by rambo and tainer
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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Bridging the solution divide: comprehensive structural analysesof dynamic RNA, DNA, and protein
assemblies bysmall-angle X-ray scattering
By Rambo and Tainer
![Page 2: By Rambo and Tainer](https://reader035.vdocuments.net/reader035/viewer/2022081513/56814e5e550346895dbbfd52/html5/thumbnails/2.jpg)
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
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Instrumentation
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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
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SAXS Theory
• Three things to examine – SAXS profile in
reciprocal and real space
– Gunier Plot (not shown)
– Kratky Plot
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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
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Idealized Data
• Measurements at different range of concentration
• X-ray sensitivity can be detected by changes in scattering by repeat exposures
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Real Data
• Raw shattering curves for all samples. 1st exposure.
• See that with increasing concentration, sample is increased. Better signal at high concentration.
804/21/23
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Guinier Plot
• Non linear dependence of log(I(q)) indicates presence of aggregation
• Presence of aggregation means no data
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Gunier Real Data
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Radius of Gyration
• Radius of gyration is calculated by taking I(0) at q= 0.
• Needs to be compared against a set of standards
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Interparticle Interference
• Increasing concentration can reveal concentration dependence
• Visible in decrease in intensity at small q.
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P(r) Distribution in Real Space
• From this distribution you can tell two things– Dmax– Some general
information about shape
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Real Data
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Dmax = 110Dmax = 115
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Kratky Plot
• Kratky plot also is an indication of protein folding/ unfolding
• Globular proteins macromolecules follow Porod’s law and are bell shaped
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Kratky Plot Real Data
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No Atomic Structure
• Without previously known structure can still make shape prediction
• Programs such as GASBOR and DAMMIF allow for low resolution structure
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Atomic Structure Solved
• Calculate curve from known data and compare to experimental data
• Disagreement– Investigate alternate
states– Investigate mixture
of states– Investigate flexibility
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Gasbor Ub-PCNA
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Conformation Assembly
• Use of a variety of software to find best fit
• X2 vs Rg gives good idea about entire ensemble
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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
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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.
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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
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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
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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
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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.
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Questions?
Froliche Weinachten!
(Merry Christmas in German)
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