Purification of human ATP13A2 for cryo-EM analysis

Thaw Sf9 cell pellets at room temperature (typical size around 10 g from 0.7L of culture)

All subsequent steps performed at 4°C

Resuspend each pellet in 30 mL Lysis Buffer (use 3x volume of cell pellet, 40 mL total volume)

Homogenize pellet with Dounce homogenizer, 100 plunges tight on ice

Pour lysate into pre-chilled 50 mL centrifuge tubes

Spin in centrifuge at 4000x g,4°Cto remove unbroken cells

Transfer supernatant to pre-chilled ultracentrifuge rotor tubes

Spin lysate in ultracentrifuge at 100000x g,4°C to pellet membranes (Beckman Type 45 Ti rotor)

Resuspend membrane pellet in Lysis Buffer and final volume 1% DDM/0.2% CHS (1X pellet, 7X Lysis Buffer, 2X 5% DDM/1% CHS)

DDM: n-dodecyl-β-D-maltopyranoside (Anatrace)

CHS: cholesteryl hemisuccinate (Anatrace)

Solubilize by rotating end-over-end for 2h 30m 0s at 4°C

Clarify lysate in ultracentrifuge at 100000x g,4°C

Equilibrate 1 mL Sepharose beads conjugated with anti-GFP nanobody with Wash Buffer

Add beads to gravity column and wash with 10 mL wash buffer

After ultracentrifugation is complete, transfer supernatant into 50 mL falcon tube

Add 1 mL equilibrated anti-GFP nanobody beads to tube

Incubate by rotating end-over-end for 2h 30m 0s at 4°C

Transfer to gravity column and let flow-through drain

Wash beads with 30 column volumes of Wash Buffer

Add 5 mL Wash Buffer to gravity column and 10 µg/mL HRV 3C protease

Incubate by rotating end-over-end overnight at 4°C

Equilibrate Superose 6 Increase 10/300 GL column with Wash Buffer

Concentrate the protein to 0.5 mL using an Amicon Ultrafilter (cut-off 100kDa)

After concentration, spin protein at 17000x g,4°C

Injected sample into FPLC

Collect peak fractions and concentrate to approximately 5-7 mg/mL for cryo-EM grid preparation