To purify the aCcr1 (SiRe_0197) and its mutant proteins from E. coli, cells harbouring plasmids pET22b-aCcr1-C-His (for the wild type cellular protein), pET22b-aCcr1-R2A-C-His, pET22b-aCcr1-K7A-C-His, pET22b-aCcr1-R27A-C-His (for the DNA binding site deficient cellular protein mutants) and pET22b-ATV_gp29-C-His and pET22b-SMV3_gp63-C-His (for virus-derived aCcr1 homologs) were grown in 2 L of LB medium at 37°C with shaking until the optical density OD600 reached 0.4∼0.6, when 1.0 mM IPTG was added into the cultures and the cells were then grown at 37°C for 4 h with shaking. The cells were harvested by centrifugation at 7000 g for 10 min and then resuspended in the lysis buffer A (50 mM Tris-HCl [pH 7.4], 200 mM NaCl and 5% glycerol). Then, the cells were crushed with an ultrasonic crusher at 40% power, working at 5 s intervals for 5 s until the cell lysate became clear and cell debris was removed by centrifugation at 12 000 g for 15 min. The supernatant was incubated at 70°C for 20 min, centrifuged at 12 000 g for 15 min again, and then filtered through a membrane filter (0.45 μm). The samples were loaded on to a Ni-NTA agarose column (Invitrogen) pre-equilibrated with buffer A. Finally, the target protein was eluted with buffer A containing 300 mM imidazole. The eluted sample was analysed using a 18% SDS-PAGE gel. The protein samples were concentrated by ultrafiltration using an Amicon Ultra-3KDa concentrator (Millipore). For further purification, size exclusion chromatography was performed using a Superdex 200 increase 10/300 column (GE Healthcare). The protein concentration was determined by the Bradford method using bovine serum albumin as the standard. To assay the oligomeric status, 20 μl of wild-type aCcr1 protein (1 mg/ml) were incubated with increasing concentrations of glutaraldehyde (0.01–0.16%) on ice at 4°C for 15 min. The reaction was then stopped by the addition of SDS-PAGE loading buffer, after which the samples were electrophoresed by 20% SDS–PAGE, and the gel was stained with Coomassie blue R-250.