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4.2.2.1 P relim inary Data C ollection of M utant P o rp h o b ilin o g en D ea m in a se

The previous characterisation of the diffraction pattern of native protein crystals provided information regarding the unit cell. The crystals were known to be orthorhombic, with unit cell dimensions of a=88Â b=76Â, c=50Â, a=90\ p=90°, y=90°. Data needed only be collection over a 90“ rotation as the cell was found to have two planes of symmetry.

The data for both mutant D84E and the mutant ES2 complex were initially collected at Birkbeck College using the Mar research 180mm image plate fitted to a Siemens generator. However, due to the small size of the crystals of the m utant protein it was necessary to collect a higher resolution data set using synchrotron radiation at the Daresbury Laboratories. Data were collected for both the mutant D84E and the mutant ES2 complex at Station 9.5 (a similar source to Station 9.6). The monochromator was calibrated to a bromine absorption edge. The cooled crystals (4°C) diffracted out to a resolution of 2.0Â and data were collected over 90°.

4.2.2.2 C ry o -co o lin g of C rystals o f M utant P o rp h o b ilin o g en D ea m in a se C rystals

Subsequently, data were collected for cryo-cooled crystals of D84E and D84E soaked with substrate. It was predicted that D84E soaked with substrate may also provide a means of obtaining an enzyme-intermediate model. It was interesting to note that D84E crystals did not shatter upon the addition of substrate, in contrast to the native protein, and this may be a tribute of the reduced activity of the m utant enzyme. A colour change accompanied the addition of substrate, causing the crystals, but not the surrounding solution, to change from colourless to pink. The low temperature (lOOK) involved with cryo-crystallography allows the lifetime of the protein crystal to be increased by reducing radiation damage during data collection (Gamblin and Rodgers, 1993). It has also been suggested that cooling may increase the internal order of the parts of the protein which are mobile at room tem perature (Petsko,

Chapter 4 ; Crystallisation of the Asp-84 Mutants

1995). For porphobilinogen deaminase this w ould include the substrate rings and the mobile loop. Synchrotron radiation data for D84E and D84E soaked w ith substrate were collected at Stations 9.5 and 9.6 respectively. As cryo- cooling is a technique not previously applied to porphobilinogen deaminase, the collection of data for the D84E crystal soaked w ith substrate is described in further detail to highlight some of the associated problems.

The D84E substrate-soaked crystals selected for data collection were rapidly cooled in liquid ethane and stored in liquid nitrogen (lOOK). The presence of a cryostream was employed and its orientation optim ised to allow the tem perature of the crystal to be m aintained once m ounted. The

cryostream consisted of low tem perature nitrogen gas at fixed pressure surrounded by an outer coaxial sheath of dry air. The flow rate of the w arm gas sheath was adjusted visually to minimise turbulence of the cold gas stream. Ideally, the loop containing the crystal w ould be m ounted parallel w ith the cold gas stream to prevent disruption at the edge of the gas stream; in this case data was to be collected through a 90° rotation and therefore this condition could not be met continuously.The presence of a dry air sheath is also helpful in preventing ice formation on the crystal.

Preliminary data were collected for the m ounted crystal of substrate- soaked D84E and the X-ray diffraction pattern w as studied. Notable ice rings were observed to have formed on the diffraction pattern, and exam ination of the crystal revealed that there had been a large build-up of ice on the crystal. Careful removal of the ice from the crystal w ith tweezers and further

collection of data from the crystal showed that the crystal had been spoilt by the ice formation. Data collection from the first crystal w as therefore

abandoned.

The pressure of the cryostream dry air sheath was readjusted and a second crystal m ounted. On data collection it appeared that the stream w as now at a more suitable flow rate and no further ice rings were observed on the diffraction pattern. Data was collected over 90° to a resolution of 2.2Â. As data was collected over a period of about 10 hours, the crystal was m onitored throughout for ice build up. Figures 4.4 and 4.5 show the crystal w ith and w ithout ice formation.

Data collection from cryo-cooled crystals was therefore viable b u t it was necessary to strike a balance betw een m aintaining low tem perature and preventing interference w ith the diffraction pattern. Achieving this balance was not only time-consuming, especially w hen time was at a prem ium , but was at the cost of the first choice of crystal. However, these may be teething problem s which w ould not be present for further data collection. The

effectiveness of the technique w ould lie w ith the structure m odelled from the data.

4.2.2.3 R a w D ata C o llec ted for M u tan t P o r p h o b ilin o g e n D e a m in a se

The X-ray diffraction oscillation images for the m utant D84E and the D84E ES2 complex were collected, the image for the D84E ES2 complex being show n in figure 4.6. The X-ray photograph shows a two-dimensional section through a three-dimensional array of spots. The intensity of each spot w as recorded and this information provided the basic experimental data of X-ray crystallographic analysis.

Chapter 4 ; Crystallisation of the Asp-84 Mutants

Figure 4.4 The Cryo-Cooled D84E Substrate-Soaked Crystal

The incorrect adjustment of the cryostream has led to ice formation on the crystal.

crystal

Figure 4.5 The Cryo-Cooled D84E Substrate-Soaked Crystal

The optimal adjustment of the cryostream has prevented ice formation on the crystal.

crystal

Figure 4.6 Oscillation Images of the Mutant D84E ES? Complex

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The summary of results for the data collection of the m utant D84E and D84E ES? complex is shown in tables 4.1 and 4.2.