Crystallization and structure determination

Engineered pro-activin A precursor and complex with truncation of the Lys-rich region (K259-D282) were concentrated to 15 mg ml⁻¹ and used to set up 96-well crystallization plate using Mosquito Crystal (TTP Labtech) crystallization robot. Crystals of engineered pro-activin A precursor were obtained from sitting drops consisting of 200 nl of protein and 200 nl of crystallization solution. Crystallographic data were collected at beamline I04 at Diamond Light Source synchrotron using a mini-kappa goniometer to improve the separation of diffraction spots on the detector. Multi-wavelength anomalous dispersion data were collected under three wavelengths (0.9793, 0.9795 and 0.9720 Å) from the crystal of selenomethionine-labelled pro-activin A precursor with truncation of Lys259-Asp282, grown in 11.4% PEG 400, 12.9% PEG 2000MME and 100 mM HEPES pH 7.1 at the protein concentration of 15 mg ml⁻¹. A single-wavelength anomalous dispersion data set was collected at a wavelength of 0.9793 Å from the crystal of the same protein grown in 25% PEG 1000 and 100 mM MES pH 6.5. A native data set was collected from a crystal of cleaved pro-activin A complex with truncation of K259-D282, grown in 20% PEG 3350 and 200 mM calcium chloride.

Raw data were indexed and integrated using XDS software⁴³ and scaled using Aimless in CCP4 suite⁴⁴. Experimental phasing was performed using autoSHARP⁴⁵ and molecular replacement was performed using Phaser in CCP4 suite⁴⁶. The model was manually corrected using Coot 0.8.1 (ref. ⁴⁷) and refined using Refmac5 (ref. ⁴⁸) and Phenix⁴⁹. The dihedral angles of 97.6% of all amino-acid residues in the structure of unprocessed pro-activin A precursor are in the favoured region and none of the residues in the non-allowed region. The dihedral angles of 97.1% of all amino-acid residues in the structure of pro-mature activin A complex are in the favoured region and none in the non-allowed region. All the data collection, data reduction, structure determination and refinement statistics are shown in Table 1.