Ni nta agarose beads

Protein purification and microtubule cosedimentation assays were performed as previously described (Talapatra et al., 2015 (link)). His-tagged proteins were purified using Ni-NTA–agarose beads (GE Healthcare) according to the manufacturer’s guidelines. Proteins were then purified using gel filtration chromatography preequilibrated in gel filtration buffer (for full-length Kif18b: 25 mM Hepes, pH 7.5, 150 mM NaCl, 300 mM KCl, 5 mM β-mercaptoethanol, 1 mM MgCl₂, 1 mM Na-EGTA, and 1 mM ATP; for Kif18b microtubule-binding domain: 50 mM Hepes, pH 7.5, 150 mM NaCl, and 5 mM β-mercaptoethanol). Analytical gel filtration chromatography was performed using either a Superdex 75 or a Superdex 200 10/300 GL column (GE Healthcare). For the Kif18b microtubule-binding domain, cleavage of the 6xHis tag was performed using the his-3C protease overnight at 4°C. For detection of Kif18b by Western blotting, an antibody against Kif18b_500–593 was raised in rabbit (Covance) and affinity purified.

All ATPase₃ domain proteins and substrate Esx proteins were purified by a similar method. Briefly, cells were thawed and resuspended in buffer A (20 mmol/L Hepes pH 7.0, 150 mmol/L NaCl, 5% (w/v) glycerol, 1 mmol/L MgCl₂, 5 mmol/L ATP). The resuspended cells were then lysed by passing through a French Press at 800 bar after adding 1 mmol/L PMSF. Cell debris was then removed by centrifugation at 18,000 rpm for 30 min at 4 °C. The supernatant was applied to Ni-NTA agarose beads (GE Healthcare) for 2 h at 4 °C. The beads were rinsed with buffer A containing 30 mmol/L imidazole. For MtEccCb1-ATPase₃, MtEccC2-ATPase₃ and Esx proteins, the N-terminal tag was cleaved by 3C protease and then eluted. For MtEccC3-ATPase₃ and MtEccC5-ATPase₃, the recombinant protein was eluted from the beads with buffer A containing 300 mmol/L imidazole. Then the sample was concentrated and purified using a 5mL Hitrap Q HP (GE life science) column followed by size exclusion chromatography (SEC) using a Superdex 75 HR 10/30 (GE life science) column. The peak fractions were pooled and concentrated to approximately 10 mg/mL using a 10 kDa cut-off spin concentrator (Millipore). The separately purified MtEccCb1-ATPase₃ and MtEsxB were mixed in a 1:1 molar ratio, incubated and purified again by gel filtration. The fractions containing complex were pooled and concentrated for crystallization.

The DNA fragment containing the ETAE_2186 gene, amplified by PCR from the vector pET28a-his-ETAE_2186 (forward primer: 5’-GCGCGGATCCGTAGAGCCG GCCCTATAGCGACG-3’, reverse primer: 5’-GCGCCTCGAGTTAGCGGGTCAGA AAGTCAG-3’) was inserted into the pET28b-His Sumo vector via the restriction sites BamH1 and Xho1. The ligated plasmid was then transformed into E. coli BL21(DE3) cells. The resulting strain was grown to mid-log phase and then induced with 0.1 mM IPTG at 16°C for 16 h. Cells were collected by centrifugation and the pellet was resuspended in lysis buffer (50 mM Tris-HCl, pH8.0, 400 mM NaCl, 10% glycerol, 2 mM 2-mercaptoethanol and protease inhibitor). Resuspended cells were lysed by sonication and cleared by high speed centrifugation at 40,000 g for 1 h. The protein was purified using Ni-NTA agarose beads (GE), followed by enzyme digestion with Ulp1 to remove the His-Sumo tag. The digested product was further purified by gel-filtration chromatography (Hiload Superdex 75) and passed through Ni-NTA beads again to remove residual His-Sumo contamination. The buffer for gel-filtration chromatography buffer contains 25 mM Tris-HCl, pH8.0, 150 mM NaCl. The purified protein was concentrated to 22 mg/ml and store at -80°C.

Cell pellets were thawed and resuspended in buffer A containing 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.4, 100 mM NaCl, and then lysed by passing through a French Press at 1200 bar three times. Cell debris and non-lysed cells were removed by centrifugation at 14,000 rpm for 10 min at 4°C. The supernatant was collected and ultra-centrifuged at 36,900 rpm and 4°C for 2 hr. The membrane fraction was solubilized by addition of 1% (w/v) lauryl maltose neopentyl glycol (LMNG) in buffer A and incubated for 2 hr at 4°C with slow stirring. The suspension was ultra-centrifuged, and the supernatant was applied to Ni-NTA agarose beads (GE Healthcare) at 4°C. The beads were further washed in buffer A with 50 mM imidazole and 0.004% (w/v) LMNG. The buffer was exchanged to buffer B (20 mM MOPS, pH 7.4, 100 mM NaCl, and 0.1% [w/v] digitonin) and then washed in resin in batch mode. The protein was eluted from the beads with buffer B containing 500 mM imidazole. Protein was then concentrated and loaded onto a Superdex 6 increase (10/300GL, GE Healthcare) column equilibrated in buffer B. Peak fractions were pooled and concentrated to ~8 mg/mL for electron microscopy studies. The protein sample was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the bands were then identified through mass spectrometry.