1. X-Ray Crystallography Susan Ahrens February 3, 2004

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

3. 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

4. X-ray Crystallography Diffraction patterns Use for determining protein structure

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

6. Membrane proteins

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

8. Membrane Proteins Ostermeier and Michel 1997 Detergents:

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

10. 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

11. Methods of Crystallization-1997 Crystallized from a monoglyceride-based cubic phase Hexagonal plate-like crystals – 20-40 x 20-40 x 15  m³ Microfocus beamline ID13 was used Space group=P63 Unit cell= – a=b=61.76Å, c=104.13Å –  =  =90° and  =120 °

12. Crystallization, data collection and processing Landau and Rosenbusch 1997

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

14. 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

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

16. Methods of Crystallization 1999 Crystallized from a monoolein-based lipidic cubic phase Hexagonal plate-like crystals – 80 x 80 15  m³ Collimated beam was used (beamline ID14- EH3) – Crystals diffracted beyond 1.9 Å

17. Data Collection Space groupP6 3 Cell parameter (Å) a,b,c 60.8, 60.8,110.52 Resolution limits (Å)38-1.9 Total # of reflections97,807 # of unique reflections17,996 Completeness (%)99.5 Redundancy5.4 R sym * (%)4.6 Belrhali et al. 1999

18. Refinement Statistics Number of Atoms Total Protein Retinal Water 1798 1752 20 26 R crys (%)22.4 R free (%)24.5 Belrhali et al. 1999

19. Electron-density map to 1.9Å Overall view of bR

20. Electron density maps assigned to lipid molecules.