title: Quantifying Nucleic Acid Ensembles with X-ray Scattering Interferometry.
abstract: The conformational ensemble of a macromolecule is the complete description of the macromolecule's solution structures and can reveal important aspects of macromolecular folding, recognition, and function. However, most experimental approaches determine an average or predominant structure, or follow transitions between states that each can only be described by an average structure. Ensembles have been extremely difficult to experimentally characterize. We present the unique advantages and capabilities of a new biophysical technique, X-ray scattering interferometry (XSI), for probing and quantifying structural ensembles. XSI measures the interference of scattered waves from two heavy metal probes attached site specifically to a macromolecule. A Fourier transform of the interference pattern gives the fractional abundance of different probe separations directly representing the multiple conformation states populated by the macromolecule. These probe-probe distance distributions can then be used to define the structural ensemble of the macromolecule. XSI provides accurate, calibrated distance in a model-independent fashion with angstrom scale sensitivity in distances. XSI data can be compared in a straightforward manner to atomic coordinates determined experimentally or predicted by molecular dynamics simulations. We describe the conceptual framework for XSI and provide a detailed protocol for carrying out an XSI experiment.
authors.affiliation: Department of Biochemistry, Stanford University, Stanford, California, USA.
authors.forename: Xuesong
authors.initials: X
authors.lastname: Shi
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, California, USA.
authors.forename: Steve
authors.initials: S
authors.lastname: Bonilla
authors.affiliation: Department of Biochemistry, Stanford University, Stanford, California, USA. Electronic address: herschla@stanford.edu.
authors.forename: Daniel
authors.initials: D
authors.lastname: Herschlag
authors.affiliation: Department of Biochemistry, Stanford University, Stanford, California, USA. Electronic address: harbury@stanford.edu.
authors.forename: Pehr
authors.initials: P
authors.lastname: Harbury
mesh_terms: D004247:DNA / Q000737:chemistry*; D005583:Fourier Analysis; D005822:Genetic Vectors / Q000737:chemistry*; D006046:Gold / Q000737:chemistry; D007368:Interferometry / Q000295:instrumentation / Q000379:methods*; D053768:Metal Nanoparticles / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D015335:Molecular Probes / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D010957:Plasmids / Q000737:chemistry*; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction; D014965:X-Rays
chemical_list: D015335:Molecular Probes; D009841:Oligonucleotides; D006046:Gold; D004247:DNA
keywords: Au; DNA; Dynamics; Ensemble; Nucleic-acids; RNA; SAXS; X-ray interferometry; XSI