Expression and purification for cryo-EM samples of FIP200NTD:ATG13(363-517)-ULK1(MIT) complexes

Transfect HEK GNTi cells at concentration of 2 × 10^6 cells/ml

Dilute PEI with Warm Hybridoma-SFM(1X)

In a separate tube, dilute DNA with Hybridoma-SFM(1X)

Add PEI to DNA dilution. Incubate mixture for 0h 30m 0s at 37°C

Add mixture to cells. Let cells grow for 48h 0m 0s

Harvest Cells 500rpm,0h 0m 0s, 4°C, 0h 10m 0s

Wash pellet with cold PBS. Store pellet at-80°C until purification.

Cell pellets were lysed at room temperature for 20 min with lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 10% Glycerol) with 5 mM EDTA, 1% Triton X-100 and protease inhibitor cocktail (Thermo Scientific) for 0h 15m 0s

Clarify lysate for 17000rpm,0h 0m 0s for 0h 35m 0s at 4°C.

Wash strep-tactin resin (IBA Lifesciences, Germany) into lysis buffer (without Triton). Load clarified lysate onto resin

Rock supernatant with equilibrated Strep-Tactin Sepharose resin for 1h 0m 0s at 4°C

Wash with 5CV lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA, 10% Glycerol)

Elute with lysis buffer plus 4 mM desthiobiotin for STREP resin

His6-TEV cleavage at 4°C 1h 0m 0s,

Concentrate elution and inject onto pre-equilibrated Superose 6 Increase 10/300 GL column (Cytiva)

(25 mM HEPES pH 7.5, 150 mM NaCl, 1 mM MgCl2, 1 mM TCEP)

Pool peak fractions, concentrate, snap freeze, and store at -80°C