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Receptor was prepared by using the Protein Preparation and Grid Preparation tools in the Schrödinger Maestro interface.
C.H. performed single particle cryo-EM grid preparation, data collection and map generation with input from R.K.H. and Z.Y.
J.W. carried out protein expression and purification, grid preparation and optimization, electron microscopy data collection, image processing and reconstruction, atomic model building and refinement, and thermal shift assays.
The time between elution of purified LRRK2 or LRRK1 and cryo-EM grid preparation was kept to a minimum (60 minutes).
7.5 µL of sample solution (0.1 w/v%, diluted from 1 w/v% immediately before grid preparation) were placed on 300-mesh copper grids with lacey carbon support, blotted, and plunge frozen into liquid ethane.
The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step.
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To avoid errors that could be induced by NC ripening or other changes during storage, PL measurements and TEM grid preparations were carried out immediately after the synthesis.
For cryo-grid preparation, the Quantifoil 1.2/1.3 holy carbon grids were baked at 50°C in an oven for 2 weeks.
For cryo-grid preparation, aliquots of 3.5 µL of purified mTOR1 complex at a concentration of ~1.5 mg/mL were applied to glow-discharged holey carbon grids (Quantifoil Cu, R1.2/1.3, 400 mesh).
As in the ELSA life history interview, LHH participants filled out the life grid in preparation for the follow-up interview.
The TEM images provided in Supporting Information Figure S1 depict ENP aggregates (distinguished by their fractal geometry and sinter necks) as well as agglomerates (particle units composed of several aggregates that might be formed due to van der Waals forces encountered during particle suspension in ethanol for TEM grid sample preparation).
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