A test of macromolecular crystallization in microgravity: large well ordered insulin crystals.

Crystals of insulin grown in microgravity on Space Shuttle Mission STS-95 were extremely well ordered and unusually large (many >2 mm). The physical characteristics of six microgravity and six earth-grown crystals were examined by X-ray analysis employing superfine phi slicing and unfocused synchrotron radiation. This experimental setup allowed hundreds of reflections to be precisely examined from each crystal in a short period of time. The microgravity crystals were on average 34 times larger, had sevenfold lower mosaicity, had 54-fold higher reflection peak heights and diffracted to significantly higher resolution than their earth-grown counterparts. A single mosaic domain model could account for the observed reflection profiles in microgravity crystals, whereas data from earth crystals required a model with multiple mosaic domains. This statistically significant and unbiased characterization indicates that the microgravity environment was useful for the improvement of crystal growth and the resultant diffraction quality in insulin crystals and may be similarly useful for macromolecular crystals in general.

Synchrotron X-ray reciprocal-space mapping, topography and diffraction resolution studies of macromolecular crystal quality.

A comprehensive study of microgravity and ground-grown chicken egg-white lysozyme crystals is presented using synchrotron X-ray reciprocal-space mapping, topography techniques and diffraction resolution. Microgravity crystals displayed reduced intrinsic mosaicities on average, but no differences in terms of strain over their ground-grown counterparts. Topographic analysis revealed that in the microgravity case the majority of the crystal was contributing to the peak of the reflection at the appropriate Bragg angle. In the ground-control case only a small volume of the crystal contributed to the intensity at the diffraction peak. The techniques prove to be highly complementary, with the reciprocal-space mapping providing a quantitative measure of the crystal mosaicity and strain (or variation in lattice spacing) and the topography providing a qualitative overall assessment of the crystal in terms of its X-ray diffraction properties. Structural data collection was also carried out at the synchrotron.

The high-mosaicity illusion: revealing the true physical characteristics of macromolecular crystals.

Typical measurements of macromolecular crystal mosaicity are dominated by the characteristics of the X-ray beam and as a result the mosaicity value given during data processing can be an artifact of the instrumentation rather than the sample. For physical characterization of crystals, an experimental system and software have been developed to simultaneously measure the diffraction resolution and mosaic spread of macromolecular crystals. The contributions of the X-ray beam to the reflection angular widths were minimized by using a highly parallel, highly monochromatic synchrotron source. Hundreds of reflection profiles over a wide resolution range were rapidly measured using a charge-coupled device (CCD) area detector in combination with superfine phi-slicing data collection. The Lorentz effect and beam contributions were evaluated and deconvoluted from the recorded data. Data collection and processing is described. From 1 degrees of superfine phi-slice data collected on a crystal of manganese superoxide dismutase, the mosaicities of 260 reflections were measured. The average mosaicity was 0.0101 degrees (s.d. 0.0035 degrees ) measured as the full-width at half-maximum (FWHM) and ranged from 0.0011 to 0. 0188 degrees. Each reflection profile was individually fitted with two Gaussian profiles, with the first Gaussian contributing 55% (s.d. 9%) and the second contributing 35% (s.d. 9%) of the reflection. On average, the deconvoluted width of the first Gaussian was 0.0054 degrees (s.d. 0.0015 degrees ) and the second was 0.0061 degrees (s. d. 0.0023 degrees ). The mosaicity of the crystal was anisotropic, with FWHM values of 0.0068, 0.0140 and 0.0046 degrees along the a, b and c axes, respectively. The anisotropic mosaicity analysis indicates that the crystal is most perfect in the direction that corresponds to the favored growth direction of the crystal.

Cryo-trapping the six-coordinate, distorted-octahedral active site of manganese superoxide dismutase.

Superoxide dismutase protects organisms from potentially damaging oxygen radicals by catalyzing the disproportionation of superoxide to oxygen and hydrogen peroxide. We report the use of cryogenic temperatures to kinetically capture the sixth ligand bound to the active site of manganese superoxide dismutase (MnSOD). Synchrotron X-ray diffraction data was collected from Escherichia coli MnSOD crystals grown at pH 8.5 and cryocooled to 100 K. Structural refinement to 1.55 A resolution and close inspection of the active site revealed electron density for a sixth ligand that was interpreted to be a hydroxide ligand. The six-coordinate, distorted-octahedral geometry assumed during inhibition by hydroxide is compared to the room temperature, five-coordinate, trigonal bipyramidal active site determined with crystals grown from practically identical conditions. The gateway residues Tyr34, His30 and a tightly bound water molecule are implicated in closing-off the active site and blocking the escape route of the sixth ligand.

The effect of temperature and solution pH on the nucleation of tetragonal lysozyme crystals.

Part of the challenge of macromolecular crystal growth for structure determination is obtaining crystals with a volume suitable for x-ray analysis. In this respect an understanding of the effect of solution conditions on macromolecule nucleation rates is advantageous. This study investigated the effects of supersaturation, temperature, and pH on the nucleation rate of tetragonal lysozyme crystals. Batch crystallization plates were prepared at given solution concentrations and incubated at set temperatures over 1 week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions. Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable, however, was pH; crystal numbers changed by two orders of magnitude over the pH range 4.0-5.2. Crystal size also varied with solution conditions, with the largest crystals obtained at pH 5.2. Having optimized the crystallization conditions, we prepared a batch of crystals under the same initial conditions, and 50 of these crystals were analyzed by x-ray diffraction techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.

Stationary crystal diffraction with a monochromatic convergent X-ray source and application for macromolecular crystal data collection.

A diffraction geometry utilizing convergent X-rays from a polycapillary optic incident on a stationary crystal is described. A mathematical simulation of the resulting diffraction pattern (in terms of spot shape, position and intensity) is presented along with preliminary experimental results recorded from a lysozyme crystal. The effective source coverage factor is introduced to bring the reflection intensities onto the same scale. The feasibility of its application to macromolecular crystal data collection is discussed.

Partial improvement of crystal quality for microgravity-grown apocrustacyanin C1.

The protein apocrustacyanin C(1) has been crystallized by vapour diffusion in both microgravity (the NASA space shuttle USML-2 mission) and on the ground. Rocking width measurements were made on the crystals at the ESRF Swiss-Norwegian beamline using a high-resolution psi-circle diffractometer from the University of Karlsruhe. Crystal perfection was then evaluated, from comparison of the reflection rocking curves from a total of five crystals (three grown in microgravity and two earth controls), and by plotting mosaicity versus reflection signal/noise. Comparison was then made with previous measurements of almost 'perfect' lysozyme crystals grown aboard IML-2 and Spacehab-I and reported by Snell et al. [Snell, Weisgerber, Helliwell, Weckert, Hölzer & Schroer (1995). Acta Cryst. D51, 1099-1102]. Overall, the best diffraction-quality apocrustacyanin C(1) crystal was microgravity grown, but one earth-grown crystal was as good as one of the other microgravity-grown crystals. The remaining two crystals (one from microgravity and one from earth) were poorer than the other three and of fairly equal quality. Crystal movement during growth in microgravity, resulting from the use of vapour-diffusion geometry, may be the cause of not realising the 'theoretical' limit of perfect protein crystal quality.

Partial improvement of crystal quality for microgravity-grown apocrustacyanin C1.

The protein apocrustacyanin C1 has been crystallized by vapour diffusion in both microgravity (the NASA space shuttle USML-2 mission) and on the ground. Rocking width measurements were made on the crystals at the ESRF Swiss-Norwegian beamline using a high-resolution psi-circle diffractometer from the University of Karlsruhe. Crystal perfection was then evaluated, from comparison of the reflection rocking curves from a total of five crystals (three grown in microgravity and two earth controls), and by plotting mosaicity versus reflection signal/noise. Comparison was then made with previous measurements of almost 'perfect' lysozyme crystals grown aboard IML-2 and Spacehab-1 and reported by Snell et al. [Snell, Weisgerber, Helliwell, Weckert, Holzer & Schroer (1995). Acta Cryst. D51, 1099-1102]. Overall, the best diffraction-quality apocrustacyanin C1 crystal was microgravity grown, but one earth-grown crystal was as good as one of the other microgravity-grown crystals. The remaining two crystals (one from microgravity and one from earth) were poorer than the other three and of fairly equal quality. Crystal movement during growth in microgravity, resulting from the use of vapour-diffusion geometry, may be the cause of not realising the 'theoretical' limit of perfect protein crystal quality.

Crystallization of chicken egg-white lysozyme from ammonium sulfate.

Chicken egg-white lysozyme was crystallized from ammonium sulfate over the pH range 4.0-7.8, with protein concentrations from 100 to 150 mg ml(-1). Crystals were obtained by vapor-diffusion or batch-crystallization methods. The protein crystallized in two morphologies with an apparent morphology dependence on temperature and protein concentration. In general, tetragonal crystals could be grown by lowering the protein concentration or temperature. Increasing the temperature or protein concentration resulted in the growth of orthorhombic crystals. Representative crystals of each morphology were selected for X-ray analysis. The tetragonal crystals belonged to the P4(3)2(1)2 space group with crystals grown at pH 4.4 having unit-cell dimensions of a = b = 78.71, c = 38.6 A and diffracting to beyond 2.0 A. The orthorhombic crystals, grown at pH 4.8, were of space group P2(1)2(1)2 and had unit-cell dimensions of a = 30.51, b = 56.51 and c = 73.62 A.

CCD video observation of microgravity crystallization of lysozyme and correlation with accelerometer data.

Lysozyme has been crystallized using the ESA Advanced Protein Crystallization Facility onboard the NASA Space Shuttle Orbiter during the IML-2 mission. CCD video monitoring was used to follow the crystallization process and evaluate the growth rate. During the mission some tetragonal crystals were observed moving over distances of up to 200 micrometers. This was correlated with microgravity disturbances caused by firings of vernier jets on the Orbiter. Growth-rate measurement of a stationary crystal (which had nucleated on the growth reactor wall) showed spurts and lulls correlated with an onboard activity: astronaut exercise. The stepped growth rates may be responsible for the residual mosaic block structure seen in crystal mosaicity and topography measurements.

Lysozyme crystal growth kinetics monitored using a Mach-Zehnder interferometer.

A Mach-Zehnder interferometer has been developed for the monitoring of the kinetics of the diffusion process in protein crystal growth. This device can be used in conjunction with the ESA Advanced Protein Crystallization Facility (APCF), which allows experiments under microgravity conditions (e.g. on board the NASA Space Shuttle). Experimental trials on the ground have been carried out with the interferometer using the engineering model of the APCF and a protein dialysis reactor. Chicken egg-white lysozyme crystal growth, as a test, has thereby been monitored directly. The changes of concentration in the solution over time have been determined via the refractive index measurements made and subsequently correlated with visual monitoring of crystal growth in a repeat experiment.

Improvements in lysozyme protein crystal perfection through microgravity growth.

Microgravity offers an environment for protein crystallization where there is an absence of convection and sedimentation. We have investigated the effect of microgravity conditions on the perfection of protein crystals. The quality of crystals for X-ray diffraction studies is characterized by a number of factors, namely size, mosaicity and the resolution limit. By using tetragonal lysozyme crystals as a test case we show, with crystal growth in two separate Space Shuttle missions, that the mosaicity is improved by a factor of three to four over earth-grown ground control values. These microgravity-grown protein crystals are then essentially perfect diffraction gratings. As a result the peak to background of individual X-ray diffraction reflections is enhanced by a similar factor to the reduction in the mosaicity. This then offers a particularly important opportunity for improving the measurement of weak reflections such as occur at high diffraction resolution. These microgravity results set a benchmark for all future microgravity and earth-based protein crystallography procedures.