1. Volume 25, Issue 5, Pages 806-815.e3 (May 2017)
Full-length, Oligomeric Structure of Wzz Determined by Cryoelectron Microscopy Reveals Insights into Membrane-Bound States 
Richard F. Collins, Vasileios Kargas, Brad R. Clarke, C. Alistair Siebert, Daniel K. Clare, Peter J. Bond, Chris Whitfield, Robert C. Ford 
Structure 
Volume 25, Issue 5, Pages e3 (May 2017)
DOI: /j.str
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2. Structure 2017 25, 806-815.e3DOI: (10.1016/j.str.2017.03.017)
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3. Figure 1 Cryo-EM of the Solubilized WzzBST in DDM
(A) An area of a micrograph with WzzB particles (white circles). Minor clustering was observed and particles tend to adopt a back-to-back association arrangement. Scale bar, 50 nm.
(B) A representative particle set.
(C) Different views after applying a reference-free 2D projection classification. Side views show clearly a bell-shaped periplasmic region with additional density at the bottom corresponded to the transmembrane region and detergent.
(D) Refined electron density map of WzzB colored according to local resolution values (cooler colors represent higher resolution). Cross-section of the density area shows the micelle formation and minor density in the center. Scale bar, 7.5 nm.
(E) Histogram plotting the range of local resolution identified in the map.
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4. Figure 2
Rigid-Body Fitting of the Periplasmic WzzB Protomers into the Cryo-EM Map
(A) Dimensions of the refined WzzB. The fitted protomers of WzzB (PDB: 4E29) provided correlation coefficient values of 0.82 and 0.92, before and after the MD flexible fitting, respectively. L5 and L6 loops were poorly fitted due to missing density in this area. The α3 helix was also found to be poorly fitted in the surrounding area.
(B) Side view cross-section of the map shows the fitting of the protomers and the ellipse-like shaped densities in the TM region, corresponding to DDM micelles. The juxtamembrane and TMDs were also resolved (red and blue arrows, respectively).
(C) Top view showing the orientation of the α3 helix and L4 loop in the fitted monomers.
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5. Figure 3
CG-MD Simulations of the Full-Length WzzB Protomer and Fitting into the Density Map
(A) Left, representative frames (opaque and transparent) of the MD trajectory showing the full-length WzzB embedded into a POPC membrane, of which the phosphate groups (brown beads) define the membrane thickness. The periplasmic domain (purple) fluctuated by an angle subtended by the (indicated) α3 helix (φ) of 0–47° perpendicular to the membrane plane (z axis). The α/β domain that is proposed to restrict complete collapse of the periplasmic region onto the membrane is indicated by the black arrows close to the membrane surface. Right, plot showing the angle (black) between a vector defined by the periplasmic α3 helix and z axis over time in microseconds. Average angle using a window of 50 frames is shown in orange color.
(B) Left, C12 density map with a highlighted protomer structure (purple). Right, a frame from the MD trajectory fitted into the protomer map. Red represents the charged residues. The TMD of the protein is well docked in the map showing that density corresponded to full-length protomers as solved by cryo-EM.
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6. Figure 4
Representative Simulation Snapshot of the TMDs of the Full-Length Dodecameric WzzBST
(A) Phosphate groups are presented in brown transparent surface. Dark gray is TMD1, yellow is TMD2. Negatively charged residues (Asp and Glu) are presented in red, positively charged (Arg, Lys) in blue.
(B) Residues involved in the interaction interface between the TM helices. Cyan shows leucine residues, and blue corresponds to valines. Prolines in the TMH2 loop, which are thought to orient the TMH2, are displayed in brown. See also Figures S1–S3.
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7. Figure 5
Atomistic MD Simulation of the Dodecameric WzzBST in a Lipid Bilayer
(A) Side views (left, center) and top view (right) of the dodecamer after 30 ns. The complex lost its symmetry and dissociation events were observed between the helical domains of the periplasmic region, while L4 loops became clustered together (right).
(B) Inter- and intramolecular contacts between residues in the complex. The long α3 helices were able to bend/kink in different regions.
(C) Distance over time between donor and acceptor atoms in key residues indicated by arrows in (B). See also Figure S4.
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8. Figure 6
Model of the Proposed O-Antigen Chain-Length Determination Mechanism
Wzy (red, represented by the acyl-CoA dehydrogenase, PDB: 1EGC) interacts with Wzz (gray and cyan) during the polymerization of the Oag (yellow). The TM region of Wzy docks in between two TMDs of Wzz (cyan) and the periplasmic domain is responsible for polymerization of the Oag chain. “Stopwatch” mutations in Wzz that cause Wzy to dissociate from the complex too early result in prematurely truncated polysaccharide chains (arrows, central Wzy molecule). “Ruler” mutations affect the interaction of the polysaccharide chain with the periplasmic barrel and this occurs either on the surface of the Wzz barrel (right) or on the inside (left) after insertion through the periplasmic ring. The stopwatch component of the model will be independent of the oligomeric state of Wzz, but the ruler component may be oligomer dependent if the polysaccharide chain must wrap around the entire barrel (right) or fill the internal cavity (left) before chain polymerization is terminated.
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