Taking a look at the calibration of a CCD detector with a fiber-optic taper.

At the Structural Biology Center beamline 19BM, located at the Advanced Photon Source, the operational characteristics of the equipment are routinely checked to ensure they are in proper working order. After performing a partial flat-field calibration for the ADSC Quantum 210r CCD detector, it was confirmed that the detector operates within specifications. However, as a secondary check it was decided to scan a single reflection across one-half of a detector module to validate the accuracy of the calibration. The intensities from this single reflection varied by more than 30% from the module center to the corner of the module. Redistribution of light within bent fibers of the fiber-optic taper was identified to be a source of this variation. The degree to which the diffraction intensities are corrected to account for characteristics of the fiber-optic tapers depends primarily upon the experimental strategy of data collection, approximations made by the data processing software during scaling, and crystal symmetry.

Development of a real-time timing-shutter performance monitor for protein crystallography.

In order to accurately monitor shutter timing events and long-term shutter performance, a timing-shutter monitor has been developed. This monitor uses a photodiode to capture X-ray-induced fluorescence from the shutter blade in synchrony with goniometer rotation to measure shutter opening and closing delay times, as well as the total time that X-rays are exposed to the sample during crystallographic data frames.

Characteristics and crystal structure of bacterial inosine-5'-monophosphate dehydrogenase.

IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the first step unique to GTP synthesis. To provide a basis for the evaluation of IMPDH inhibitors as antimicrobial agents, we have expressed and characterized IMPDH from the pathogenic bacterium Streptococcus pyogenes. Our results show that the biochemical and kinetic characteristics of S. pyogenes IMPDH are similar to other bacterial IMPDH enzymes. However, the lack of sensitivity to mycophenolic acid and the Km for NAD (1180 microM) exemplify some of the differences between the bacterial and mammalian IMPDH enzymes, making it an attractive target for antimicrobial agents. To evaluate the basis for these differences, we determined the crystal structure of the bacterial enzyme at 1.9 A with substrate bound in the catalytic site. The structure was determined using selenomethionine-substituted protein and multiwavelength anomalous (MAD) analysis of data obtained with synchrotron radiation from the undulator beamline (19ID) of the Structural Biology Center at Argonne's Advanced Photon Source. S. pyogenes IMPDH is a tetramer with its four subunits related by a crystallographic 4-fold axis. The protein is composed of two domains: a TIM barrel domain that embodies the catalytic framework and a cystathione beta-synthase (CBS) dimer domain of so far unknown function. Using information provided by sequence alignments and the crystal structure, we prepared several site-specific mutants to examine the role of various active site regions in catalysis. These variants implicate the active site flap as an essential catalytic element and indicate there are significant differences in the catalytic environment of bacterial and mammalian IMPDH enzymes. Comparison of the structure of bacterial IMPDH with the known partial structures from eukaryotic organisms will provide an explanation of their distinct properties and contribute to the design of specific bacterial IMPDH inhibitors.