X-Ray Crystallography Susan Ahrens February 3, 2004

Outline X-ray crystallography review Problems in crystal formation – Membrane Proteins Diffraction Data – Bacteriorhodopsin – Bermuda grass pollen (BG60)

X-ray Crystallography Protein crystals – Symmetry and optical properties Exposure to X-rays – Beam of high energy electrons strike a target – Wavelength expressed in Å – Sources include: Chromium, Iron, Cobalt, Copper, Molybdenum

X-ray Crystallography Diffraction patterns Use for determining protein structure

Crystal Formation Proper crystal formation is imperative for accurate X-ray data Soluble proteins Membrane proteins – Amphipathic nature poses problem

Membrane proteins

Membrane Proteins Crystallization strategy – Form 2D crystals in the plane of the membrane – Type I – Crystallize proteins within their detergent micelle – Type II

Membrane Proteins Ostermeier and Michel 1997 Detergents:

Membrane Proteins Lipidic Cubic Phases Rummel et. al 1997 Landau and Rosenbusch 1996

Bacteriorhodopsin Proton pump that converts energy of light into a proton gradient that drives ATP synthesis Characterized by Landau and Rosenbusch in 1997 and 1999 Trimer of 7 transmembrane helices

Methods of Crystallization-1997 Crystallized from a monoglyceride-based cubic phase Hexagonal plate-like crystals – x x 15  m³ Microfocus beamline ID13 was used Space group=P63 Unit cell= – a=b=61.76Å, c=104.13Å –  =  =90° and  =120 °

Crystallization, data collection and processing Landau and Rosenbusch 1997

Crystal packing along the c axis. The protein-protein interactions are limited in extent and invovle mainly loops AB and BC.

bR trimers in the (a,b) plane solid lines= cytoplasmic side dashed lines= extracellular side A helices of a bR monomer perpendicular to the membrane plane. Green= x-ray analysis Red= EM

Electron density maps (2F obs -F calc ) from x-ray analysis to 2.5Å resolution

Methods of Crystallization 1999 Crystallized from a monoolein-based lipidic cubic phase Hexagonal plate-like crystals – 80 x  m³ Collimated beam was used (beamline ID14- EH3) – Crystals diffracted beyond 1.9 Å

Data Collection Space groupP6 3 Cell parameter (Å) a,b,c 60.8, 60.8, Resolution limits (Å) Total # of reflections97,807 # of unique reflections17,996 Completeness (%)99.5 Redundancy5.4 R sym * (%)4.6 Belrhali et al. 1999

Refinement Statistics Number of Atoms Total Protein Retinal Water R crys (%)22.4 R free (%)24.5 Belrhali et al. 1999

Electron-density map to 1.9Å Overall view of bR

Electron density maps assigned to lipid molecules.