Figure 3. Structure of Ca²⁺-free BEST1₃₄₅.

(a) Overlay comparison of the Ca²⁺-free conformation of BEST1₃₄₅ (yellow) with the Ca²⁺-bound closed conformation of BEST1₃₄₅ (green). One (Ca²⁺-free) or two (Ca²⁺-bound) channel subunits in ribbon are shown from the side with the approximate boundaries of the bilayer indicated. The side chains of labeled residues are shown. The boxed area highlights the location of the Ca²⁺-clasp. (b) Density for the Ca²⁺-clasp is missing in the absence of Ca²⁺. The structure of the Ca²⁺-clasp region from the Ca²⁺-bound closed conformation (green) is shown in comparison with the cryo-EM density in this region in the Ca²⁺-free map, showing that the density for the Ca²⁺ ion and surrounding protein residues are missing in the absence of Ca²⁺. Ca²⁺ is depicted as a green sphere and two aspartate residues that coordinate Ca²⁺ as part of the Ca²⁺ clasp are shown as sticks. (c) Refined cryo-EM maps of two conformations (blue, gray) of Ca^2+-free BEST1₃₄₅ that were identified using 3D classification. The maps are aligned according to their membrane-spanning regions, with the relative rotation between the cytosolic regions indicated.

Figure 3—figure supplement 1. Cryo-EM workflow for the BEST1₃₄₅ Ca²⁺-free dataset.

A detailed description can be found in Methods.

Figure 3—figure supplement 2. Cryo-EM structure determination of: Ca²⁺-free BEST1_345.

(a–d) Structure determination of the consensus Ca²⁺-free BEST1₃₄₅ conformation. (a) Angular orientation distribution of particles used in the final reconstruction. The particle distribution is indicated by color shading, with blue to red representing low and high numbers of particles. (b) Gold-standard Fourier shell correlation (FSC) curve of the final 3D reconstruction. The resolution is 3.0 Å at the FSC cutoff of 0.143 (dotted line). A thin vertical line indicates that only special frequencies to 1/ (5 Å) were used to determine particle alignment parameters during refinement. (c) Local resolution of the map estimated using Relion and colored as indicated. (d) Model validation. Comparison of the FSC curves between the model and half map 1 (work), model and half map 2 (free) and model and full map are plotted. (e) Table of data processing and model statistics.

Figure 3—figure supplement 3. Map of Ca²⁺-free BEST1₄₀₅.

Density for the Ca²⁺-clasp and the inactivation peptide is missing in the absence of Ca²⁺ for BEST1_405. The X-ray structure of Ca²⁺-bound BEST1₄₀₅ (Kane Dickson et al., 2014) is shown as purple ribbons for comparison with the cryo-EM density of Ca²⁺-free BEST1₄₀₅. Ca²⁺ is depicted as a green sphere and a Ca²⁺ clasp and an inactivation peptide are annotated.