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. 2020 Nov 30;9:e62772. doi: 10.7554/eLife.62772

Figure 2. OraiH206A–Fab complex.

(A and B) Overall structure of the complex is shown from the perspective of the membrane (A) and from the extracellular side (B). The cryo-EM map (left) and the cartoon representation of the structure (right) are shown. Each Fab is colored individually; the α-helices of Orai are colored as indicated. Horizontal bars in (A) denote approximate boundaries of the plasma membrane.

Figure 2—source data 1. Cryo-EM data collection, refinement, and validation statistics.

Figure 2.

Figure 2—figure supplement 1. Flowchart for cryo-EM data processing of the OraiH206A–Fab complex.

Figure 2—figure supplement 1.

Details can be found in the Materials and methods.
Figure 2—figure supplement 2. 3D reconstructions of OraiH206A–Fab complexes containing between one and three Fab molecules.

Figure 2—figure supplement 2.

3D reconstructions of complexes containing between one and three Fabs are shown in the upper panels (Chimera representations). Slices of the Fab and transmembrane portions of the 3D reconstructions are shown in the middle and lower panels, respectively (the views are from the extracellular side of the reconstruction in these panels). The halo in the lower panels is due to the amphipols surrounding the transmembrane portion of Orai. Complexes containing two Fab molecules have Fabs arranged in two alternative configurations (1,4) or (1,3).
Figure 2—figure supplement 3. Cryo-EM structure determination and density.

Figure 2—figure supplement 3.

(A) Densities (blue mesh) of indicated regions of Orai in the Orai–Fab complex are shown in the context of the atomic model (cartoon and stick representation). A superposition of the M1–M2 turrets from two neighboring subunits is also shown (lower left panel, main chain RMSD = 0.39 Å). (B) Local resolution of the map estimated using the Local Resolution Estimation function of Cryosparc2 (colored as indicated). An overall view (left panel) and a cross-section (right panel) are shown. (C) Angular orientation distribution of the particles used in the final reconstruction. (D) Gold-standard FSC curve of the final 3D reconstruction. The resolution is 3.3 Å at the FSC cutoff of 0.143. (E) FSC curve showing the correlation of the atomic model and cryo-EM map.
Figure 2—figure supplement 4. The Orai–Fab interface.

Figure 2—figure supplement 4.

(A) Cartoon representations of the antibody (violet) and two neighboring subunits of Orai (labeled A and B and colored according to transmembrane α-helices as in Figure 2) are shown with the corresponding cryo-EM density (semitransparent surfaces). Amino acids in the Fab–Orai binding interface are drawn as sticks (with carbon atoms colored violet for the Fab and gray for Orai, and with oxygen and nitrogen atoms colored red and blue, respectively). Dashed lines indicate hydrogen bonds. Two perspectives are shown (left and right panels). (B) Analogous depictions as in (A) with the density removed for clarity.