GoKawiilScFks1 expression and purification
ScFks1 was expressed and purified from S. cerevisiae BY4741 fks2 deletion strain18 harbouring the genomic copy of the fks1 gene engineered to have a C-terminal HRV3C protease site, ten amino acid linker, 3× Flag tag, and 10× His-tag. The fks1 gene was modified by homologous recombination by following a standard procedure42. To express ScFks1, yeast cells were grown in YPD medium and then collected by centrifugation. The resulting cells were used to prepare membrane fractions as described previously18. To purify ScFks1, the membrane fractions were passed through a French Press cell twice at 18,000 psi. Then, buffer A (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol) with 1.5% N-dodecyl β-d-maltoside (DDM) and 0.15% CHS (w/v) was added slowly while stirring then stirred overnight at 4 °C. The resulting mixture was clarified by centrifugation at 15,000g for 30 min at 4 °C, and the supernatant was then treated with anti-Flag affinity resin (Pierce). The slurry was mixed for 1 h, and the resin was then packed in a column and washed with 12.5 column volumes of buffer A with 1.5% DDM and 0.15% CHS (w/v). The resin was then washed with another 12.5 column volumes of buffer A with 0.05% LMNG and 0.005% CHS (w/v) or 0.1% CHAPS and 0.01% CHS (w/v) buffer. Bound proteins were eluted by washing with buffer containing 200 µg ml−1 Flag peptide. Eluates were concentrated to <0.5 ml, and purified with size-exclusion chromatography (Superose 6 Increase 10/300 GL column, GE Healthcare) equilibrated in buffer B (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA) with 0.005% LMNG and 0.0005% CHS (w/v) or 0.1% CHAPS and 0.01% CHS (w/v). The peak fractions were pooled and concentrated for cryo-EM sample preparation. For the structural and functional characterization of S643P-Fks1, the S643P mutation was introduced to the genomic copy of fks1 described above using the CRISPR–Cas9 genome editing using a reported protocol43. The mutant was expressed and purified following the same protocol as for wild-type ScFks1.
ScRho1 expression and purification
S. cerevisiae Rho1 (ScRho1) with N-terminal 6×His-tag and tobacco etch virus protease site was expressed in Escherichia coli Rosetta 2 (DE3) cells (Novagen) harbouring the pET28a plasmid with the rho1 gene codon optimized for expression in E. coli. The E. coli cells were grown in Luria Bertani medium with chloramphenicol (25 mg l−1) and kanamycin (50 mg l−1) at 37 °C to an optical density at 600 nm (OD 600 ) of around 1.0. Protein expression was induced by adding 0.5 mM isopropyl β-d-1-thiogalactopyranoside, and the cultures were continued for 20 h at 15 °C. Cells were then collected by centrifugation and resuspended in buffer C (50 mM Tris, pH 7.5, 200 mM NaCl, 10% glycerol) containing 2 mM β-mercaptoethanol (BME) and lysed by passing them through a French Press cell operating at 18,000 psi. The lysate was then clarified by centrifugation at 25,000g for 25 min at 4 °C. The supernatant was then applied to a column packed with cobalt-iminodiacetate resin (Prometheus), the column was washed with 12.5 column volumes of buffer C with 5 mM imidazole, and Rho1 was eluted with 5 column volumes of buffer C with 250 mM imidazole. After adding 2 mM BME, the eluate was concentrated and buffer-exchanged using PD10 columns (GE Healthcare) equilibrated with 50 mM Tris, 200 mM NaCl, 2 mM BME and 30% glycerol. Protein concentrations were determined using the Bradford method, with bovine serum albumin as a concentration standard. For cryo-EM sample preparation, Rho1 was further purified by size-exclusion chromatography (Superdex 200 Increase, Cytiva) equilibrated in 20 mM Tris, 150 mM NaCl, 2 mM BME. The peak fractions were pooled and concentrated for cryo-EM sample preparation.
ScFks1 activity assays
In vitro radioactive GS assays were performed as reported previously18. To test the effects of detergents on GS activity, ScFks1 was mixed with four times the final reaction concentration of detergents. The mixture was then mixed with an equal volume of 125 µM ScRho1 and incubated on ice for 1 h. Reactions were initiated by mixing with equal volumes of the ScFks1–Rho1 solution and a UDP-Glc (4 mM) solution as described previously18.
Cryo-EM sample preparation
All cryo-EM samples in this study were prepared on freshly glow-discharged UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil), using a Leica EM GP2 plunge freezer with the chamber set at 30 °C and 85% humidity. For the ScFks1 sample in LMNG/CHS, peak fractions from the size-exclusion chromatography in LMNG/CHS were concentrated to approximately 10 mg ml−1 and doped with 0.1% CHAPS/0.02% CHS. Then, 5 mg ml−1 CFN dissolved in water with 10% dimethylsulfoxide was mixed with ScFks1 at 1:20 (v/v) and incubated for 2 min. A 3-μl sample of ScFks1 was applied to the grid, incubated in the chamber for around 60 s then blotted for 1.5–2 s followed by plunge-freezing in liquid ethane cooled by liquid nitrogen. For the ScFks1 sample in CHAPS/CHS, peak fractions from the size-exclusion chromatography in CHAPS/CHS were concentrated to around 12–15 mg ml−1. Purified Rho1 was mixed with ScFks1 at a 1.5:1 molar ratio, and GTPγS was added at a 1.5-fold molar concentration of Rho1. 1.5 mM EDTA, 50 mM KF and 7.5% glycerol were further included in the reaction mixture. The sample was incubated with 12.5 mM UDP-Glc and 0.5 mg ml−1 CFN on ice for 30 min. During grid freezing, 3 μl of ScFks1 was applied to the grid, incubated in the chamber for approximately 60 s and blotted for 1.5–2 s followed by plunge-freezing in liquid ethane cooled by liquid nitrogen. The S643P sample was prepared similarly, with a longer reaction time (2 min).
Cryo-EM data collection
The LMNG/CHS doped with CHAPS/CHS sample was collected with a Titan Krios microscope (Thermo Fisher) operating at 300 kV and equipped with a K3 detector (Gatan) and a GIF energy filter with slit width of 20 eV in super-resolution mode using SerialEM (v.4.2)44. Videos were collected at a nominal magnification of ×105,000 with a super-resolution pixel size of 0.4135 Å per pixel at the specimen level. Each video contained 50 frames over a 2.9-s exposure time, using a dose rate of around 17.2 e− pixel−1 s−1, resulting in the total accumulated dose of approximately 50 e− Å−2. The nominal defocus range was set from −0.8 to −2.2 μm.
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