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By Rambo and Tainer

Explore comprehensive structural analyses using SAXS in bridging the gap between existing techniques for studying nucleic acids and proteins assemblies. Learn about theory, data analysis, and future applications of SAXS in biological research.

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By Rambo and Tainer

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Presentation Transcript

  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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