Fig 1.
Cryo-EM structures of the ClyA pore complexes.
(A) Top view (or bottom view) of the dodecamer, the tridecamer and the tetradecamer from 2D class averages of the ClyA pore complexes. The numbers of symmetric features are indicated. (B) The final three-dimensional reconstruction electron microscopy density maps of the dodecamer, the tridecamer, and the tetradecamer at the resolution of 2.8 Å, 3.2 Å and 4.3 Å. Top views and side views are shown. One protomer from each oligomer is shown in purple, blue or orange for clarity.
Fig 2.
Structure comparison of the ClyA dodecamer, tridecamer, and tetradecamer complexes.
(A) Illustration of secondary elements within one protomer from the dodecamer. Five α-helices (αA1, αB, αC, αF, and αG) within one protomer (left) are colored in blue, cyan, green, orange and red (right). The corresponding amino acid boundaries are indicated, with the short loops divided and assigned to the linked helices for simplicity. The turning point of αC and αF is indicated by an arrow. The same coloring paradigm is applied in Figs 2D, 2E and S7. (B) Comparison of one protomer from the dodecamer (Prot_12, in cyan) with that from the crystal structure (Prot_cry, in magenta). The PDB accession code for the crystal structure is 2WCD. The two mercury atoms in the crystal structure are shown as grey balls. Cα atoms of R291 are indicated as balls. (C) Comparison of Prot_12 with a protomer from the tridecamer (Prot_13, in blue) and the tetradecamer (Prot_14, in orange). (D, E) Extensive hydrogen bonds and salt bridges mediate the interaction between neighboring protomers in the dodecamer and the tridecamer. Residues involved are labeled and shown as sticks. Residues from different protomers are distinguished with or without “*”.
Fig 3.
Characterization of a ClyA intermediate state.
(A) SEC profile of ClyA in the soluble state and an intermediate state. Digitonin (1%) was incubated with ClyA overnight before gel filtration to obtain the intermediate state sample. The elution volumes for the two states were 17.7 mL and 14.4 mL (Superose 6 10/300 GL, GE Healthcare), respectively. (B) Negative staining images of ClyA in the form of soluble state and intermediate state. Fractions indicated in (A) were applied to negative staining (“-” indicates the soluble protein, “+1” or “+2” indicates the sample with digitonin). (C) Native PAGE analysis of ClyA in the soluble state (lane 1, 5), the intermediate state (lane 2, 6), and the state of mature pore complex (lane 3, 4, 7, 8). (D) Fluorescence emission spectra of the ClyA monomer, the intermediate state (IntS) and the pore complexes. Wavelengths of 316, 323, and 330 nm are indicated for clear visualization of peak shift.
Fig 4.
ClyA forms pores on cholesterol-containing liposomes.
(A) POPC-DOPS, POPC-DOPS-cholesterol or BTLE liposomes were incubated ON with 5 μM ClyA (-tag) at 4°C (pH 7.4) and examined with negative staining. ClyA forms pore structures on POPC-DOPS-cholesterol and BTLE liposomes, but not on POPC-DOPS liposomes. Representative pores are indicated with white arrows. Regions within the cyan boxes are enlarged for clear visualization. Crescent structures are indicated with yellow arrows. (B) Model for the assembly of ClyA pore complexes from the soluble monomer. The corresponding transmembrane elements in the soluble monomer and one protomer are colored purple.