Amicon ultra 50k

Myosin II was extracted and purified from rabbit skeletal muscle44 (link). Heavy meromyosin (HMM) was obtained by enzymatically digesting myosin with α-chymotrypsin45 (link). Amine-modified DNA oligo (100 μM; Hokkaido System Science; Oligo D* in Supplementary Data) was mixed with 50 mM sulfo-SMCC (Thermo Scientific) and incubated for 1 h at room temperature. Excess SMCC was removed three times by gel filtration (Micro BIO-SPIN P-6, Biorad). HMM was mixed with SMCC-oligo at the stoichiometry 1:10, incubated overnight at 4 °C and then mixed with N-ethylmaleimide at 1:2 for 30 min at 4 °C. Excess SMCC-oligo was removed twice by ultrafiltration (Amicon Ultra 50 K, Merck Millipore) and Oligo D*-labelled HMM was aliquoted and stored at −80 °C until use. Labelling efficiency was estimated to be ∼50% by a gel-shift assay. Immobilization was confirmed by the in vitro motility assay46 (link).

The purified QsGH13 was concentrated to 10 mg ml^–1 using Amicon Ultra 50K ultrafiltration devices (Merck Millipore, Darmstadt, Germany). The crystallization of QsGH13 was prepared by handing drop vapor diffusion methods by mixing 1 μl of QsGH13 (10 mg ml^–1) with an equal volume of reservoir solution at 4°C. It was grown in a condition of 0.1 M Tris–HCl (pH 8.5), 0.2 M NaCl, and 20% PEG 3,350. The crystals were briefly soaked with the cryoprotectant solution, and then flash cooled directly in liquid nitrogen at −173°C. The x-ray diffraction data were collected at the BL17U beamline of SSRF (Shanghai Synchrotron Radiation Facility, Shanghai, China). The data were processed and scaled using XDS (Kabsch, 2010 (link)). The collected data and processing details are shown in Table 1.
The structure of QsGH13 was determined by molecular replacement method with Phaser (McCoy et al., 2007 (link)), using the crystal structure of α-glucosyltransferase XgtA from Xanthomonas campestris WU-9701 (PDB entry: 6AAV, 51% identity to QsGH13) (Watanabe et al., 2020 (link)) as a search model. The refinement data of QsGH13 was performed using REFMAC and Phenix (Liebschner et al., 2019 (link)). Coot was used to further modify and adjust the structure (Emsley and Cowtan, 2004 (link)). The refinement statistics data of QsGH13 are shown in Table 1.

Expression of the SdrD protein was assessed by immunoblot analysis. Lysates of S. aureus subsp. aureus NCTC8325-4 and the isogenic NCTC8325-4 ΔsdrD mutant were prepared for Western blotting as described previously (4 (link)). To assess the release of SdrD, the culture supernatant was filter sterilized (pore size, 0.22 μm) to remove intact bacterial cells and concentrated using an Amicon Ultra 50K centrifugal filter device (Millipore Corp., USA). Expression of SdrD in the bacterial pellet and supernatant was evaluated by immunoblotting using SdrD A region-specific (a kind gift from Elisabet Josefsson) (primary) and polyclonal swine anti-rabbit immunoglobulin (Dako, Denmark) (secondary) antibodies.

Sf9 cells seeded in roller bottles at a density of 400,000 cells/mL in serum-free medium were infected at a MOI of 2 PFU per cell. After 3-day incubation at 28°C, supernatants were collected and stored at −80°C before use. Supernatants were then concentrated and diafiltered against TS buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl) using Centromate cassettes (Pall, 0.1 m²/30 kDa) before loading on a 5 ml column of anti-FLAG M2 Affinity gel (Sigma) equilibrated with TS buffer. The FLAG-tagged protein was eluted with 30 ml of 100 µg/ml FLAG peptide (in TS buffer). Fractions containing the purified protein were concentrated (AMICON Ultra 50 k, Millipore) and analyzed by PAGE and western blotting.