Extended Data Fig. 3: PhPINK1(D357A) oligomerization enables EM studies.
a, Elution profile of the PhPINK1(D357A) mutant during purification by SEC. Data shown is from a representative experiment of three runs. b, Thermal denaturation studies of purified PhPINK1 (amino acids 115–575) variants. The crystallized D334A mutant has a melting curve profile and melting temperature similar to WT PhPINK1. PhPINK1(D357A) shows an unusual profile with a high secondary melting temperature. Technical duplicates were measured in three independent experiments. Average melting temperatures are indicated. c, Negative stain EM analysis of PhPINK1(D357A) revealed a highly ordered oligomer suitable for cryo-EM studies. Minimal processing was performed in RELION (v.3.1)51, and a subset of the resulting 2D classes is depicted as insets. Scale bar, 50 nm. Negative staining for this sample was performed once, before advancing to cryo-EM analysis. d, Flowchart for cryo-EM analysis as described in Methods. e, 2D classification of particles reveal a 2D-crystalline arrangement of PhPINK1(D357A) oligomers in some areas of the grid. f, Final cryo-EM density maps (coloured by local resolution) for the PhPINK1(D357A) dodecamer (left) at 2.48 Å and the extracted dimer (right) at 2.35 Å. g, Examples of map quality for the final 2.35 Å density covering the PhPINK1(D357A) dimer.
