Extended Data Fig. 2. Isolation and biochemical characterization of the inhibitor-bound ribosome-Sec61 (RNC) complexes.
(a) Photocrosslinking experiment showing competitive binding of KZR-8445 with photo-cotransin (CT7) in the final purified sample. Highlighted sample (gray) was used for cryo-EM analysis. Gels are representative examples of n = 3 independent experiments. (b) Flowchart of purification of KZR-8445-bound RNC–Sec61 complex. Isolated sheep pancreatic rough ER microsomes were supplemented with 1 µM inhibitor and incubated on ice for 30 min followed by solubilization with 1% LMNG for 1 h. Solubilized material was clarified by centrifugation followed by gel filtration. (c) Gel filtration profile of clarified detergent-solubilized sample fractions with measured absorbance at A260. Western blot analysis demonstrates presence of Sec61 and ribosomes in the peak fractions confirming integrity of the purified complex. Gels are representative examples of n = 3 independent experiments.