Extended Data Fig. 1: Optimization of LRRK2 constructs and cryo-EM analysis of a LRRK2^RCKW trimer.

a, We systematically scanned domain boundaries (amino acid numbers of boundaries noted above domain names) to generate LRRK2 constructs that expressed well in baculovirus-infected insect cells and yielded stable and soluble protein. These attempts included full-length LRRK2, the kinase domain alone or with the WD40 domain, and other isolated domains. In this approach, only the GTPase domain on its own expressed well. Next, we gradually shortened LRRK2 from its amino terminus. Red asterisks indicate constructs that were soluble. b, After identifying domain boundaries yielding constructs that expressed soluble protein, additional fine tuning of boundaries was performed. A Coomassie-stained SDS–PAGE gel shows systematic N-terminal truncations at the ROC domain resulting in the identification of a construct with the highest expression levels: amino acids 1327–2527 (red asterisk, ‘LRRK2^RCKW’ here). c, A Coomassie-stained SDS–PAGE gel of purified LRRK2^RCKW after elution from an S200 gel filtration column. As predicted by its primary structure, LRRK2^RCKW runs at approximately 140 kDa. d, Electron micrograph of LRRK2^RCKW. e, 2D class averages of the LRRK2^RCKW trimer. f, 2D/3D classification scheme used to obtain the 3.5 Å structure of the LRRK2^RCKW trimer. g, h, Fourier shell correlations (from Cryosparc) (d) and Euler angle distribution (e) for the LRRK2^RCKW trimer.