Fig. 2: Cryo-EM map and model of RcpAstrep-TadDFLAG complex with C13 symmetry.
From: Assembly mechanism of a Tad secretion system secretin-pilotin complex
a Purification and visualisation of RcpA-TadD complex. (Left) SDS-PAGE analysis of RcpAstrep-TadDFLAG purified by double pulldown affinity chromatography reveal RcpAstrep in both monomeric (43 kDa with C-terminal strep tag) and an SDS-resistant form consistent with secretin assembly. TadDFLAG migrated as two bands consistent with full-length (27 kDa with C-terminal FLAG tag) and a clipped form (~26 kDa). (Right) Typical cryo-EM micrograph showing RcpAstrep-TadDFLAG particles with associated class averages beneath. The purification was repeated at least three times independently with similar particles obtained. b, c Side and central section view of the RcpAstrep-TadDFLAG complex cryo-EM map at overall 2.7 Å resolution. No map was resolved for the RcpA N-domain. Map contoured at 6σ. d Fit of the secretin model within the RcpAstrep-TadDFLAG complex map with selected regions zoomed to show side chain detail and build. Map contoured at 5σ. e TadD map obtained with a symmetry expansion and focussed refinement strategy (termed TadDFRmap). Map contoured at 6.5σ. f Fit of the TadD model within TadDFRmap. g Zoomed regions of TadDFRmap as boxed in f showing side chain detail and build.
