These amino acids are buried in by α0 in M2-M5 but exposed in M6 where α0 is melted. ( D) Surface representation of individual subunits highlighting amino acids in the LD likely to engage the hydrophobic substrate. Positive charges are colored in blue, negative charges in red, and neutral side chains in white. ( B and C) The electrostatic potential surface of the Δ30-Msp1 E214Q structure shows that Msp1 displays a positively charged surface. On the right, the structure is displayed at σ = 3.8, showing the density of the central hub (cyan) emerge where the α0s of M1-M5 converge in a staggered alignment. On the left, the structure is displayed at σ = 5.5, showing the fishhook motifs of different subunits radially organized with their N-termini pointing to the center of the spiral. M1-M5 shows significant density for the entire fishhook motif (α0 and the L1), whereas M6 shows density for L1 but not for α0. ( A) Cryo-EM map of Δ30-Msp1 E214Q showing the arrangement of the fishhook motifs in the spiral. Model building for the larger oligomer showed potential clash between subunits, which is further described in Figure 1-figure supplement 6. ( E) Angle distribution of particles of the hexamer and the larger oligomer species generated by cryoSPARC. ( D) Fourier Shell Correlation (FSC) plots of the 3D reconstructions of the Δ30-Msp1 E214Q hexamer: masked (red), unmasked (blue) and map to model (green). The side facing the cytosol is better resolved than the side facing the membrane. The core of the protein complex including the central pore and the nucleotide binding pockets are among the best resolved regions. ( C) Local resolution maps of the hexamer and the larger oligomer structures generated with cryoSPARC. The final refinement yielded reconstructions of 3.5 and 3.7 Å for the hexamer and the larger oligomer species respectively. In order not to lose good particles along the many rounds of refinement, these two structures were used as input models to perform another round of heterogeneous refinement against the particle stack from the first round of heterogeneous refinement (indicated with *). Rounds of homogeneous and heterogeneous refinement was performed to identify the hexamer and the larger oligomer species at 3.89 Å and 3.80 Å each. ( B) Data processing scheme showing that the RELION software was used for 2D classification, and the cryoSPARC software was used to generate the ab initio 3D models. ( A) Representative micrograph showing the quality of the data used to generate the 3D reconstruction. ![]() ( D) Fourier Shell Correlation (FSC) plots of the 3D reconstructions of Δ30-Msp1: Δ30-Msp1 (closed) masked (dark blue), Δ30-Msp1 (closed) unmasked (orange) and Δ30-Msp1 (closed) map to model (light blue) Δ30-Msp1 (open) masked (red), Δ30-Msp1 (open) unmasked (green) and Δ30-Msp1 (open) map to model (black). ![]() ( C) Local resolution maps of the open and the closed conformations show that the core of the protein complex including the central pore and the nucleotide binding pockets are the best resolved regions. Angle distributions are shown under the two structures, respectively. The open and the closed conformations were individually refined in cryoSPARC to generate the final structures of 3.7 Å and 3.1 Å, respectively. To identify different conformations, we performed another round of 3D classification without alignment. The first rounds of 3D classification generated a consensus model of 3.7 Å resolution where the mobile subunit (M1) has poor density. ( B) Data processing scheme showing the 2D and 3D classification done using the RELION software. ( A) Representative micrograph showing the quality of data used for the final reconstruction of the Δ30-Msp1 (open) and Δ30-Msp1 (closed) structures.
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