Fig. 2: Measurement of interhelical distances and water accessibility of membrane-bound ETM.

a, Schematic of mixed [¹⁹F]ETM and [¹³C]ETM in a five-helix bundle. b, 2D ¹³Cα-F REDOR spectra of ERGIC-membrane-bound ETM. The control spectrum (S₀, black) shows the signals of all residues, whereas the difference spectrum (ΔS, red) shows the signals of residues that are close to fluorinated Phe on a neighboring helix. c, 2D ¹³C-¹⁹F correlation spectrum allows assignment of the –118 ppm peak to Phe20 due to a cross-peak with Ala22, whereas the −113 ppm peak is assigned to Phe23 and Phe26 on the basis of correlations with Phe23 and Phe26 and with Val24 and Val25. A 1D ¹H-¹⁹F cross-polarization (CP) spectrum is shown on the left. d, 2D NHHC correlation spectrum of mixed [¹³C]ETM and [¹⁵N]ETM, measured using 0.5 ms (red) and 1 ms (black) ¹H mixing. All peaks arise from interhelical contacts. Selected assignments are given. e, Residue-specific water accessibilities of ERGIC-bound ETM obtained from the intensity ratios of water-edited spectra measured with 9 ms and 100 ms ¹H mixing. Higher values (blue) indicate greater water accessibility. f, Water-edited intensities of ETM (black) and influenza BM2 (blue) obtained as the peak intensity ratios of the 9-ms and 100-ms spectra. Closed and open symbols indicate resolved and overlapped peaks, respectively. Error bars indicate the random uncertainty, which is propagated from the signal-to-noise ratios of the two spectra. ETM shows lower water-edited intensities than does BM2, indicating that the ETM pore is drier than the closed BM2 pore. g, Water- and lipid-edited ¹³C spectra of membrane-bound ETM. The Phe signals are high in the lipid-edited spectra but very low in the water-edited spectra, indicating that the three Phe residues are poorly hydrated and point to the lipids or the helix–helix interface.