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.

Figure 1.. Cryo-EM of OmcE filaments.

(a) Representative cryo-EM image of the purified OmcE filaments (black arrow) from the OmcS-H357M producing variant strain of G. sulfurreducens, taken from the 35,976 images recorded. Lacey carbon grids were used for the cryo-EM imaging (carbon film indicated by white arrow). The sample was treated with DNase I prior to freezing. Scale bar, 200 Å. (b) A two-dimensional class average of the OmcE filament, showing the rise of 34 Å between adjacent subunits. (c) The surface of the reconstruction (transparent gray) with backbone trace of the OmcE subunits. A zoomed region of the circle is shown on the right: the hemes and their coordinating histidines are labeled. (c) The heme array in OmcE, with the minimum observed edge-to-edge distances indicated between adjacent hemes. The heme from the adjacent subunit is shown in blue.

Figure 2.. OmcE versus OmcS, very different protein structures but almost identical heme packing

(a) The backbone traces of single OmcE and OmcS subunits. The heme molecules are shown as sticks. (b) Alignment of OmcE and OmcS proteins. The RMSD of the aligned atom pairs and total atom pairs are shown. (c) The comparison of heme chains in OmcE and OmcS filaments. The OmcS filament is aligned to the OmcE subunit on the left containing hemes 1–4. (d) The bis-histidine coordinations for the six hemes aligned in (c). The iron atoms in heme are shown as orange circles, and the imidazole side chains of the histidines are represented with blue circles.

Figure 3.. Orientation plot for heme-c pairs

(a) One heme in a pair can be aligned to the other heme by a rotation and a translation. The rotation angle 𝜃 is shown. For example, as shown in Fig. 2c, the rotation angle 𝜃 between heme 1 and heme 2 is ~130°, between heme 2 and heme 3 is ~175°, and between heme 3 and heme 4 is ~140°. (b) The heme (heme c only, ligand ID: HEC) pairs in all PDB structures were analyzed. The minimum distances refer to the smallest distance between the heme pairs, regardless of the atom type. The angle θ was determined from the alignment rotation matrix between heme pairs. For example, θ=0° means two hemes are perfectly parallel, while θ=180° means two hemes are perfectly antiparallel (flipped) during the alignment. All heme pairs with a minimum distance less than or equal to 6 Å are shown. The heme pairs in the OmcE and OmcS filaments are highlighted in green and red, respectively.

Figure 4.. Mixtures of OmcS variant (H357M), extracellular T4P, DNA, and OmcE filaments

(a) Representative cryo-EM image of filaments sheared and precipitated from an overproducing OmcS-H357M variant strain of G. sulfurreducens, taken from the 17,105 images recorded. The sample was not treated with DNase I prior to freezing, and was not centrifuged to remove aggregates as in Fig. 1. Other than OmcS filaments, other species of filaments were observed and labeled correspondingly: OmcE filaments (black arrow), OmcS filaments (blue arrows), PilA-N-C type IV pili (orange arrows), and extracellular B-form DNA (white arrows). Scale bar, 200 Å. (b) Two-dimensional averages of the four different filaments in (a), showing that these different filaments are easily distinguishable. (c) A ~4.1 Å cryo-EM reconstruction of the Type IV pili, with ribbon model of the PilA subunits containing two chains, PilA-N (cyan) and PilA-C (orange). (d) A ~10 Å cryo-EM reconstruction of DNA filaments showing a clear pitch of ~ 36 Å with ribbon model of B-DNA.