By Rambo and Tainer

Bridging the solution divide: comprehensive structural analysesof dynamic RNA, DNA, and protein

assemblies bysmall-angle X-ray scattering

By Rambo and Tainer

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.

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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

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

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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