Core Center Resources:
Resources for Structural Biology & Crystallography
X-Ray Crystallography – John Pascal, Ph.D., KCC, TJU
The X-ray Crystallography shared resource provides KCC investigators the ability to obtain structural information directly relevant to their research interests. This includes the determination of de novo structures, structural consequences of point mutations, co-crystallization with small molecules including inhibitors and potential therapeutics, and multi-component systems including proteins, DNA and/or RNA. Although structure determination is a highly specialized, labor-intensive endeavor, the information obtained from these investigations frequently addresses seemingly disparate biochemical data and invariably improves future research efforts. Significant progress has been made to improve access to the facility for the KCC community. For instance, a liquid handling robot now tests for crystallization feasibility with significantly less material and personal effort, making preliminary screening experiments cost effective. This has encouraged a number of KCC members to consider employing structural biology in their program. The Department of Biochemistry and Molecular Biology recognizes the significance of structural biology to the university, and has made an additional hire in structural biology at the assistant professor level. With the increase of users and the new hire, we anticipate that a number of new structures will be forthcoming, most with immediate relevance to molecular origin of cancer.
Equipment
The X-ray Crystallography Shared Resource is located in a dedicated 1200 square foot laboratory located on the 8th floor of the Bluemle Life Sciences Building.
- The facility houses two X-ray systems: a Rigaku RU200 generator with an R-AXIS II detector with MSC Blue Confocal Optics on the right port and a state of the art RAXIS IV++ detector with MSC Blue Confocal Optics on the left port, and a Oxford Diffraction UltraPX Ultra diffractometer with a 2.2 kW sealed tube X-ray source, a four circle diffractometer, a Onyx CCD camera and a Cryojet.
- In addition, the facility houses a Matrix Technologies HydraII plus 1 crystallization robot capable of 96 and 384 well trials and dispensing volumes as low as 200 nL.
- Incubators are multiple temperatures, microscopes with photographic capabilities, and crystal manipulation tools.
- The facility has several PCs running linux as well as a Mac dual G5. Each computer contains the relevant software for all aspects of structure determination.
Macromolecular X-ray Crystallography – Brian Bahnson, Ph.D., Chemistry/Biochemistry, UD
The X-ray Crystallography facility offers state-of-the-art instrumentation to solve structures of biological macromolecules from single crystals, and is open to university researchers and collaborators.
Equipment
- Rotating Anode X-ray Generator: Rigaku/MSC RU-H3R rotating anode X-ray generator offering a high brilliance 300 micron X-ray beam. The generator located in 314 Drake Hall is equipped with Osmic optics, which give the beam a more tightly focused and monochromatic beam.
- R-axis IV Image Plate Area Detector: Rigaku/MSC R-axis IV image plate area detector is located in 314 Drake Hall. The detector collects images that are 3,000 square pixels with a 100 micron pixel resolution. Data to a resolution of 1.4 Ã… has been collected on this X-ray system.
- X-stream Cryo-System for Data Collection at –180 °C: Data are typically collected under cryo conditions using this self-contained X-stream cryo-system that includes an air compressor, gas separation filter bank, He-refrigerated cryo system, and nozzle. Data is collected with an inverse phi, which allows easy mounting of liquid nitrogen frozen samples.
- Data Workstation: Dell Precision workstation (Linux Redhat 7.5, HKL2000, and CrystalClear software) for data processing.
The protein crystallography core is funded by a NIH COBRE (Center of Biomedical Research Excellence) grant from NIH (Abraham Lenhoff, Chemical Engineering, P.I.) Dr. Brian Bahnson, Core Director, works closely with multidisciplinary research groups to make optimal use of the macromolecular crystallography facilities for preliminary studies of specific research projects. Outside industry users will be accommodated on a fee-for-service basis when scheduling permits.
Structural Biology – Steve Bai, Ph.D., Chemistry/Biochemistry, UD
The NMR facility offers a suite of spectrometers in Brown Laboratory, and is open to university researchers and collaborators. Instruments are available for liquid-state and solid state NMR spectroscopy.
- Bruker AMX360 spectrometer (011A BRL). This spectrometer equipped with a 5-mm QNP probe and a 5-mm broadband probe. Variable temperature ranges from -70 C to +150 C.
- Bruker AV400 spectrometer (208 LDL). The AV400 is scheduled to be installed Spring 2005. AV400 will be equipped with an auto-sampler, which handles 60 samples. Remote access capability will facilitate use by other institutions of higher education in the region.
- Bruker DRX-400 spectrometer (021 BRL). This spectrometer equipped with a 5-mm QNP probe and a 5-mm broadband probe. Variable temperature ranges from -70 C to +150 C.
- Bruker AV600 spectrometer (015BRL). The AV600 spectrometer is equipped with a 5-mm inverse triple resonance CryoProbe, a conventional 5-mm inverse triple resonance probe (ATM PTXI probe), and a 5-mm broadband probe.
- Data Workstation: Dell Precision workstation (Linux Redhat 7.5 and Bruker xwinnmr 3.5) for off-line data processes
In addition, the facility provides spectrometer training and sample services to campus researchers, who may request instrument time or training via an online reservation system. Outside industry users will be accommodated on a fee-for-service basis when scheduling permits.