MAD PHASING USED IN THE STRUCTURE DETERMINATION OF DESULFOFERRODOXIN. Ana Coelho^1,2, Pedro M. Matias¹, Maria A. Carrondo^1,3, Vilmos Flp⁴, Ana Gonzalez⁵ and Andy Thompson⁶, ¹ITQB, Universidade Nova de Lisboa, 2780 Oeiras, Portugal; ²Universidade de vora, 7000 vora, Portugal; ³IST, Universidade Tcnica de Lisboa, 1000 Lisboa, Portugal; ⁴LMB and OCMS, University of Oxford, Oxford OX1 3QU, UK; ⁵ESRF, BP-220, 38043 Grenoble Cedex France; ⁶EMBL Grenoble Outstation, BP-156, 38042 Grenoble Cedex France

Multiwavelength anomalous data collected at ESRF, BL- 19, were used to solve the structure of desulfoferrodoxin (DFX), isolated from the sulphate reducing bacteria D. desulfuricans ATCC 27774. This non-heme iron protein is a 13.4 kDa monomer with 125 residues and two iron centres. The two midpoint redox potentials for this protein (4 and 240 mV) permit its separation in three oxidation states. The crystals of the fully oxidized form belong to space group R32 (a=112.5, c=63.2, Z=1). The MAD method was tried due do the failure in finding suitable heavy atom derivatives to be used with the MIR method. The crystal used for data collection was frozen and mounted with the c axis perpendicular to the spindle. Data were collected at 3 wavelengths near the iron absorption edge and scaled against a data set collected at 1.09. For each data set the R_merge is less than 4%, the multiplicity is around 4.5 and the completeness is greater than 96%. The iron atom positions were determined from an anomalous difference map and used for phase refinement, giving a figure of merit of 0.7 at 2.8 . The electron density maps obtained were improved by solvent flattening before model building. Refinement is in progress.