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By Rambo and Tainer

Bridging the solution divide: comprehensive structural analyses of dynamic RNA, DNA, and protein assemblies by small-angle X-ray scattering. By Rambo and Tainer. Introduction. Importance of development of techniques that probe nucleic acid or protein-nucleic acid complex

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By Rambo and Tainer

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  1. Bridging the solution divide: comprehensive structural analysesof dynamic RNA, DNA, and protein assemblies bysmall-angle X-ray scattering By Rambo and Tainer

  2. Introduction • Importance of development of techniques that probe nucleic acid or protein-nucleic acid complex • Three Predominant Techniques Used in Structural Biology • Macromolecular X-ray Crystallography (MX) • Nuclear Magnetic Resonance (NMR) • Electron Microscopy (EM) • These techniques have limitations for macromolecules with functional flexibility and intrinsic disorder

  3. Instrumentation

  4. Sample Preparation • Stresses high purity, high homogeneity similar to crystallography • Amount needed is 15 μL with protein concentration ranging from 0.1- 10 mg/ml. • Typically 2-5 mg/ml is best higher concentration yields better signal but can lead to aggregation

  5. SAXS Theory • Three things to examine • SAXS profile in reciprocal and real space • Gunier Plot (not shown) • Kratky Plot

  6. SAXS Profile • Transformation of the scattering data I(q) yield P(r) a histogram of interatomic vectors • Calculate a structure based on a atomic resolution macromolecular structure

  7. Idealized Data • Measurements at different range of concentration • X-ray sensitivity can be detected by changes in scattering by repeat exposures

  8. Real Data • Raw shattering curves for all samples. 1st exposure. • See that with increasing concentration, sample is increased. Better signal at high concentration.

  9. Guinier Plot • Non linear dependence of log(I(q)) indicates presence of aggregation • Presence of aggregation means no data

  10. Gunier Real Data

  11. Radius of Gyration • Radius of gyration is calculated by taking I(0) at q= 0. • Needs to be compared against a set of standards

  12. Interparticle Interference • Increasing concentration can reveal concentration dependence • Visible in decrease in intensity at small q.

  13. P(r) Distribution in Real Space • From this distribution you can tell two things • Dmax • Some general information about shape

  14. Real Data Dmax = 110 Dmax = 115

  15. Kratky Plot • Kratky plot also is an indication of protein folding/ unfolding • Globular proteins macromolecules follow Porod’s law and are bell shaped

  16. Kratky Plot Real Data

  17. No Atomic Structure • Without previously known structure can still make shape prediction • Programs such as GASBOR and DAMMIF allow for low resolution structure

  18. Atomic Structure Solved • Calculate curve from known data and compare to experimental data • Disagreement • Investigate alternate states • Investigate mixture of states • Investigate flexibility

  19. Gasbor Ub-PCNA

  20. Conformation Assembly • Use of a variety of software to find best fit • X2 vs Rg gives good idea about entire ensemble

  21. SAM-I: Comparison of Crystal Against SAXS • Crystal structure was determined in presence of ligand and poorly fit SAXS data • SAXS guided hypothesis about conformational switching as mechanism

  22. Abscisic Acid Hormone Receptor PYR1 • Crystalized with open-lid and closed-lid conformations. • Crystal contacts show three possible dimers α-α, β-β, α-β • SAXS profile distinguishes between three conformations.

  23. VS Ribozyme Solution Structure • Ab initio modeling which lead to identificaiton of helical regions based on helical secondary structure • Resulting model was converted to residue specific model

  24. Erp72 Solution Structure • Parts previously solved by NMR and MX but solution structure unknown • Ab initio modeling allowed for putting together of parts into correct orientation in solution

  25. p53-Taz2-DNA complex • Parts had been solved previously • Core and tetramerization solved by MX • Taz2 by NMR • Used in rigid body analysis and protein with and without DNA to model

  26. Future of SAXS • Data analysis are contuining to be developed computational tools • Synchotron-based facilities can extend SAXS into high throughput region • Can answer fundamental questions in DNA repair, modeling of large multidomain macromolecular machines and suggests flexibility are criticical for biological funcitions.

  27. Questions? Froliche Weinachten! (Merry Christmas in German)

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