The X-ray Crystallography and Macromolecular Characterization Shared Resource Facility at the Kimmel Cancer Center (KCC), dedicated to supporting cutting edge structural and molecular biology research applied to cancer biology, is one of the 11 NCI-supported Core Facilities at Thomas Jefferson University (TJU). Because of its potential to enhance existing NIH-funded research programs and further develop research at TJU research centers, X-ray crystallography is one of the techniques identified by Dr. Tykocinski, our Dean of Jefferson Medical College, for strategic development of our research mission. Our existing two X-ray generators are between 11 and 22 years old, limiting our current ability to carry out cutting edge structural work. The older diffractometer (installed in Oct 1991) was decommissioned to users in Sept 2010 due to constant failure in the rotating anode. The younger unit, a sealed X-ray tube operational since Feb 2002, is also prone to frequent service failures, costly maintenance and provides a very weak X-ray beam. Therefore, as part of a cost-sharing plan to establish a state-of-the-art center for structural biology at TJU, we request funds to replace both aged diffractometers with a Rigaku MicroMax-007HF microfocus rotating anode X-ray generator dual equipped for data collection from macromolecular crystals and Small Angle X-Ray Scattering (SAXS). We have identified a group of 9 major core users and 2 minor users from both inside TJU and in nearby institutions who eagerly support this proposal. If funded, TJU will leverage this support with ~$280,000 in parts derived from our current Rigaku diffractometer, and funds supporting 30% of a Ph.D.-level facility manager, Dr. Anshul Bhardwaj. The KCC will also infuse four years of additional funds to cover the remaining 70% of Dr. Bhardwaj salary and equipment maintenance fees. In the planning stages, our preliminary tests indicated that the Rigaku MicroMax-007HF is ideally suited for our needs. This system allows us to re-utilize perfectly functional parts of our current generator (i.e. R-AXIS IV++ imaging plate area detector, XStream 2000 cryogenic system, etc.) in the left port, while the right port will host the new BioSAXS-1000 unit. With a unique 70 um circular focal spot, the MicroMax-007HF is perfectly suited for rapid in house screening of small crystals. Preliminary tests have also shown that the Rigaku BioSAXS-1000 unit provided SAXS data of comparable quality as beamline X9 at the National Synchrotron Light Source (NSLS). Additionally, the routine maintenance needs of the proposed Rigaku system are lower than that of our combined existing equipment. By increasing X-ray beam intensity and expanding our capabilities for in solution SAXS analysis, the proposed instrumentation will significantly enhance the competitiveness of current NIH-funded research projects and expand the body of users to our NCI-funded facility, with the ultimate goal of improving the quality of our biomedical research, and strengthening the liaison between basic research, cancer research and its clinical applications.
The X-ray Crystallography and Macromolecular Characterization Shared Resource Facility at the Kimmel Cancer Center, at Thomas Jefferson University is one of the 11 NCI-supported Core Facilities dedicated to developing cutting edge research in structural and molecular biology applied to cancer biology. The current crystallography instrumentation hosted in this core facility is between 11 and 22 years old and has shown an increase in down time and a decrease in performance, which hampers access by our facility users and limits the quality of acquired X-ray data. We request matching funds to acquire a hybrid X-ray home lab source for macromolecular crystallography and Small Angle X-ray Scattering (SAXS) that will be maintained as a shared resource, and integrated with the goals of our existing NCI-funded Cancer Center, whose primary mission is to improve public health.
|Lokareddy, Ravi K; Sankhala, Rajeshwer S; Roy, Ankoor et al. (2017) Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation. Nat Commun 8:14310|
|Wu, Wei; Sankhala, Rajeshwer S; Florio, Tyler J et al. (2017) Synergy of two low-affinity NLSs determines the high avidity of influenza A virus nucleoprotein NP for human importin ? isoforms. Sci Rep 7:11381|
|Sankhala, Rajeshwer S; Lokareddy, Ravi K; Begum, Salma et al. (2017) Three-dimensional context rather than NLS amino acid sequence determines importin ? subtype specificity for RCC1. Nat Commun 8:979|
|Bhardwaj, Anshul; Sankhala, Rajeshwer S; Olia, Adam S et al. (2016) Structural Plasticity of the Protein Plug That Traps Newly Packaged Genomes in Podoviridae Virions. J Biol Chem 291:215-26|
|Riccio, Amanda A; Cingolani, Gino; Pascal, John M (2016) PARP-2 domain requirements for DNA damage-dependent activation and localization to sites of DNA damage. Nucleic Acids Res 44:1691-702|
|Sankhala, Rajeshwer S; Lokareddy, Ravi K; Cingolani, Gino (2016) Divergent Evolution of Nuclear Localization Signal Sequences in Herpesvirus Terminase Subunits. J Biol Chem 291:11420-33|
|Khasnis, Mukta D; Halkidis, Konstantine; Bhardwaj, Anshul et al. (2016) Receptor Activation of HIV-1 Env Leads to Asymmetric Exposure of the gp41 Trimer. PLoS Pathog 12:e1006098|
|Kralt, Annemarie; Jagalur, Noorjahan B; van den Boom, Vincent et al. (2015) Conservation of inner nuclear membrane targeting sequences in mammalian Pom121 and yeast Heh2 membrane proteins. Mol Biol Cell 26:3301-12|
|Sun, Jim; Siroy, Axel; Lokareddy, Ravi K et al. (2015) The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD. Nat Struct Mol Biol 22:672-8|
|McNulty, Reginald; Lokareddy, Ravi Kumar; Roy, Ankoor et al. (2015) Architecture of the Complex Formed by Large and Small Terminase Subunits from Bacteriophage P22. J Mol Biol 427:3285-3299|
Showing the most recent 10 out of 12 publications