Small angle X-ray scattering from biological macromolecules in - PowerPoint PPT Presentation

Small angle X-ray scattering from biological macromolecules in solution Al Kikhney

European Molecular Biology Laboratory Austria Belgium Croatia Denmark Finland France Hamburg/DESY Germany • EBI/Hinxton Greece • Iceland Heidelberg Ireland • Israel Italy Luxembourg Grenoble/ESRF Netherlands • Norway Portugal Monterotondo Spain • Sweden Switzerland United Kingdom + Australia Melbourne ↓

DESY, Hamburg, Germany Since 1974 EMBL Macromolecular crystallography DORIS III Small angle X-ray scattering 2 MX beamlines 1 BioSAXS beamline Meyer-Klaucke team

Biological SAXS at EMBL

Biological SAXS at EMBL Group leader: Dmitri Svergun Staff: M. Petoukhov, C. Blanchet, D. Franke, A. Kikhney Staff with joint appointments: P. Konarev Postdocs: M. Gräwert, C. Jeffries, G. Schenk, A. Spilotros, A. Tuukkanen PhD students: G. Tria, M. Kachala, E. Valentini Trainees: M. Franklin, D. Ruskule Major Tasks  Running EMBL SAXS beamline P12 at Petra-III  User support and collaborative projects  Development of data analysis methods  Education and training (including regular courses)

Small Angle X-ray Scattering solution X- rays → 2 θ s Homogeneous and monodisperse solution X-ray detector solvent |s| = 4π sinθ / λ s – scattering vector 1-2 mg purified material 2θ – scattering angle concentration from 0.5 mg/ml, λ – wavelength exposure times: a few seconds/minutes I(s) – intensity

Small Angle X-ray Scattering Exposure beamstop X-ray detector

Small Angle X-ray Scattering Exposure beamstop

Small Angle X-ray Scattering Exposure Log I(s) a.u. s, nm -1

Shape and size Log I(s) lysozyme a.u. apoferritin s, nm -1

SAXS studies of biological macromolecules R g MM Volume Shape Rigid body modelling Missing fragments Oligomeric Flexible mixtures systems

SAXS studies of biological macromolecules R g MM Volume ATSAS Shape Rigid body modelling software package Missing www.embl-hamburg.de/biosaxs/software.html fragments Oligomeric Flexible mixtures systems

solution solution Crystal vs.

solution solution Crystal vs.

solution Crystal vs. • Thousands of reflections • A few Shannon channels • 3D, high resolution • 1D, low resolution

solution Crystal vs. SAXS Not limited by molecular mass No need to grow crystals Applicable under nearly any physiological conditions No crystallographic packing forces are present Observe responses to changes in conditions Quantitative analysis of complex systems and processes

Problems • Sample preparation • Experiment • Data processing • Unambiguous interpretation

Outline • SAXS experiment • Primary data reduction and analysis • Overall parameters • Ab initio modelling • Complementary methods • Rigid body modelling • Mixtures and flexible systems