Extended Data Fig. 2: Effects of temperature and membrane composition on ETM structure.

a, ¹³C and ¹⁵N CP-MAS spectra of ERGIC-bound ETM. The spectra show high sensitivity and resolution, indicating a well ordered and rigid protein. b, ¹³C and ¹⁵N CP-MAS spectra of ETM in DMPX membranes from 303 K to 263 K. The spectral intensities and linewidths are insensitive to temperature, indicating that the protein is mostly immobilized at ambient temperature. c, ¹³C direct-polarization (DP) spectra of DMPX-bound ETM. The E8 sidechain carboxyl chemical shift changes between high and low pH, indicating that this residue is protonated at low pH. d-f, 2D ¹⁵N-¹³C (left) and ¹³C-¹³C (right) correlation spectra of ETM at high and low temperatures and in ERGIC versus DMPX membranes. Yellow rectangles highlight peaks with clear chemical shift or intensity changes. d, 2D spectra of ERGIC-bound ETM (orange) at 293 K and DMPX-bound ETM at 303 K (green). The chemical shifts are similar, indicating that the protein conformation is unaffected by the presence of POPS, POPI and cholesterol. T11 and L12 signals are not detected in the ERGIC sample at this temperature, suggesting that the N-terminus is mobile under these conditions. e, 2D spectra of ERGIC-bound ETM at 293 K (orange) and 263 K (blue). Moderate chemical shift changes are observed for C-terminal residues from T35 to R38, while the I13 signal is not visible at low temperature. f, 2D spectra of DMPX-bound ETM at 303 K (green) and 263 K (purple). The C-terminal residues exhibit temperature-dependent chemical shifts, similar to the ERGIC-bound peptide. The N-terminal residues of T9 to I13 do not exhibit signals at 263 K, indicating that the N-terminus undergoes intermediate-timescale motion at this temperature. Thus, the C-terminal conformation is temperature-dependent while the N-terminus is dynamic at high temperature.