledgement
This work was funded by support from the Howard Hughes Medical Institute, NIH
1F32GM143836 (R.A.W.), and NIH 1K99GM152765 (R.A.W.). We thank Dr. Douglas Rees, Welison Floriano, Dr. Songye Chen, and Dr. Julian Braxton for invaluable discussions. Bottom412 up mass spectrometry of protein samples was performed at the Beckman Proteome Exploration Laboratory supported by the Arnold and Mabel Beckman Foundation. Generous support of the Beckman Institute for the Caltech Cryo-EM Resource Center was essential for the performance of this research. We thank Momoko Shiozaki, Xiaowei Zhao, Nikki Jean, and Rui Yan at the HHMI Janelia CryoEM Facility for help in sample preparation, microscope operation, and data collection for cryoET.
Contributions
R.A.W. designed experiments. R.A.W., A.O.M., and Y.S. performed sample preparation, biochemical analyses, electron microscopy data collection and analyses, and model building and refinement. T.Z. assembled curated AlphaFold databases. R.A.W. supervised all research. R.A.W. wrote the manuscript, and all authors contributed to revisions.
Supplemental Material
-
Supplementary Information[supplements/636493_file02.pdf]
Files
| Name | Size | Download all |
|---|---|---|
|
md5:c35ab5e5b130b94bda56253f6092373b
|
9.9 MB | Preview Download |
|
md5:3c757f77ef6a82f712cb14ebc60bfcdc
|
7.5 MB | Preview Download |
Additional details
- Howard Hughes Medical Institute
- National Institutes of Health
- 1F32GM143836
- National Institutes of Health
- 1K99GM152765
- Caltech groups
- Division of Chemistry and Chemical Engineering (CCE)
- Publication Status
- Submitted