Derivative screening.



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Derivative screening.

After the crystals had been soaking in their respective heavy atom solutions for a week the prospective derivatives were screened by collecting diffraction data to a maximum resolution of Å on the X31 beam-line. The X-ray energy for each set was tuned to be above the absorption edge of the heavy atom in question so as not to necessitate the use of the calibrator for the screening process. In the majority of cases this meant that a complete set of diffraction data could be obtained in less that four hours depending on the angular setting of the crystals.

To test for the presence of heavy atoms, native data were collected to Å resolution and used along with phases from a refined model of the hen egg-white lysozyme [8] structure found in the Protein Data Bank at Brookhaven to calculate isomorphous and anomalous difference Fourier maps (see Sec. gif).

The model was first subjected to eight cycles of least squares refinement against the native data to ensure that no large differences existed between the two and the resulting phases used in the Fourier synthesis. Data on the iridium derivative crystals were collected after soaking for 10 days. The Bijvoet difference Fourier map calculated with data lying between and Å is shown in Fig. gif. The map represents a projection down the z-axis through of the unit cell, an asymmetric unit. There is evidence in the map for the presence of anything up to ten anomalously scattering iridium sites in the asymmetric unit, i.e. ten iridium atoms per lysozyme molecule. The isomorphous difference Fourier maps calculated against the native data also supported the presence of multiple iridium sites. The predicted Iridium site of Blake [11] was not present in the difference Fourier maps.

  
Figure: Difference Fourier synthesis calculated with the coefficients from the iridium derivative data and phases from a native lysozyme model obtained from the Protein Data Bank. The map for only th of the unit cell along the z direction is shown. Five strong peaks in the map are clearly visible and several weaker peaks are also present. The map is contoured at intervals starting at and the largest peak reaches .

Anomalous and isomorphous difference Fouriers calculated for the other four potential derivatives , , and displayed no evidence for bound heavy atoms. In each case no significant peaks were observed in the Fourier synthesis above a cut off level.



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Next: X-ray energy selection Up: MAD experiments on Previous: Crystallisation and derivative



Gwyndaf Evans
Fri Oct 7 15:42:16 MET 1994