Overcoming the challenges of twinning and pseudo symmetry in solving the structure of flavodoxin from Desulfovibrio desulfuricans 29577 by x-ray crystallography

In solving the structure of flavodoxin from Desulfovibrio desulfuricans 29577, crystallographic challenges of twinning and pseudo translational symmetry were encountered. The effects of both challenges were investigated to determine how they complicate structure solution and to develop insights into dealing with these factors for future studies.;Flavodoxins are small, single domain flavoproteins that bind a single flavin mononucleotide (FMN) and participate in electron-transfer pathways at low redox potentials. The structure of oxidized flavodoxin from D. desulfuricans 29577 was solved in two crystal forms at 2.0 A and 2.5 A resolution, and that of the semiquinone form of flavodoxin was determined at 2.5 A resolution. The overall topology of this class of proteins, a five-stranded parallel beta sheet flanked by two alpha helices on each side, was conserved. The FMN is bound at the periphery and forms stacking interactions with Trp60 and Tyr98 and a number of hydrogen bonds with the protein which are conserved between the oxidized and semiquinone states. Two conformations for the Trp60-containing loop were determined, straight and bent, indicating a high degree of flexibility of the loop that may be a starting point for elucidating the residues involved in protein partner recognition.;Each of the crystal forms oxidized flavodoxin represented crystallographic challenges. One crystal exhibited almost perfect merohedral twinning, but early detection and application of the appropriate twin law allowed successful structure solution and refinement. The presence of pseudo translational symmetry and a high copy number in the second crystal prevented phasing by multiwavelength anomalous dispersion and complicated phasing by molecular replacement by essentially reducing the signal to noise ratio in both cases. Ultimately, the structure was solved using an alternative approach; the refined structure from the twinned crystal was used as a perfect search model to identify initial molecular replacement solutions, and the final copies were then placed using the pseudo translational symmetry operators. Although, this structure was not twinned, application of a twin law, -h, -k, l, was essential for refining the model to reasonable R-factor values. Twinning and pseudo symmetry complicated structure solution, but alternative approaches to phasing and refinement allowed successful structure determination.