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Within this pore, the central -sheet has straightened and opened by *70? as measured from your fitting, and TMH1 and TMH2 are absolutely unwound into -hairpins to variety a -barrel spanning the membrane bilayer (Fig. 3A?C). The pore channel is hence shaped by a 52-stranded -barrel that is 80 ?in internal diameter and about one hundred ?in height.PLOS Biology | DOI:10.1371/journal.pbio.February 5,five /Conformation Changes through Pore Development by a Perforin-Like ProteinFigure 3. Construction of your pleurotolysin pore. (A) Slash away facet and (B) tilted surface sights in the cryo-EM reconstruction of the pleurotolysin pore together with the fitted atomic structures. (C) Phase from the pore map corresponding to only one subunit with pore model fitted into the density. The PlyB crystal structure is superposed to point out a 70?opening with the MACPF -sheet (crimson) and motion with 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 involving TMH2, the HTH region, and PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/591453 the underlying sheet in the PlyB crystal structure. The posture from the TMH2 helix lock (pink spheres) and TMH2 strand lock (grey spheres) are demonstrated. The very conserved "GG" motif (296?ninety seven) while in the HTH area is represented as yellow spheres. doi:ten.1371/journal.pbio.1002049.gThe PlyB C-terminal trefoil sits in the cavity formed by a V-shaped wedge of density making contact with the membrane (Figs. 3C and 4A). This density might 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 within the density. Having said that, inside the crystal framework of PlyA, we mentioned two distinct V-shaped dimers (termed N-dimer and C-dimer) during the asymmetric device (S3A and S3D Fig.). The two types fitted adequately into EM density, putting either the PlyA N-terminus (N-dimer) or C-terminus (C-dimer) in proximity into the membrane surface. We analyzed the orientation of PlyA by adding a hexahistidine tag into the N-terminusPLOS Biology | DOI:10.1371/journal.pbio.February 5,6 /Conformation Modifications through Pore Formation by a Perforin-Like ProteinFigure four. Validation on the orientation of PlyA. (A) Proposed orientation of PlyA dimer (pink) and interface with PlyB C-terminal trefoil (eco-friendly). Trp 6 is demonstrated as purple spheres. (B) Western blot displaying PlyA binding to crimson blood cells when untagged or C-terminally tagged although not when N-terminally tagged. doi:ten.1371/journal.pbio.1002049.g(Fig. 4A and 4B), which abrogated membrane binding of PlyA to crimson blood cells while a Cterminal tag had no impact on binding (Fig. 4B). Also, mutation of Trp 6 (W6E), situated in the PlyA N-dimer interface, reduced membrane binding and resulted in 100-fold lessen pore-forming activity (Fig. 4A, denoted as purple spheres; S4A and S4B Fig.). These data support an Ndimer-like arrangement of PlyA molecules (Fig. 4A), regular with all the identified orientation of actinoporins around the membrane floor [29]. The ensuing in good shape of 26 PlyA and 13 PlyB subu.