Extended Data Fig. 1. Properties of purified ELOVL7.

a, SDS-PAGE gel of purified ELOVL7. Data shown is from a single purification but similar results were observed for multiple purifications / modifications. b, SEC profile and MALS analysis showing that OGNG-solubilised protein exists as a dimer in solution. Experiment carried out for tag-cleaved ELOVL7 purifications with and without IAM modification. Similar results were seen for each sample. c, Representation of head-to-tail dimer present in the crystal. d, ELOVL7 head-to-tail dimer packing within the crystal lattice. e, f, Intact mass analysis of ELOVL7 protein at various stages during purification. Deconvoluted mass spectra are shown for ELOVL7 protein purified from (e) Sf9 (n=3 or 4) and (f) Expi293F cells (n=2). For protein purified after expression in insect cells, the samples are shown after immobilized metal affinity chromatography (IMAC), after cleavage of the tag and size exclusion chromatography (SEC) and after treatment with iodoacetamide (IAM). The expected mass of the untagged ‘native’ enzyme (E) based on the sequence is 34222.38 Da. The observed mass peaks (Sf9, 34132.78 Da; Expi 34133.36 Da) correspond to the loss of the N-terminal methionine (-131.20 Da) and acetylation of the resulting new N-terminus (+42.04 Da). All samples were run in their reduced state. The modified material (E*) appears as an adduct with an average mass shift of +1073.6 Da. The addition of 113.55 Da upon treatment with IAM suggests modification of two cysteine residues. g-i, Deconvoluted intact mass spectra for the untagged g, WT (n=3), h, H150A (n=2) and i, H181A mutants (n=2). The expected mass decrease of a His-to-Ala mutation is 66.06 Da. Evidence of in vivo modification (E*) is observed for the H181A mutant but not for H150A. For intact mass experiments, theoretical and experimental masses along with mass errors are given in Supplementary Table 1.