2.1. Crystal growth

Crystals of Enterococcus casseliflavus glycerol kinase were obtained by the hanging-drop technique from a solution containing 29% polyethylene glycol 400, 0.1 M sodium acetate pH 4.5, 0.1 M calcium acetate and 10% glycerol. Crystals appeared in varying sizes from small needles to large rod-like crystals that were 0.7 mm long and 0.25 mm thick. The space group was determined as P2₁2₁2, with a solvent content of 56%, resulting in an occupancy of one dimer per asymmetric unit. The unit-cell parameters were a = 93.77, b = 195.26, c = 55.31 Å. This crystallization condition is different from that first found for the glycerol kinase crystals used in the original study (Yeh & Hol, 1998) as are the space group and unit-cell parameters, although the solvent content remains very similar.

2.2. Flash-annealing

Annealing experiments were performed with a new device, the Cryo-Shutter, shown in Fig. 1, which was fabricated at Brookhaven National Laboratories. The Cryo-Shutter is a simple device that allows blocking for programmed amounts of time of the 100 K nitrogen stream that surrounds the sample. The shutter is controlled by EPICS (Experimental Physics and Industrial Control System) and activated by air pressure. The opening and closing time of the Cryo-Shutter is of the order of milliseconds, which is insignificant compared with the total annealing time. The main advantage of the shutter is that it allows a controlled instant re-cooling of the crystals and avoids significant turbulence induced by traditional annealing `devices' such as a piece of hard thin plastic.

Figure 1 Image of the Cryo-Shutter in the (a) open and (b) closed state.

Although the glycerol kinase crystals can be mounted directly into cryoloops and flash-cooled in a 100 K nitrogen gas stream without apparent icing problems, it was found that this concentration of cryoprotectant (29% PEG 400) was `borderline' upon closer inspection. Apparently, at this concentration of PEG 400 the crystallization solution exhibited varying minute degrees of opacity upon cryocooling. The amount of opacity and icing, as indicated by a higher background of diffraction between 3.4 and 3.8 Å, was dependent on the age of the crystals and the mounting technique (i.e. the length of time a drop was open prior to mounting in a loop and cooling). We attributed this to dehydration, effectively increasing the concentration of PEG 400 to a concentration that prevented `residual' icing, which correlated with the increased visual transparency of the cryocooled solution. We initially added an additional 4% PEG 400 to the artificial mother liquor (29% PEG 400, 0.1 M sodium acetate pH 4.5, 0.1 M calcium acetate and 10% glycerol) and used this solution for mounting and cryocooling. However, we found that we could grow the crystals in the presence of 33% PEG 400, which allowed us to mount the crystals and directly flash-cool them.

For flash-annealing, the Cryo-Shutter controller software was set to a 4 s annealing cycle. The annealing was repeated two times for each crystal as it was found from previous experiments that between two and three cool–thaw cycles improved the mosaicity and resolution, whereas greater numbers of cool–thaw cycles eventually degraded diffraction qualities. Thawing and re-cooling of the buffer and crystal were observed on a monitor during the flash-annealing process and the crystal remained transparent throughout the flash-annealing process.

2.3. Fine φ-sliced data collection and processing

A highly parallel unfocused beam from a bending magnet on beamline X6a at the National Synchrotron Light Source (NSLS) was used to minimize beam divergence. The beam properties were calculated from the source size and slit apertures using the SHADOW software (Cerrina et al., 1998). The vertical and horizontal beam divergences were calculated to be 52.2 and 33.4 µrad, respectively, with primary slits set to 1.2 × 0.8 mm just before the Si(111) channel-cut monochromator (Allaire et al., 2003) located at 7.8 m from the source. The secondary slits were set to 0.5 × 0.5 mm 16.9 m from the source and Δλ/λ was calculated to be 2 × 10⁻⁴.

Data were collected with a Quantum-4 CCD detector (ADSC) using rotation-camera geometry as described previously (Bellamy et al., 2000; Borgstahl et al., 2001; Snell et al., 2001). To determine the crystal orientation, two orthogonal 3–6° swaths of coarse data were collected (Δφ = 1° with 20 s exposure) and processed with MOSFLM (Powell, 1999). From each swath, a 1.5° range was then selected and fine φ-­sliced data corresponding to that range were collected for mosaicity measurements. Data were collected on three glycerol kinase crystals (denoted xtl1, xt2 and xtl3). Each crystal was cryocooled directly in the nitrogen cryostream at 100 K, data were collected (data denoted as `cryo' and `cr') and then the crystal was flash-annealed (data denoted as `flash-annealed' and `FA'). The same data-collection parameters were used for each data set. Fine φ-sliced data were collected as stills spaced by 0.003° with 2 s X-ray exposures and a total of 500 frames were collected for each crystal at 100 K. The crystal-to-detector distance was 225 mm and the resolution at the edge of the detector was 2.3 and 1.8 Å at the corners. The wavelength was 1.033 Å and the beam was collimated to 0.141 mm diameter. Each data set took less than 20 min per crystal, therefore decay in the beam over time was ignored in the analysis. Crystal decay, as indicated by the DENZO scale factor per frame, was negligible. The fine φ-­sliced data were processed and analyzed using BEAM-ish 2.0 (Lovelace et al., 2000). The program BEAM-ish deconvolutes the reflection mosaicity, η, using the measured reflection full-width at half-maximum, φ_R, as described by Bellamy et al. (2000) and Helliwell (1988). To reduce noise, the data points were averaged over 0.006°, with two slicing events in a 0.003° window. In order to be accepted for profile analysis the reflections had to have I_max > 100 and I/σ(I) > 5. Gaussian curves were fit to the reflection profiles using a genetic algorithm (Wormington et al., 1999) and the inter-process communication feature of BEAM-ish 2.0 was used to search for identical reflections across multiple data sets (Lovelace et al., 2000).

3.1. Effects of flash-annealing on mosaicity, peak intensity and resolution

Annealing approaches have been proposed by several groups (Yeh & Hol, 1998; Bunick et al., 1998; Harp et al., 1998, 1999; Kriminski et al., 2002) to overcome the disadvantage of increased mosaicity as a result of cryocooling or to generally improve data quality. In the current study, mosaic domain theory was applied to the interpretation of the reflection profiles (Vahedi-Faridi et al., 2003) measured from the glycerol kinase crystals. A total of three glycerol kinase crystals (xtl1, xtl2 and xtl3) were examined to study the effects of flash-annealing on crystal quality. 500 images each with an oscill­ation angle of 0.003° were collected before (denoted as `cryo' and `cr') and after annealing (denoted as `flash-annealed' and `FA') to obtain reflection profiles and to deconvolute reflection mosaicity and peak-intensity values. Mosaicity data were measured from the full-width at half-maximum (FWHM) and full-width at quarter-maximum (FWQM) of reflection profiles. Table 1 shows a summary of the averaged diffraction statistics for each of the three crystals examined. Flash-annealing resulted in a noticeable overall decrease of the averaged reflection mosaicities, ranging from 26.3% (xtl1) to a decrease of 38.2% for xtl3 (see Fig. 2a). The averaged maximum pixel count for all reflections decreased slightly, as indicated by the change in peak area. However, the overall decrease in maximum intensities can be attributed to the fact that a filter was imposed for reflections to be included in the statistical analysis [I/σ(I) > 5 and I_MAX > 100; see §2.3]. The improvement in crystal quality as a result of flash-annealing allowed many more reflections to pass this filter (e.g. an increase of 48 reflections for xtl3). Most of these additional reflections have a peak count slightly above 100, thus contributing to the small overall decrease in the averaged values. The peak count is also referred to as `maximum intensity' in the figures and is the count at the peak of the rocking curve. Fig. 2(b) shows a plot of the maximum peak intensities as a function of resolution for crystal xtl3. The plot shows `annealed' low-intensity reflections at a resolution greater than 2.5 Å, showing an increase in diffractive resolution as a result of flash-annealing.

Figure 2 Crystal-quality comparison of glycerol kinase before (cryocooled, red crosses) and after flash-annealing (flash-annealed, blue diamonds) by plotting individual reflection (a) mosaicity and (b) peak count (`maximum intensity') against resolution.

Detailed comparisons of orientation matrices and observed reflection profiles showed that crystals xtl1 and xtl2 underwent a noticeable shift in position within the cryoloop during the flash-annealing process. As a result, identical reflections were not recorded for these crystals within the two measured 1.5° swaths. Crystal xtl3, however, moved to a far lesser degree, allowing the comparison of 79 identical reflections before and after flash-annealing (see Table 2). The averaged mosaicity decrease for all 79 reflections is 25.7%. We integrated the peak areas of seven reflections to quantify the change before and after flash-annealing (Table 3, column ΔArea). For the seven reflections, all but one exhibited a small decrease in peak areas; however, for the one reflection (3, −11, −69) whose peak area increased, the gain after annealing was significant (+50.8%). Contrary to the averaged value for all reflections, the averaged maximum peak intensity counts in the most intense pixels for all identical reflections actually increased by 28.8%. The increase of 46% in the standard deviation for the reflection mosaicities indicates that although reflection profiles become narrower overall as a result of flash-annealing, the mosaicity distribution for individual reflection increases overall. This fact is evident in Fig. 2(a), showing annealed reflections with smaller mosaicities but wider mosaicity distribution as a function of resolution. Figs. 3(a) and 3(b) show a plot of the mosaicity and peak-count change (`maximum intensity change', in percent) as a function of resolution. Approximately 93% of all observed identical reflections showed a decrease in their reflection mosaicities along with an increase in the corresponding peak intensities.

Figure 3 Crystal-quality comparison of glycerol kinase as a result of flash-annealing by plotting identical reflection (a) mosaicity change and (b) peak-count change (`maximum intensity change') against resolution.

3.2. Effect of flash-annealing on reflection profiles and domain structure

The availability of a large number of identical reflections allows detailed characterization of the annealing effects on the reflection profiles and domain structure. Fig. 4 shows 14 representative reflection profiles corresponding to seven identical reflections from crystal xtl3. In general, the cryocooled reflection profiles had a similar number of mosaic domains as the `annealed' reflections (Figs. 4). All of the `annealed' peak profiles were described by two Gaussian curves. Thus, `annealed' reflections still retain a dual-domain conformation with a small shoulder adjacent to the main peak. Most cryocooled profiles were also described by two Gaussians, with a few exceptions which were fitted with an additional third small Gaussian (Figs. 4d and 4g). The most noticeable effect of flash-annealing is the gain in maximum peak height along with a decrease in peak width for one fitted Gaussian at the expense of the second (Table 3). These plots suggest that the decreased mosaicity can arise from improved alignment of non-resolvable domains or a decrease in the variation of lattice spacings. Overall peak profiles are much more homogeneous and symmetrical. As a result of the enhancement of a single domain after flash-annealing, the signal-to-noise ratio of the data is improved by increasing the peak height of the reflections, making the weaker and higher resolution data easier to measure.

Figure 4 Seven representative reflection profiles out of 79 available identical reflections for crystals xtl3 (see Table 3). The red diamonds mark the measured individual raw data points. The black graph represents a summation of the fitted individual Gaussians (red, purple and green).

Another mechanism for decreased mosaicity is through improved domain misalignment and this may be exhibited through decreased peak separation of the Gaussian profiles of reflections after annealing. Our results indicate that two are approximately the same, three are reduced and two are increased (Table 3). Consequently, within the subset of reflections that we analyzed there appears to be a random distribution, although a trend may possibly be detected with a larger database for analysis.