Table 1

Summary of structure determination. Crystals were in the space group P21 (a = 92.99 Å,b = 106.29 Å, c = 95.54 Å, β = 99.29°) with one complete transporter (BtuC2D2) per asymmetric unit. Data were processed with DENZO and SCALEPACK (48). For phasing, a combination of methods was applied using the programs XtalView (49), SOLVE (50), SHARP (51), and programs from the CCP4 suite (52). The locations of the vanadates were determined by inspection of the Patterson maps calculated at 5 Å resolution. Subsequently, several gold sites were located, and the phases were improved by combined solvent flattening and two-fold noncrystallographic averaging. The obtained phases were used to calculate anomalous cross-Fourier maps of data collected from a selenomethionine crystal at the peak wavelength. In two rounds, 34 selenium sites were located and their parameters refined. A model of the protein was built into the solvent-flattened and averaged maps using O (53). Refinement was carried out using CNS (54), and except for regions involved in lattice contacts, strict noncrystallographic symmetry was imposed. A model from the solved structure of cyclotetravanadate (55) was placed into the density, and its location was improved by rigid-body refinement. Group B factors were refined throughout, and the occupancy of side chains on the surface of the protein was set to zero if no density was visible. Model quality was verified with PROCHECK (56), and all residues are in the most favored or additionally allowed regions of the Ramachandran plot. MAD, multiple wavelength anomalous diffraction; rmsd, root mean square deviation.

Data setNativeNa3Au(S2O3 )2o-vanadateSeMet MADSeMet/o-vanadate
Beam lineAPS 19BMAPS 19IDAPS 19IDSSRL 9-2SSRL 9-2SSRL 9-2SSRL 9-2
Wavelength (Å)0.97771.03940.97920.91840.97980.98001.7712
Resolution (Å)30–4.050–4.050–4.030–3.530–3.530–3.730–3.2
Unique reflections16265157501582823662234331986830660
Completeness* 98.3 (83.0)98.6 (80.3)99.3 (92.7)99.5 (94.2)99.1 (89.4)99.0 (91.4)99.5 (94.3)
I/σ(I)*26.1 (8.3)23.9 (8.3) 19.0 (10.5) 22.1 (6.8)21.7 (7.6) 19.6 (9.0) 30.1 (4.1)
Rsym (%)*4.4 (18.0) 7.5 (24.5) 9.5 (18.9)  6.1 (21.7) 8.1 (26.7)  5.7 (15.6) 6.7 (44.8)
Phasing from SeMet MAD data setRefinement against SeMet/o-vanadate data
Resolution (Å)25–3.5Resolution (Å)15–3.2
Number of selenium sites34Reflections used27122
Isomorphous phasing power from peak (centric/acentric)2.75/4.14Test reflections2944
Anomalous phasing power from peak (acentric)2.37Number of nonhydrogen atoms 7950
Isomorphous phasing power from inflection (centric/acentric)2.33/3.66Rwork(%)26.2
Anomalous phasing power from inflection (acentric)1.96Rfree(%)28.6
Figure of merit (centric/acentric)0.53/0.65Average B factor (Å2)101.6
rmsd bond length (Å)0.005
rmsd bond angle (°)1.12
  • * Numbers in parentheses refer to data in the highest resolution shell.

  • This number does not include atoms whose occupancy has been set zero.