Supplementary Figure 3: Purification of PI4KIIIα complexes for in vitro kinase assay and representative electron density maps for the FAM126A-N/TTC7B crystal structure.
From: The leukodystrophy protein FAM126A (hyccin) regulates PtdIns(4)P synthesis at the plasma membrane
(a) 3xFLAG-tagged PI4KIIIα (wild-type or a kinase-dead point mutant) were expressed in Expi293 cells alone or co-expressed with TTC7B or both TTC7B and FAM126A-N. The kinases (or kinase complexes) were purified by anti-FLAG affinity chromatography and analyzed by SDS-PAGE, staining with Coomassie blue. Arrowhead indicates Hsp70, which partially co-purified with all samples (and whose identity was verified by mass spectrometry). (b) Top, Experimental electron density map, contoured at 1.0σ, into which the initial model was built. The map was calculated with phases from a SAD experiment after density modification and sharpened using B-factors (−30 Å2). The initial model before refinement is shown in green. Bottom, 2Fo-Fc map of the same region, contoured at 1.0σ, with B-factor sharpening (−30 Å2). The refined model is shown in green. (c) Point mutations in FAM126A that underlie HCC are indicated in the TTC7B/FAM126A-N structure. Most of the disease-causing mutations in FAM126A result in premature termination; two known disease-causing missense mutations (L53P and C57R), indicated here, likely cause FAM126A misfolding.
