Table 1

Summary of x-ray data collection and MIR phase determination. X-ray data used for this structure determination were collected at ID2/BL4 and ID14/EH3 at ESRF. Intensity data were obtained using Mar IP detectors (ID2/BL4) or Mar CCD detector (ID14/EH3). Data were processed with DENZO and SCALEPACK (35). Initial phases were obtained for theP6522 crystals by multiple isomorphous replacement (MIR). MIR phasing and refinement of the heavy atom positions were carried out using the program MLPHARE (36). MIR phases were calculated up to 4.0-Å resolution. The overall figure of merit was 0.51. Density modification and phase extension to 3.0-Å for the P6522 crystal form was performed with the program DM (37). The initial free-R factor was 0.524 and was reduced to 0.268 after 200 cycles. The best result was obtained with a solvent content of 64%. Using this electron density, the structure of the P65 crystal form was solved using the program AMORE (38). Multicrystal averaging between two crystal forms was performed using the program DMMULTI (37), after which the free-R factor for theP6522 and P65 forms were 47% and 33%, respectively. Atomic models of the bc1 complex were built using the program O (39). In the initial electron density map, the electron density for the ISP for both crystal forms was ambiguous. The density for the ISP appeared in different positions in the different crystal forms after the first cycle of refinement. Simulated annealing (β-test version of CNS; A. Brünger, personal communication) followed by positional refinement using the program REFMAC (40) resulted in a structural model with anR-factor of 28.5% and 32.0% forP6522 and P65 forms, respectively, for the data between 30- and 3.0-Å resolution. The freeR-factor within the same resolution range was 34.5% and 36.5% for P6522 and P65forms, respectively (from 2% of the data). The rms deviations from standard values of bond lengths and angles forP6522 and P65 forms were 0.012 Å and 3.4°, 0.013 Å and 3.6°, respectively. All the figures here were created using the programs O (39), MOLSCRIPT (41), and RASTER3D (41).

Crystaldmin(Å)ObservationsCompleteness (%)Rmerge* (%)Rderiv (%)Sites (n)RcStation
Diffraction Data
P6522 form
Native (initial phasing)4.089,52132,69484.05.5ID14
(Me)3PbAc (24 hour)4.081,64131,02577.79.813.930.83ID2
(Me)3PbAc (48 hour)4.065,72227,34469.37.811.130.84ID2
EMTS (12 hour)4.097,69633,87185.49.115.9110.82ID2
EMTS (18 hour)4.082,47731,15379.39.115.8110.76ID2
EMTS (co-crystallized)4.034,15018,97648.24.511.270.87ID14
Native (high resolution)3.0223,18279,39688.97.0ID2
Antimycin A3.3138,91155,17079.010.5ID2
P65 form
MIR Phase Determination
Resolution (Å)
Figure of merit0.440.540.520.570.500.39
  • * Rmerge = ΣhΣi|Ii(h) – 〈I(h)〉|/ΣhΣiIi(h), where Ii(h), is the ith measurement.

  • Rderiv = Σ∥FPH| – |FP∥/Σ|FP|, where FPH and FP are protein and heavy-atom derivative structure factors, respectively.

  • Rc = Σ∥FPH-FP|-FH(calc)∥/Σ|FPH-FP|,FPH and FP are defined above, FH(calc) is the calculated heavy-atom structure factor. Summation is done using centric reflections only.