journal menu
abstracts
Group A streptococcus (GAS) is a significant human pathogen, renowned for its rapidly and highly desctructive ability to infect a wide variety of tissues. Clinical manifestation of GAS infection ranges from mild pharyngitis to severe life-threating disease such as necrotizing fasciitis. Unlike other gram-positive bacteria, GAS does not produce catalase, but has an ability to resist killing by reactive oxygen species through unknown novel mechanisms. Our previous studies have discovered that the peroxide response regulator (PerR) is crucial for GAS to cope with oxidative stress and it directly regulates the expression of an iron-binding protein Dpr [1,2]. PerR is a member of Fur (ferric uptake regulator) family which is known to be dimeric, metal-binding regulators. Currently, no structural information is available to understand how the similar structures of the Fur family regulators recognize divergent DNA sequences. To study how PerR interacts with dpr promoter DNA, we have conducted a series of mutagenesis, biochemical and structural studies by combining protein crystallography and small-angle X-ray scattering (SAXS). We have determined the PerR crystal structure to 1.6 Å resolution and identified the DNA-binding residues, which suggest PerR binds to the dpr promoter through a winged-helix motif. By performing SAXS studies, we confirmed that the PerR crystal structure reflects its conformation in solution. Furthermore, SAXS analysis allowed us to resolve the molecular architecture of PerR-DNA complex, in which two 30 bp DNA fragments wrap around two PerR homodimers by interacting with the adjacent positively-charged winged-helix motifs. Our results have revealed the PerR-DNA interaction model and illustrated the DNA-binding mode of PerR that is distinct from all other regulators in Fur family [3].
abstracts
The Molecular Biology Consortium Beamline 4.2.2 at the Advanced Light Source has recently installed a new, high-speed, large surface-area CMOS detector for macromolecular crystallography by funds from NIH grant S10OD012073. The detector is 25 x 28 cm and has a readout speed greater than 25 frames per second. This allows shutterless operations and the collection of fine-sliced data. Presented is a description of the hardware and software integration of the detector into the controls system, and the implementation of a streamlined and intuitive collection interface. The interface is implemented in TCL/TK for integration into the beamline's custom Blu-Ice/EPICS environment and remote operations of the beamline with the new detector are routine. The increased sensitivity of the detector and shutterless operations allow for shorter exposure times per image, and up to 3X decrease in time per dataset. Optimum data collection and processing strategies to maximize the benefits of shutterless operations are discussed.
