Fig. 1: Visualization of GspDα multimers within E. coli cells and determination of the overexpression and membrane location of GspDα.
From: Membrane translocation process revealed by in situ structures of type II secretion system secretins
a The tomogram z slice view of E. coli overexpressing GspDα. b Segmentation of the tomogram shown in (a), with OM colored purple, PG colored yellow, IM colored dark red, and GspDα on IM colored blue. c Zoom in tomogram z slice view of top view GspDα particles. d Zoom in tomogram z slice view of side view GspDα particles, indicated by white arrowheads with blue outline. e–g Tomogram z slices showing tilted GspDα on the inner membrane or GspDα with only one side connecting the inner membrane (white arrowheads with blue outline). The outer and inner membranes are indicated by the purple line and the dark red line, respectively. h A tomogram z slice view of E. coli overexpressing GspDα and with D-methionine added (the GspDα particle on the OM is indicated by a white arrowhead with a green outline). i Segmentation of the tomogram shown in h, with OM colored purple, PG colored yellow, IM colored dark red, GspDα on IM colored blue, and GspDα on OM colored green. j Immunoblotting results of GspDα overexpression by using anti-His tag antibodies. BL21 (DE3) cells (WT) without plasmid transformation were used as the control. k, Inner and outer membrane fractions of the GspDα overexpressing cells treated with/without D-methionine were separated by sucrose density gradient centrifugation. The membrane location of GspDα was immunoblotted with anti-His tag antibodies. l Outer membrane proteins of the GspDα overexpressing cells treated with/without D-methionine were extracted using a bacterial membrane protein extraction kit. The total GspDα and outer membrane GspDα were examined by western blot analysis using anti-His tag antibodies. The expression and membrane location experiments of GspDα were repeated three times independently with similar results. IM inner membrane, OM outer membrane, PG peptidoglycan, D-Met D-methionine, WT wild type. Source data are provided as a Source Data file.
