Fig. 4. LRRK1^RCKW is structurally similar to LRRK2^RCKW but does not bind to microtubules.

a, Cryo-EM map (5.8 Å) of a LRRK1^RCKW monomer, with domains colored according to the scheme shown above. b, The molecular model for LRRK2^RCKW (PDB: 6VNO) is shown as a calculated 6-Å density (molmap command in ChimeraX), in the same orientations used for LRRK1^RCKW in a. c,d, Close-ups of the LRRK1^RCKW map shown in a, with the AlphaFold model of LRRK1 docked into it. These close-ups highlight the difference in length in the αC helix between LRRK1 and LRRK2 (c), and a difference at the C-terminal helix emerging from the WD40 domain between our experimental map of LRRK1^RCKW and the AlphaFold model of LRRK1 (d). e, Representative images of Alexa Fluor 488-labeled microtubules (cyan) incubated with 50 nM of either LRRK2^RCKW (magenta, top) or LRRK1^RCKW (magenta, bottom). f, Quantification of data in e, as outlined in Figure 3c. Data are mean ± s.d., n = 8 fields of view. g, Microtubule pelleting assay for 200 nM LRRK2^RCKW or LRRK1^RCKW with increasing tubulin concentrations. Data are mean ± s.d., n = 4. The solid lines represent a hyperbolic curve fit to the data. h, Example kymographs for single-molecule kinesin motility assays alone or in the presence of 100 nM of either LRRK2^RCKW or LRRK1^RCKW. i, Quantification of data in h as percentage of motile kinesin events per microtubule. Data are mean ± s.d. ****P < 0.0001, Kruskal–Wallis test with Dunn’s post hoc for multiple comparisons. j, Cumulative distribution of run lengths for kinesin in the absence or presence of 100 nM LRRK2^RCKW or LRRK1^RCKW. The run lengths were not significantly different between 0 nM and 100 nM LRRK1^RCKW conditions (Kruskal–Wallis test with Dunn’s post hoc for multiple comparisons). Data are from two biological replicates, with three or two technical replicates of each experiment.

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