1 55 mm filter papers

Quantifoil 1.2-1.3 400-mesh grids were used for sample freezing and glow discharged for ~30s at -20 mA immediately prior to plunge-freezing. Grids were frozen on a Vitrobot Mark IV, with the humidity set to 100%, and using Whatman 1 55 mm filter papers. The final conditions used for γTuRC WT filament freezing was 40 mM HEPES pH 7.5, 2 mM MgCl2, 1 mM EGTA, 1 mM GDP, 100 mM KCl, 1 mM DTT and 2.5% v/v glycerol. The final conditions used for γTuRC SS filament freezing was 40 mM HEPES pH 7.5, 2 mM MgCl2, 1 mM EGTA, 0.5 mM GTP, 100 mM KCl, 1mM oxidized glutathione and 2.5% v/v glycerol.

Prior to grid preparation γTuSC aliquots were centrifuged in a benchtop centrifuge (Eppendorf 5415D) at 16'000 g for 15 minutes and transferred to a new tube. The sample concentration was assessed on a nanodrop, and diluted to a final concentration of ~1 μM (O.D. at 280nm wavelength of 0.28-0.35) such that the final buffer conditions were 40 mM HEPES pH 7.5, 2 mM MgCl2, 1 mM EGTA, 1 mM GDP, 100 mM KCl and 2.5% v/v glycerol. Data used for initial model generation and refinement had final buffer conditions of 40 mM HEPES pH 7.5, 1 mM MgCl2, 1 mM EGTA, and 100 mM KCl. C-flat 1.2-1.3 4C grids were used for sample freezing and glow discharged for ~30s at -20 mA immediately prior to plunge-freezing. Grids were frozen on a Vitrobot Mark II or Mark IV, with the humidity set to 100%, and using Whatman 1 55 mm filter papers.