Crystal structure determination

Crystals were obtained by sitting-drop vapor diffusion at +20°C. The crystals of dArc2-NL were grown by mixing 150 nl of dArc2-NL at 8 mg/ml with 150 nl of reservoir solution (200 mM ammonium chloride, 100 mM sodium acetate (pH 5), 20% PEG 6000). The crystals of dArc2-CL were made by mixing 200 nl of protein at 15 mg/ml with 100 nl of reservoir solution (1.25 M ammonium sulphate, 100 mM Tris (pH 8.5), 200 mM lithium sulphate). The crystals of dArc1-CL were made by mixing 150 nl of the protein at 12 mg/ml with 150 nl of a reservoir, consisting of 100 mM MIB buffer (malonic acid, imidazole, boric acid) (pH 5) and 25% PEG 1500. The crystals of dArc1-CL used for phasing were grown by mixing 2 μl of the purified protein at 12 mg/ml with 2 μl of a reservoir solution, consisting of 20% PEG 3350, by hanging-drop vapour diffusion at +20°C. These crystals were soaked in a solution of 20% PEG 3350 with 500 mM NaI for 20 s.

Crystals were mounted in loops and snap-cooled in liquid nitrogen. X-ray diffraction data for dArc2-NL were collected on the I03 beamline at Diamond Light Source (Oxfordshire, UK), while the data for dArc1-CL and dArc2-CL were collected on the P11 beamline [25] at PETRAIII/DESY (Hamburg, Germany). All data were processed using XDS [26].

Phasing of dArc2-NL was done with molecular replacement in AMPLE [27] and ab initio models generated by QUARK [28], using the CCP4 online server [29, 30]. The phasing of dArc1-CL was done via iodine single-wavelength anomalous dispersion (SAD) and the Auto-Rickshaw pipeline [31], with the combined use of SHELX [32], PHASER [33], PARROT [34], and BUCCANEER [35]. The resulting near-complete model was taken as a template for molecular replacement in PHASER [33], using atomic-resolution data from a native crystal with a different space group. The phasing of dArc2-CL was done using the dimeric structure of dArc1-CL as a search model in PHASER [33]. All structures were refined with phenix.refine [36], and model building was done in Coot [37]. The quality of the structures was assessed using MolProbity [38]. Data processing and refinement statistics are given in Table 1.

The values in parentheses refer to the highest-resolution shell.