Supplementary Figure 6: Paramagnetic relaxation enhancement (PRE) broadening profiles for the collection of intra- and intermolecular distance restraints.

(a-h) Paramagnetic relaxation enhancement (PRE) broadening profiles versus the amino acid sequence are shown for various MTSL-Cys variants of rdHDL as indicated. Intensity ratios were calculated by comparing peak intensities from a sample tagged with paramagnetic MTSL and the corresponding peak intensities from a sample tagged with a diamagnetic MTSL analog (control). (a) The measurements of intra-molecular PRE broadening were established using a sample with ¹⁵N-²H-MTSL-Cys67-MSPΔH5 assembled with ¹⁴N-¹H-wildtype-MSPΔH5 in a ratio of 1:4. (b)-(h) Intermolecular PRE broadenings to probe the intermolecular arrangement of two MSPΔH5 copies in an rdHDL particle were established by assembling ¹⁴N-¹H-MTSL-Cys-variant-MSPΔH5 and ¹⁵N-²H-wildtype-MSPΔH5 in a ratio of 3:1. (i) Cartoon representation of the rotamer cloud of MTSL. The center of the rotamer cloud was calculated as the population-weighted mean coordinate of the nitroxide moiety that is represented by a dummy C^α atom whose distance is fixed to the protein backbone by 4 restraints (N, C’, C^α and O of the respective cysteine-mutated residue). Intra- and Intermolecular distance restraints were calculated between amide protons and the C^α dummy atom representing the N-O nitroxide moiety. Error bars were calculated with Gaussian error propagation using the signal-to-noise ratios from the dia- and paramagnetic samples.