X-ray or gamma ray systems or devices – Specific application – Diffraction – reflection – or scattering analysis
Reexamination Certificate
2001-06-28
2002-12-24
Church, Craig E. (Department: 2882)
X-ray or gamma ray systems or devices
Specific application
Diffraction, reflection, or scattering analysis
Reexamination Certificate
active
06498829
ABSTRACT:
TECHNICAL FIELD
The present invention generally relates to a method to measure the physical characteristics of crystals, and, in particular, to a method and apparatus which simultaneously measures the diffraction resolution and mosaic spread of macromolecular crystals.
BACKGROUND OF THE INVENTION
The use of reflective mosaicity has been used since 1981 (Shaikevitch et al., 1981) as an indicator of macro-molecular crystal perfection. Subsequently, synchrotron radiation was used to minimize the geometric and spectral contributions of the X-ray source to the experimental data (Colapietro et al., 1992; Helliwell, 1988). Mosaicity analysis of chicken egg-white lysozyme, apocrustacyanin C
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and thaumatin crystals established a physical basis for the improvements seen in some microgravity-grown samples. In these samples, a reduction in the mosaic spread produced a corresponding increase in the signal-to-noise ratio of the reflection (Ng et al., 1997; Snell et al., 1995, 1997). The minimum mosaicities recorded were 0.005° for lysozyme, 0.030° for apocrustacyanin C
1
, and 0.018° for thaumatin measured at the full-width at half-maximum (FWHM). These values were obtained by deconvoluting the spectral and geometric contributions of the X-ray beam from the recorded rocking width, which allowed a quantitative comparison between samples (Colapietro et al., 1992) independent of the instrument measuring them.
Successful measurement of mosaicity requires that the geometric and spectral parameters of the instrumentation do not mask the crystal characteristics. Synchrotron radiation is a useful tool for these studies, as it can provide a highly parallel and (when a suitable monochromator is used) a highly monochromatic beam (Helliwell, 1992; Margaritondo 1995). Specially configured in house x-ray sources could also be used.
Previous efforts have been devoted to reflections recorded individually with a scintillation counter mounted in the equatorial (vertical) plane by rotating the crystal about the horizontal axis which minimized the Lorentz effect and eliminated the contribution from the horizontal beam divergence of the synchrotron beam (Colapietro et al., 1992; Fourme et al., 1995; Helliwell, 1998; Ng et al. 1997; Snell et al., 1995). An algorithm and software for mosaic spread analysis using data from an area detector was developed in 1998 (Ferrer et al., 1998).
There is still a need for a method to accurately measure the physical characteristics of crystals where the measuring instrument itself does not mask the measurements being made.
There is a particular need for a method to simultaneously measure the diffraction resolution, mosaicity, and intensity data of macromolecular crystals.
SUMMARY OF INVENTION
The present invention addresses these needs. 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. The present invention is an improved method for mosaic spread analysis which uses superfine &PHgr;-slicing data collection, unfocused monochromatic radiation, and a suitable fast readout area detector, such as a charge-coupled device (CCD) X-ray area detector.
According to the invention, a fast readout area detector is used to rapidly record many reflections simultaneously. Suitable available protein crystallography software is used to assign indices which identify each reflection and to obtain standard crystallographic statistics, such as l/&sgr;(l) and diffraction resolution. Because the data are not all on the equatorial plane, horizontal divergence is a contributor to the recorded rocking width in addition to the vertical divergence and spectral spread contributions present in other methods. These effects are deconvoluted from the data so that the true crystal mosaicity is evaluated. The fast readout area detector makes the superfine &PHgr;-slicing technique efficient and practical. The development of the crystal-quality evaluation method and the deconvolution of the beam divergence, spectral divergence and Lorentz effects from the measured rocking widths of the reflections are described herein. It is to be understood that the crystal rotations used in super fine &PHgr; slicing can range from less than about 0.0001° to greater than about 1°, and that all such measurements are within the scope of the present invention. The processing is also described, along with examples of the technique using single crystals of manganese superoxide dismutase (MnSOD), insulin and lysozyme.
The present invention provides a method to physically characterize crystals by simultaneously measuring the diffraction resolution and mosaic spread of macromolecular crystals. The contributions of the X-ray beam to the reflection angular widths are minimized by using a highly parallel, highly monochromatic X-ray source. Many, tens to thousands, of reflection profiles over a wide resolution range are rapidly measured using an area detector (e.g. charge-coupled device (CCD)) in combination with superfine &PHgr;-slice data collection. The Lorentz effect and beam contributions are evaluated and deconvoluted from the recorded data. For example, from 1° of superfine &PHgr;-slice data collected on a crystal of manganese superoxide dismutase, the mosaicities of 260 reflections were measured. The average mosaicity was 0.0101° (s.d. 0.0035°) measured as the full-width at half-maximum (FWHM) and range from 0.0011 to 0.0188°. 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° (s.d. 0.0015°) and the second was 0.0061° (s.d. 0.0023°). The mosaicity of the crystal was anisotropic, with FWHM values of 0.0068, 0.0140° and 0.0046° 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.
Methods according to this aspect of the present invention can be used, for example, to inspect a crystal's structure and its perfection. This method is of particular benefit in studying practical techniques of sample preparation for structural crystallography and for the use of crystals as a device rather than merely as a step to a structural solution.
In particular, the present invention relates to a method to simultaneously measure diffraction resolution and mosaic spread of a macromolecular crystal including the steps of (a) minimizing contributions of an x-ray beam to any reflection angular widths in the crystal by using a highly parallel, highly monochromatic x-ray source; (b) rapidly measuring multiple reflection profiles in the crystal over a wide resolution range using a suitable fast readout area detector in combination with superfine oscillation &PHgr;-slicing imaging data collection; and (c) evaluating and deconvoluting the Lorenz effect and beam contributions from the recorded data.
In certain aspects, the method further includes the step of: (d) determining the direction in which the crystal is most imperfect.
In still further aspects, the method further includes the step of: (e) measuring accurate intensities through file slicing, deconvolution and profile fitting.
The superfine oscillating &PHgr; slice data can be collected on a crystal where from about 0.0001° to about 1° of the super fine oscillation data is collected and where multiple reflection profiles are measured as the full-width at half-maximum (FWHM) or as the full width at quarter maximum (FWQM). Each reflection profile is fitted with at least one mathematical function that fits the recorded data. Preferably, the mathematical function is at least one Gaussian profile, is at least one Lorentzian profile, or other mathematical function.
Also the fast readout area detector is composed of a charge coupled device(s).
Mosaicity is determined fro
Borgstahl Gloria E. O.
Lovelace Jeffrey J.
Snell Edward H.
Church Craig E.
Emch, Schaffer, Schaub & Porcello & Co., L.P.A.
The University of Toledo
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