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Cryo-EM structure of an extracellular Geobacter OmcE cytochrome filament reveals tetrahaem packing

Abstract

Electrically conductive appendages from the anaerobic bacterium Geobacter sulfurreducens were first observed two decades ago, with genetic and biochemical data suggesting that conductive fibres were type IV pili. Recently, an extracellular conductive filament of G. sulfurreducens was found to contain polymerized c-type cytochrome OmcS subunits, not pilin subunits. Here we report that G. sulfurreducens also produces a second, thinner appendage comprised of cytochrome OmcE subunits and solve its structure using cryo-electron microscopy at ~4.3 Å resolution. Although OmcE and OmcS subunits have no overall sequence or structural similarities, upon polymerization both form filaments that share a conserved haem packing arrangement in which haems are coordinated by histidines in adjacent subunits. Unlike OmcS filaments, OmcE filaments are highly glycosylated. In extracellular fractions from G. sulfurreducens, we detected type IV pili comprising PilA-N and -C chains, along with abundant B-DNA. OmcE is the second cytochrome filament to be characterized using structural and biophysical methods. We propose that there is a broad class of conductive bacterial appendages with conserved haem packing (rather than sequence homology) that enable long-distance electron transport to chemicals or other microbial cells.

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Fig. 1: Cryo-EM of OmcE filaments.
Fig. 2: OmcE versus OmcS, very different protein structures but almost identical haem packing.
Fig. 3: Orientation plot for HEC pairs.
Fig. 4: Mixtures of OmcS variant (H357M), extracellular T4P, DNA and OmcE filaments.

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Data availability

The 3D reconstruction for OmcE has been deposited in the Electron Microscopy Data Bank with accession code EMD-25879, and the atomic model has been deposited in PDB with accession code 7TFS. The 3D reconstruction for PilA-N/C has been deposited in the Electron Microscopy Data Bank with accession code EMD-25881, and the atomic model has been deposited in PDB with accession code 7TGG. Other atomic models used in the study were PDB 2P0B, 2J7A and 6VK9.

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Acknowledgements

Cryo-EM imaging was done at the Molecular Electron Microscopy Core Facility at the University of Virginia, which is supported by the School of Medicine and built with NIH grant no. G20-RR31199. Mass spectrometry was performed in the Department of Chemistry Mass Spectrometry Facility at the University of California, Irvine on an instrument generously donated by Biosera. This work was supported by NIH grant nos. GM122510 (E.H.E.) and K99GM138756 (F.W.), DOE grant no. DE-SC0020322 (A.I.H., D.R.B. and E.H.E.), AFOSR grant no. FA9550-19-1-0380 (A.I.H.), NSF grant no. 2030381 (D.S.), Office of Naval Research grant no. N00014-18-1-2632 (C.H.C., K.J. and S.C.) and the SRCP Seed grant at the University of Washington Bothell (D.S). We thank B. Katz and C. Wilmot for helpful discussions.

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K.M., K.J., C.H.C. and S.C. prepared samples. F.W. performed microscopy, image analysis and model building. V.S., Z.S., D.S. and F.W. carried out analysis of existing multihaem structures. A.I.H., E.H.E. and D.R.B. directed the research. F.W., A.I.H., E.H.E and D.R.B. wrote the paper.

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Correspondence to Allon I. Hochbaum, Edward H. Egelman or Daniel R. Bond.

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The authors declare no competing interests.

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Nature Microbiology thanks Andreas Schramm and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Cryo-EM images of the purified OmcE filaments.

Representative cryo-EM images of the purified OmcE filaments (black arrow) from the pTn7m::OmcS-H41M variant strain (a, from 5,269 images recorded) and pGeo7::OmcS-H38M variant strain (b, from 442 images recorded). The two-dimensional class averages of the OmcE filaments are shown on the top right of each. The sample was treated with DNase I and clarified via centrifugation prior to freezing. Scale bar, 200 Å.

Extended Data Fig. 2 Averaged power spectra from different filaments.

All power spectra were generated from raw images aligned to the same axis.

Extended Data Fig. 3 Fourier Shell Correlation (FSC) calculations of OmcE and PilA filaments.

(a) The map:map FSC calculation of OmcE filament (0.143 cutoff). (b) The model:map FSC calculation of OmcE filament (0.38 cutoff). (c) The map:map FSC calculation of PilA filament (0.143 cutoff). (d) The model:map FSC calculation of PilA filament (0.38 cutoff). The 0.38 threshold is used as it is √0.143.

Extended Data Fig. 4 OmcE is the only good match to the map.

(a) AlphaFold2 predictions of eight tetraheme proteins in Geobacter sulfurreducens. (b) Docking of a single subunit into the cryo-EM map. The parts not fitting into the map are labeled by black arrowheads. (c) The cryo-EM map was filtered to 6 Å resolution and set to a very high threshold, so the only densities shown (magenta) are the centers of heme molecules. Bis-His coordination of hemes is shown, and only histidines in OmcE coordinate all four hemes. The poorly coordinated hemes are indicated by black arrowheads. (d) The OmcE atomic model fits into the ~4.3 Å cryo-EM map, with protein Cα trace and ligand displayed. (e) Fitting of four heme co-factors into the corresponding cryo-EM densities.

Extended Data Fig. 5 Mass spectrometry data of isolated filament samples.

a) Silver (for protein) and (b) 3,3′,5,5′- tetramethylbenzidine (for heme) stained SDS-PAGE gels of the four mutants used in this study: pTn7m::OmcS_H41M, pGeo7::OmcS_H38M, pTn7m::OmcS_H357M, pGeo7::OmcS_H357M. Approx. 10 different gels were run during the purification procedure, with the gel shown being the final one. Cryo-EM images of pTn7m::OmcS_H357M were used for OmcE structure determination. OmcE has the highest sequence coverage (c) of the four tetraheme cytochromes with similar predicted folds, OmcE, OmcP, GSU1787, GSU3221, from protein sequencing mass spectrometry of in-solution digests of the isolated filament samples. OmcP was not detected in this analysis. (d) Mass spectrum and (e) sequence matching data of pTn7m::OmcS_H357M filament isolate digest. Underlined sequences are matched peptides from the digest. Red residues are those likely to be chemically modified during sample preparation (see Materials and Methods), as indicated in (e).

Extended Data Fig. 6 Comparisons between OmcE and OmcS.

(a) All seven histidines in OmcE that coordinate heme molecules within the same subunit are highlighted with red stars. These seven histidines in OmcE have been aligned, based upon the structures, to seven histidines in OmcS, and the order of these residues in the primary sequence is the same in both proteins. The histidine in OmcE that coordinates a heme molecule in an adjacent subunit is labeled with a green star. The structurally aligned histidine in OmcS is marked with a blue star. (b,c) All volumes were filtered to 10 Å for a clear display. The density accounted for by atomic models is colored in gray, and the extra density is colored in red. The backbones of OmcE (a) and OmcS (b) filaments are shown, and they are colored in magenta and yellow, respectively. (b,c) All volumes were filtered to 10 Å for a clear display. The density accounted for by atomic models is colored in gray, and the extra density is colored in red. The backbones of OmcE (b) and OmcS (c) filaments are shown, and they are colored in magenta and yellow, respectively.

Extended Data Fig. 7 The omcE genomic region.

Organization of the omcE genomic region, showing the 17-gene cluster downstream containing glycosyltrasferases, undecaprenyl-phosphate transferases and sugar modification enzymes. Similar gene clusters exist downstream of all Geobacter omcE homologs.

Extended Data Fig. 8 Analysis of previous images shows OmcS, not PilA, filaments.

An averaged power spectrum has been computed from the filaments in Malvankar et al., mBio 6, e00084 (2015), Supplemental Figure S1, labeled ‘Transmission electron micrograph of pili of Geobacter sulfurreducens strain Aro-5’. The power spectrum shows a periodicity of ~ 1/(200 Å) marked by the yellow arrows, indicating that these filaments are actually composed of the cytochrome OmcS and are not pili.

Extended Data Fig. 9 PilA-N chain is highly hydrophobic.

(a) Transmembrane predictions of PilA-N by TMHMM2. The first ~25 residues are predicted to be signal peptide and would be cleaved in the mature protein. (b) PilA-C domains bury hydrophobic surface of PilA-N. The hydrophobic residues (Val, Leu, Ile, Met, Phe, Pro and Try) in PilA-N are shown in ball and stick representation in red. The outer globular domain (PilA-C) is shown in yellow for two neighboring subunits. It can be seen that these N-terminal hydrophobic residues get buried by the C-terminal chain when the filament is formed, suggesting that a filament composed of only PilA-N would contain too many surface-exposed hydrophobic residues to be soluble. (c) The hydrophobicity (or lipophilicity) can be compared between a model for a PilA-N only filament (left) and the observed PilA-N/C filament (right). The rather hydrophobic surface for the PilA-N model suggests that it might not be soluble.

Extended Data Fig. 10 Diameter estimations of four different filaments from atomic models.

The atomic models without hydrogens are shown with atoms represented by spheres having the appropriate van der Waals radii.

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Wang, F., Mustafa, K., Suciu, V. et al. Cryo-EM structure of an extracellular Geobacter OmcE cytochrome filament reveals tetrahaem packing. Nat Microbiol 7, 1291–1300 (2022). https://doi.org/10.1038/s41564-022-01159-z

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