For chemical cross-linking, the LRRK2 concentration was adjusted to 3 µM, and each Nb was added at a 2:1 molar ratio and incubated for 1 h at 4 °C. The cross-linking reaction was performed using the N-hydroxysuccinimide (NHS)-ester–based and collision-induced dissociation (CID)-cleavable reagent disuccinimidyl sulfoxide (Thermo Fisher Scientific) (60 (link)) at a molar excess of 60:1 (referred to the Nbs) and carried out for 30 min at room temperature. Proteins were precipitated by chloroform/methanol and subjected to tryptic proteolysis (61 ). The tryptic peptide solutions were cleaned up by StageTips and subjected to size exclusion chromatography (SEC) separation to enrich for cross-linked peptides (28 (link)). Vacuum-dried fractions were analyzed on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) using the MS2_MS3 fragmentation method (version 3.0), and the Thermo Raw files were analyzed with the MS2_MS3 workflow provided by in Proteome Discoverer 2.5 (build 2.5.0.400) using XlinkX (version 2.5) (62 (link)), as described in SI Appendix, SI Materials and Methods.