X-Ray CrystallographyX-Ray Crystallography

Susan Ahrens

February 3, 2004

OutlineOutline

X-ray crystallography reviewProblems in crystal formation

– Membrane Proteins

Diffraction Data– Bacteriorhodopsin– Bermuda grass pollen (BG60)

X-ray CrystallographyX-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 CrystallographyX-ray Crystallography

Diffraction patternsUse for determining protein structure

Crystal FormationCrystal Formation

Proper crystal formation is imperative for accurate X-ray data

Soluble proteinsMembrane proteins

– Amphipathic nature poses problem

Membrane proteinsMembrane proteins

Membrane ProteinsMembrane ProteinsCrystallization strategy

– Form 2D crystals in the plane of the membrane– Type I

– Crystallize proteins within their detergent micelle

– Type II

Membrane ProteinsMembrane Proteins

Ostermeier and Michel 1997

Detergents:

Membrane ProteinsMembrane Proteins

Lipidic Cubic Phases

Rummel et. al 1997Landau and Rosenbusch 1996

BacteriorhodopsinBacteriorhodopsin 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-1997Methods 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 °

Crystallization, data collection Crystallization, data collection and processingand processing

Landau and Rosenbusch 1997

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

bR trimers in the (a,b) planesolid lines= cytoplasmic sidedashed lines= extracellular side

A helices of a bR monomerperpendicular to the membraneplane.Green= x-ray analysisRed= EM

Electron density maps (2Fobs-Fcalc) from x-ray analysis to 2.5Å resolution

Methods of Crystallization 1999Methods 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

Data CollectionData CollectionSpace group P63

Cell parameter (Å)a,b,c

60.8, 60.8,110.52

Resolution limits (Å) 38-1.9

Total # of reflections 97,807

# of unique reflections 17,996

Completeness (%) 99.5

Redundancy 5.4

Rsym* (%) 4.6Belrhali et al. 1999

Refinement StatisticsRefinement Statistics

Number of Atoms Total

Protein

Retinal

Water

1798

1752

20

26

Rcrys (%) 22.4

Rfree (%) 24.5Belrhali et al. 1999

Electron-density map to 1.9Å

Overall view of bR

Electron density maps assigned to lipid molecules.