Figure EV1. Purification and cryo‐EM of the Sec complex.

• A
  SDS–PAGE analysis of the purification of the heptameric Sec complex containing FLAG‐tagged Sec62. After solubilization, the tagged complex was applied to anti‐FLAG‐M2 resin and the eluate was subjected to ion‐exchange chromatography using Q sepharose (S: solubilized microsomes; FT: flow‐through from the anti‐FLAG resin; 3C: eluate after 3C cleavage; E: eluate from Q sepharose; C: concentrated heptameric Sec complex sample).
• B
  Cryo‐EM map of the (heptameric) Sec complex (apo state) shown after focused refinement and post‐processing. The map is shown at contour level 0.02 except for the Sec71/72 part, which is at 0.018. Individual subunits of the Sec complex are color‐coded as in Fig 1C.
• C
  Molecular model of the Sec complex in apo state.
• D
  SDS–PAGE analysis of pull‐down assays of the purified Sec complex with the signal sequence‐containing mEGFP‐tagged ppαF or its translocation defect mutant ppαFm3. Pull downs were performed at varying molar ratios (B: bead‐bound fraction; FT, unbound flow‐through). Semi‐quantitative assessment of the Sec63 bands on the gel using ImageJ shows that the mutant signal sequence binding to the Sec complex appears weaker than the wildtype, as expected by weaker activity of the mutant in translocation (Allison & Young, 1989).
• E
  SDS–PAGE analysis of the purification of the signal sequence‐bound Sec complex. Lanes are labeled as in (A) except: FT1: flow‐through after binding of the Sec complex on beads; FT2: flow‐through after binding of ppαF‐mEGFP to the Sec complex immobilized on beads; W1‐3: washes after ppαF‐mEGFP binding.
• F
  A representative cryo‐EM micrograph as obtained from TITAN KRIOS equipped with a K2 direct electron detector. The scale bar represents 20 nm.
• G
  Selected 2D class averages. The box size was chosen to be 200 pixels resulting in a box width of 212 Å.