Ian D. Glover and Don Nguti.
Desulphoredoxin is an Fe-S protein isolated from Desulphovibrio gigas,(Mouri et al., 1977, Bruschi et al., 1979) comprised of two 36 residue monomers, each coordinating an iron atom, which form a dimers with an Mr of 7740. Each of the monomers has four Cys residues expected to coordinate the iron atom. Most biochemical and spectroscopic evidence points to a similar coordination of iron but in relation to rubredoxin, higher symmetry in Fe binding is anticipated.
Good quality crystals of desulphoredoxin were first reported in 1980 (Seiker et al., 1980), but no suitable derivatives have been prepared. As a small metalloprotein it presented a good case for structure determination using MAD methods. With two Fe atoms in a small protein significant anomalous scattering contributions are expected, the maximal anomalous diffraction ratios (Hendrickson, 1991) of 5% and 4.8% for the absorptive and dispersive contributions respectively.
Desulphoredoxin crystallises in space group P3121 (or its enantiomer) with cell dimensions a = b = 42.28Å , c = 72.46Å , and = 120o. The crystals grow to approximately 0.3mm in the largest dimensions and are relatively radiation stable. All data were collected on station 9.5(Thompson et al., 1992) at the Daresbury SRS using an 18cm diameter MAR image plate detector and a channel cut Si(111) double crystal monochromator. MAD data were collected at four wavelengths, three close to the Fe-K edge, determined from XANES scans from a crystal and a fourth, higher resolution, dataset recorded at a remote wavelength. As the data were collected at room temperature all measurements contributing to a particular phase determination were collected as close together in time as possible. Initial calibration of the incident X-ray wavelengths was performed using the iron edge in a piece of magnetic tape, and thereafter x-ray wavelengths calculated using the monochromator angle. Due to the goniometer geometry the closest possible approach of the detector limited data collected at the longer wavelengths to approximately 3Å resolution.
The XANES spectrum were recorded from a single crystal of desulphoredoxin is shown in figure 1. The spectrum was transformed using the Kramers-Kronig (Kronig & Kramers, 1928) transform, to obtain experimental values of f' and f" (table 1). The values of the anomalous scattering coefficients were used to select the nominal wavelengths, 1 at 1.744Å , the first point of inflection on the f" curve, and therefore the minimum or most negative value on the f' curve. The second wavelength, 2 was selected at 1.740Å , the maximum on the f" curve, this data set will yield the greatest Bijvoet or Friedel differences. The third wavelength, 3, was selected at 1.7285Å , remote from the edge. The fourth wavelength was collected at 0.9Å , where the incident flux on station 9.5 is significantly higher and with the same data collection geometry allowed much higher resolution data (1.8Å ) to be collected. During data collection at the longer wavelengths the monochromator second crystal was detuned to avoid harmonic contamination of the incident beam.
Dataset Wavelength (Å) f' f'' 1 1.7444 -8.091 1.993 2 1.7405 -6.096 4.337 3 1.7284 -4.054 3.975 4 0.9000 -1.100 2.900
Table 1. The anomalous scattering factors for iron in desulphoredoxin at the wavelengths selected for data collection, the first three are derived from the Kramers-Kronig transform of the recorded XANES spectrum shown in figure 1.
One crystal was used in the collection of the three near edge data sets, 1, 2 and 3, and a second crystal used to record the fourth, 0.9Å wavelength, 4 data set. The crystals were accurately aligned with the c* axis parallel to the spindle axis. In this orientation there were no mirror related reflection recorded on the same image, all mirror related reflections were recorded by inverting the crystal, i.e. recording data at and + 1800. A total of 940 ( = 3 or 40) of data were collected at wavelengths 1,2, and 3 and 700 ( = 2o) at the fourth wavelength
Initial data reduction was carried out using the MOSFLM (Leslie, 1992) suite of programs after the determination of the initial orientation matrix using REFIX. Regardless of the phasing approach to be used, MADSYS or MLPHARE, once collected the data must be scaled, both within datasets and for the MAD analysis, between datasets to reduce differences due to crystal decay, absorbtion and any variation in detector response. Scale factors were calculated initially using ROTAVATA (CCP4, 1994) which calculated a single scale factor (Fox & Holmes, 1966) that is applied to all reflections in a particular batch, usually a single image. This means that symmetry related reflection falling on consecutive batches can have very different scale factors. Since the scaling is based on all symmetry related reflections within a dataset whose intensities are expected to be equal a continuously varying scale factor may be more appropriate, such as the approach used in SCALA (P.R. Evans, this volume) where the scale factor is a continuous function of rotation angle and detector position.
Scale and temperature factors between batches within each dataset were initially calculated using ROTAVATA and applied using AGROVATA. The results are set out in detail in tables 2 and 3. Taking the three datasets collected at wavelengths close to the iron edge, the overall RSYMM values are 14.6%, 15.2% and 13.8% respectively for the 1, 2 and 3 datasests which compare very unfavourably with the dataset recorded at 0.9Å wavelength. This poor scaling is clearly seen in the tables of batch scale and temperature factors calculated by ROTAVATA which show a large variation in scale factors and very significant variation in temperature factors. This poor scaling contributes to the mediocre quality of the merged data. Few batches had low RSYMM values and the signal to noise, as judged by the value of I/(I) was poor, averaging 3.5. Contrasting with this is the 4 dataset where the scale factors follow a regular progression, the biggest variations occurring either side of a beam refill and the temperature factors vary only slightly. The RSYMM values are significantly lower and the signal to noise better with an average I/(I) of 17.5.
This variation is seen despite the fact that the data were collected from similar sized crystals of the same shape. Furthermore it should be noted that the RSYMM value of the 4 data at 3.05Å resolution is only 1.8%. The only difference between the data is that the 1, 2 and 3 data were collected at longer wavelengths and that higher absorbtion at these wavelengths is having a significant effect on the internal consistency. Data of this quality is clearly going to present problems for the subsequent MAD analysis when the expected values for the largest anomalous and dispersive diffraction ratios are 5% and 4.8% respectively.
Dataset Wavelength (Å) IMEAN/ RSYMM Nobs 1 1.7444 3.54 0.146 6857 2 1.7405 3.29 0.152 6889 3 1.7284 3.95 0.138 6766 4 0.9000 17.46 0.034 20308
Table 2. Summary of the overall batch symmetry R-factors for the four MAD datasets. Note that the fourth wavelength extends to 1.8Å resolution.
DATASET 1 DATASET 4 BATCH SCALE B SCALE B 1 1.000 0.0 1.000 0.0 2 1.445 5.0 0.965 -0.2 3 1.970 -0.7 1.007 -0.1 4 1.499 6.0 0.937 -0.6 5 1.957 7.9 0.951 -0.6 6 2.345 -2.9 0.988 -0.7 7 2.520 3.6 0.992 -0.8 8 2.201 1.4 1.032 -0.8 9 2.505 0.3 1.060 -0.8 10 2.113 4.6 1.114 -0.9 11 2.827 5.0 1.077 -0.9 12 3.504 4.0 1.099 -1.0 13 2.134 4.4 1.092 -1.1 14 2.574 5.7 1.156 -1.4 15 2.833 6.5 1.185 -0.7 16 2.883 4.3 1.235 -1.0 17 2.925 4.1 1.222 -1.3 18 3.081 3.3 1.263 -1.1 19 3.193 1.1 1.248 -1.3 20 3.385 -0.6 1.268 -1.2 21 3.758 0.3 1.298 -1.4 22 4.021 -1.2 1.305 -1.4 23 4.434 -2.6 1.412 -1.5 24 4.811 -4.1 1.380 -1.5 25 2.453 -2.8 1.404 -1.6 26 2.918 -2.6 1.423 -1.4 27 3.153 3.6 1.445 -1.9 28 1.053 -1.6 29 1.061 -1.6 30 1.069 -2.2 31 1.066 -2.0 32 1.082 -2.3 33 1.076 -2.2 34 1.073 -2.3 35 1.089 -2.2
Table 3. a)The scale and temperature factor (B) for the datasets 1 and 4, recorded at 1.7444Å and 0.900Å wavelengths calculated using the program ROTAVATA. The abrupt change in scale factors in the short wavelength data at batch 28 is due to a beam refill. b) Values for the 1 dataset after scaling using SCALA.
BATCH SCALE B 1 0.279 0.0 2 0.334 -0.47 3 0.416 -1.16 4 0.534 -3.72 5 0.333 -1.95 6 0.414 -1.45 7 0.594 -3.298 8 0.5624 -4.62 9 0.652 -5.37 10 0.504 -2.65 11 0.657 -2.87 12 0.878 -2.61 13 0.535 -1.61 14 0.583 -2.30 15 0.644 -2.87 16 0.705 -2.32 17 0.711 -3.09 18 0.771 -4.31 19 0.872 -4.69 20 0.970 -5.93 21 1.014 -7.12 22 1.149 -7.91 23 1.334 -9.61 24 1.506 -11.00 25 0.7614 -8.83 26 0.873 -10.21 27 0.940 -10.68
The program SCALA was used to calculate scale and temperature factors for each dataset prior to merging in AGROVATA. SCALA differs in methodology in that it calculates a three dimensional scale factor for each reflection taking into account rotation angle and its position on the detector. This methodology has significant benefits when applied to this case where sample absorbtion is anticipated to have a large effect on the internal consistency of the data. The results from scaling and merging with SCALA/AGROVATA (tables 3 and 4) show a very significant improvement for the data collected at long wavelengths. The signal to noise ratios have increased considerably and the consistency, typically from approx 12% to 3%. The short wavelength data, however, shows very little improvement.
Table 4. a) Summary of the overall batch symmetry R-factors for the four MAD datasets scaled using SCALA (data compared to 3.05Å resolution) and b) the merging statistics and multiplicity (Mult.) from AGROVATA.
Dataset Wavelength (Å) IMEAN/ R Nobs 1 1.7444 9.44 0.033 5820 2 1.7405 10.51 0.034 5723 3 1.7284 11.14 0.034 5446 4 0.9000 21.20 0.029 4447b)
Dataset RMERGE DMIN NUNIQUE %COMPLETE Mult. 1 0.045 3.05 1510 96.3 4.4 2 0.048 3.04 1519 96.1 4.4 3 0.036 3.03 1524 95.6 4.1 4 0.029 1.78 7155 95.5 3.1
Patterson maps calculated using anomalous differences and dispersive differences are shown in fig. 2. The anomalous scattering partial structure was interpreted in terms of two independent Fe sites. A calculated Patterson is also shown, confirming the interpretation of the anomalous scattering partial structure.
MLPHARE was used to refine each of the two Fe sites independently and then used together in phasing and site refinement. Initial real occupancies were estimated in the ratios of the real, f' components of the anomalous scattering and refined against centric data before anomalous occupancies were estimated and refined. The two sites were then refined using real and anomalous occupancies simultaneously against all data to 3.05Å resolution. The overall figures of merit were 0.82 and 0.74 for centric and acentric reflections respectively.
Parameter 1 2 3 Phasing power (acentric) 2.6 2.2 2.2 (centric) 1.6 1.3 1.3 RCULLIS (acentric) 0.53 0.59 0.59 (centric) 0.53 0.63 0.63 (anomalous) 0.70 0.70 0.80
1 2 3 4 SITE 1 Real Occupancy 0.404 0.313 0.301 0.0 Anom. Occupancy 0.909 1.197 1.051 0.339 SITE 2 Real Occupancy 0.441 0.340 0.330 0.0 Anom. Occupancy 0.862 1.086 0.962 0.327
Table 5. a)Summary of the statistics for the refinement of the two Fe sites in MLPHARE and b) real and anomalous occupancies for the two sites after refinement.
The anomalous scattering partial structure had been solved using Patterson methods and the ambiguity in the hand of the partial structure was resolved by calculating the two alternate maps, in this case by calculating the maps in the alternate space groups P3221 and P3121. The former showed clear molecular boundaries and the iron sites could be readily located along with clear density for the iron ligands. Away from the iron sites however no clear contiguous density was observed so the map was subjected to iterative cycles of density modification, solvent flattening and histogram matching using the program DM. Map improvement was monitored using the free R flag as shown in table n, and the increase in the overall figure of merit from 0.69 to 0.81 for all data accomplished with a mean change in phase angle of 15.50. The calculated electron density map had improved significantly with evidence of contiguous density, showing the iron site to be in a distorted tetrahedral geometry coordinated through four cysteinyl sulphurs and clear strands of density including the short loop between Cys 9 and 12, figure 3.
Interwavelength scaling and scattering factors.
Although the MLPHARE approach to phasing has been used in this case the MADSYS suite of programs may alternatively be used. In this case the datasets , scaled using SCALA as before, were merged to give one '+' and one '-' reflection for each hkl. After local scaling (ANOSCL) the datasets recorded at each wavelength were put on the same relative, quasi-absolute scale using WVLSCL. In the course of the program the anomalous scattering factors f' and f" are refined from the crystallographic data, giving what should be analogous results to the refinement of occupancies (both real and imaginary) from MLPHARE. The results are shown in table 6, and it is clear that the refinement of the scattering factors from WVLSCL is more satisfactory than that from MLPHARE, apparently preserving the variation in the anomalous scattering contributions at values closer to those obtained from the Kramers-Kronig transform of the observed XANES spectrum from the crystal, suggesting that the inter-wavelength scaling using in this program maintains a more consistent representation of the anomalous scattering contributions in the scaled data.
Dataset & wavelength f' f'' 4 -0.31 1.11 1 -8.03 2.92 2 -5.47 4.03 3 -5.40 3.34
Table 6. The values of the refined f' and f" contributions at the four wavelengths from WVLSCL.
Bruschi, M., Moura, I., LeGall, J., Xavier, A.V. & Seiker, L.C. (1979) Biochem. Biophys. Res. Comm. 90 596-600
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Leslie, A.G.W. (1992) In CCP4-ESF-EACMB Newsletter for Protein Crystallography. Vol 26.
Thompson, A.W., Habash, J., Harrop, S., Helliwell, J.R., Nave, C., Atkinson, P., Hasnain, S.S., Glover, I.D., Moore, P.R., Harris, N., Kinder, S. & Buffey, S. (1992) Rev. Sci. Instrum. 63 1062-1064
Figure 1.a) The fluoresence XANES spectrum recorded from a single crystal of desulphoredoxin using a single wire proportional counter on station 9.5 at Datesbury.
b) The transformed spectrum showing the values of f' and f'' in electrons as a function of incident x-ray wavelength
Figure 2. a) Anomalous difference Patterson map calculated using the 2 (maximised f'') dataset, b) Dispersive difference Patterson calculated using the difference in tructure factors between 1 and 4 c) Calculated Patterson map using refined Fe site positions.
Figure 3. The calculetd electron density map, showing 1/6th of the unit cell in c the section direction, two unit cells in each other direction.