Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source

Classen, Scott; Hura, Greg L.; Holton, James M.; Rambo, Robert P.; Rodic, Ivan; McGuire, Patrick J.; Dyer, Kevin; Hammel, Michal; Meigs, G.; Frankel, Kenneth A.; Tainer, John A.

doi:10.1107/s0021889812048698

Cited by 225 publications

(206 citation statements)

References 74 publications

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“…Details of the SAXS data collection and processing to extract interlabel distance information are provided in SI Appendix, SI Methods. Briefly, all SAXS experiments were collected at the SIBYLS beamline (beamline 12.3.1) at the Advanced Light Source at Lawrence Berkeley National Laboratory, with an X-ray energy of 8 keV (54). The CSF was derived from the experimental scattering profile by fitting of the gold label scattering G(q), by an ellipsoid of revolution in the low q region, and by fitting a constant to ensure that the CSF oscillated about zero.…”

Section: Methodsmentioning

confidence: 99%

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

Hura

Tsai

Claridge

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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DNA metabolism and processing frequently require transient or metastable DNA conformations that are biologically important but challenging to characterize. We use gold nanocrystal labels combined with small angle X-ray scattering to develop, test, and apply a method to follow DNA conformations acting in the Escherichia coli mismatch repair (MMR) system in solution. We developed a neutral PEG linker that allowed gold-labeled DNAs to be flashcooled and stored without degradation in sample quality. The 1,000-fold increased gold nanocrystal scattering vs. DNA enabled investigations at much lower concentrations than otherwise possible to avoid concentration-dependent tetramerization of the MMR initiation enzyme MutS. We analyzed the correlation scattering functions for the nanocrystals to provide higher resolution interparticle distributions not convoluted by the intraparticle distribution. We determined that mispair-containing DNAs were bent more by MutS than complementary sequence DNA (csDNA), did not promote tetramer formation, and allowed MutS conversion to a sliding clamp conformation that eliminated the DNA bends. Addition of second protein responder MutL did not stabilize the MutS-bent forms of DNA. Thus, DNA distortion is only involved at the earliest mispair recognition steps of MMR: MutL does not trap bent DNA conformations, suggesting migrating MutL or MutS/MutL complexes as a conserved feature of MMR. The results promote a mechanism of mismatch DNA bending followed by straightening in initial MutS and MutL responses in MMR. We demonstrate that small angle X-ray scattering with gold labels is an enabling method to examine protein-induced DNA distortions key to the DNA repair, replication, transcription, and packaging. D NA is frequently considered a passive component in interactions with proteins involved in DNA metabolism. Despite this view, many proteins use DNA structural features to mediate catalysis and identify damaged DNA through the effects of damage on DNA rigidity and conformation (1-6). The view of DNA as a passive element is therefore at least in part due to a paucity of robust tools to examine dynamic DNA conformational states during multistep reactions. Gold-labeled DNA enables measurement over length scales sufficient to accommodate several proteins to identify cooperative effects on DNA. Because X-rays scatter predominantly from electrons, using heavy atom labels (7-11) provides high contrast relative to organic molecules. By using labels of moderate size (∼5 nm), the scattering from gold nanocrystals dominates all other scattering signals by three orders of magnitude, thereby reducing analysis complexity while minimizing nanocrystal influence on biological macromolecules. Importantly, small angle X-ray scattering (SAXS) provides global information on conformations adopted by a population of macromolecules in almost any solution condition (12)(13)(14).Mismatch repair (MMR) is an evolutionarily conserved process that corrects mismatches generated during DNA replication (15,16). Despite the i...

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Section: Methodsmentioning

confidence: 99%

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

Hura

Tsai

Claridge

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Small angle X-ray scattering data were collected at the SIBYLS beamline [38]. Data set on 15 ll sample volumes of purified hRNF4 RING domain.…”

Section: Saxs and Crystallographic Data Collection/analysismentioning

confidence: 99%

RNF 4 interacts with both SUMO and nucleosomes to promote the DNA damage response

et al. 2014

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The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining.

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“…Solution scattering data on WT NmSOD and BaSOD were collected at SIBYLS beamline 12.3.1 at the Advanced Light Source of Lawrence Berkeley National Laboratory (63). Both samples were subjected to size exclusion chromatography using a Superdex 75 10/300 GL column (GE Healthcare) prior to small-angle X-ray scattering (SAXS) analysis.…”

Section: Methodsmentioning

confidence: 99%

Structural, Functional, and Immunogenic Insights on Cu,Zn Superoxide Dismutase Pathogenic Virulence Factors from Neisseria meningitidis and Brucella abortus

et al. 2015

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Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen and general pathogenicity factors and are therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomic details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and of SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly, and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes, and suggest general targets for antibacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors of or vaccines against these harmful pathogens. IMPORTANCEBy protecting microbes against reactive oxygen insults, SODs aid survival of many bacteria within their hosts. Despite the ubiquity and conservation of these key enzymes, notable species-specific differences relevant to pathogenesis remain undefined. To probe mechanisms that govern the functioning of Neisseria meningitidis and Brucella abortus SODs, we used X-ray structures, enzymology, modeling, and murine infection experiments. We identified virulence determinants common to the two homologs, assembly differences, and a unique metal reservoir within meningococcal SOD that stabilizes the enzyme and may provide a safeguard against copper toxicity. The insights reported here provide a rationale and a basis for SOD-specific drug design and an extension of immunogen design to target two important pathogens that continue to pose global health threats.

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Implementation and performance of SIBYLS: a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline at the Advanced Light Source

Abstract: The SIBYLS beamline of the Advanced Light Source at Lawrence Berkeley National Laboratory is a dual endstation small-angle X-ray scattering and macromolecular crystallography beamline. Key features and capabilities are described along with implementation and performance.

Cited by 225 publications

References 74 publications

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

RNF 4 interacts with both SUMO and nucleosomes to promote the DNA damage response

Structural, Functional, and Immunogenic Insights on Cu,Zn Superoxide Dismutase Pathogenic Virulence Factors from Neisseria meningitidis and Brucella abortus

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