Figure 1. Overall structure of the IR–insulin complex.
(A) 3D reconstruction of the IR dimer with four insulins bound (left) and the corresponding ribbon representation of this complex (right). (B) 3D reconstruction of IR dimer with four insulins bound after forced 3D refinement (left) and the corresponding ribbon representation (right).
Figure 1—figure supplement 1. Purification of the full-length human insulin receptor (IR).
(A) A representative size-exclusion chromatogram of IR. The peak fractions were visualized on SDS–PAGE by Coomassie staining, in the absence or presence of DTT. Most of IR was processed into α-chain and β-chain. (B) Domain organization of IR. Regions that are not built in the model are drawn with dashed lines. C647 and C860 make an intra-protomer α/β disulfide bond. C524–C524 and C683–C683 make inter-protomer disulfide bonds. (C) Binding of insulin labeled with Alexa Fluor 488 to purified IR WT or IR site two mutant (Mean ± SD). Each experiment was repeated three times. Significance calculated using two-tailed student t-test; between WT and mutants; ****p<0.0001. (D) Representative western blot images of the amount of IR in C.
Figure 1—figure supplement 2. Flowchart of cryo-EM data processing.
Figure 1—figure supplement 3. Cryo-EM analysis of the IR–insulin complex.
(A) Representative electron micrograph and 2D class averages of the IR–insulin complex. (B) Unsharpened cryo-EM map colored by local resolution. (C) The gold-standard Fourier shell correlation curve for the cryo-EM map shown in Figure 1B. (D) FSC curves for the refined model versus the summed map (black curve), the refined model versus half map 1 (red curve), and the refined model versus half map two that was not used for refinement (green curve). (E) Euler angle distribution of particles used in the final 3D reconstructions.
Figure 1—figure supplement 4. Cryo-EM density of the IR–insulin complex.
(A) Representative densities of the cryo-EM map of each domain of IR, insulins 1 and 2 are shown. (B) Cryo-EM density of the transmembrane (TM) domain. Inset, close-up view of the TM domain. The TM sequence was not assigned due to the lack of clear side-chain densities.
Figure 1—figure supplement 5. Detecting variations in insulin occupancies of IR by focused classification.
(A) Symmetry expanded focused classification on insulin two with signal subtraction of IR. (B) The classification resulted three types of insulin occupancies of IR with different populations.