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1B, and 3C, 3D). Just after refinement in the central uneven device, the pore was rebuilt with C13 symmetry in Chimera [33] to present the final pore model. On this pore, the central -sheet has straightened and opened by *70? as calculated within the fitting, and TMH1 and TMH2 are fully unwound into -hairpins to type a -barrel spanning the membrane bilayer (Fig. 3A?C). The pore channel is therefore fashioned by a 52-stranded -barrel that is 80 ?in interior diameter and over 100 ?in top.PLOS Biology | DOI:10.1371/journal.pbio.February five,five /Conformation Alterations through Pore Formation by a Perforin-Like ProteinFigure 3. Construction of the pleurotolysin pore. (A) Reduce away aspect and (B) tilted floor sights on the cryo-EM reconstruction of the pleurotolysin pore using the fitted atomic buildings. (C) Section on the pore map similar to an individual subunit with pore design equipped into the density. The PlyB crystal construction is superposed to point out a 70?opening in the MACPF -sheet (red) and motion of the HTH motif (cyan). TMH regions (yellow) are refolded into transmembrane PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/10999558 -hairpins. The PlyB C-terminal trefoil (inexperienced) sits on top of the PlyA dimer (pink). (D) Interface amongst TMH2, the HTH area, and PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/591453 the underlying sheet during the PlyB crystal construction. The situation on the TMH2 helix lock (pink spheres) and TMH2 strand lock (gray spheres) are revealed. The extremely conserved "GG" motif (296?ninety seven) within the HTH region is represented as yellow spheres. doi:ten.1371/journal.pbio.1002049.gThe PlyB C-terminal trefoil sits in the cavity shaped by a V-shaped wedge of density speaking to the membrane (Figs. 3C and 4A). This density may be accounted for by two PlyA molecules, revealing a tridecameric PlyB/2xPlyA pore assembly. The symmetrical condition of PlyA precludes discrimination of up/down orientation in the density. Even so, inside the crystal structure of PlyA, we noted two unique V-shaped dimers (termed N-dimer and C-dimer) in the asymmetric unit (S3A and S3D Fig.). Both varieties equipped sufficiently into EM density, positioning either the PlyA N-terminus (N-dimer) or C-terminus (C-dimer) in proximity for the membrane floor. We analyzed the orientation of PlyA by including a hexahistidine tag on the N-terminusPLOS Biology | DOI:ten.1371/journal.pbio.February five,six /Conformation Variations through Pore Formation by a Perforin-Like ProteinFigure four. Validation in the orientation of PlyA. (A) Proposed orientation of PlyA dimer (pink) and interface with PlyB C-terminal trefoil (green). Trp six is revealed as purple spheres. (B) Western blot showing PlyA binding to pink blood cells when untagged or C-terminally tagged although not when N-terminally tagged. doi:10.1371/journal.pbio.1002049.g(Fig. 4A and 4B), which abrogated membrane binding of PlyA to red blood cells whilst a Cterminal tag had no effect on binding (Fig. 4B). Also, mutation of Trp 6 (W6E), located in the PlyA N-dimer interface, decreased membrane binding and resulted in 100-fold lessen pore-forming action (Fig. 4A, denoted as purple spheres; S4A and S4B Fig.). These details help an Ndimer-like arrangement of PlyA molecules (Fig.