x-ray crystallography susan ahrens february 3, 2004
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X-Ray CrystallographyX-Ray Crystallography
Susan Ahrens
February 3, 2004
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OutlineOutline
X-ray crystallography reviewProblems in crystal formation
– Membrane Proteins
Diffraction Data– Bacteriorhodopsin– Bermuda grass pollen (BG60)
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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
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X-ray CrystallographyX-ray Crystallography
Diffraction patternsUse for determining protein structure
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Crystal FormationCrystal Formation
Proper crystal formation is imperative for accurate X-ray data
Soluble proteinsMembrane proteins
– Amphipathic nature poses problem
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Membrane proteinsMembrane proteins
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Membrane ProteinsMembrane ProteinsCrystallization strategy
– Form 2D crystals in the plane of the membrane– Type I
– Crystallize proteins within their detergent micelle
– Type II
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Membrane ProteinsMembrane Proteins
Ostermeier and Michel 1997
Detergents:
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Membrane ProteinsMembrane Proteins
Lipidic Cubic Phases
Rummel et. al 1997Landau and Rosenbusch 1996
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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
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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 °
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Crystallization, data collection Crystallization, data collection and processingand processing
Landau and Rosenbusch 1997
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Crystal packing along the c axis. The protein-protein interactions arelimited in extent and invovle mainly loops AB and BC.
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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
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Electron density maps (2Fobs-Fcalc) from x-ray analysis to 2.5Å resolution
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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
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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
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Refinement StatisticsRefinement Statistics
Number of Atoms Total
Protein
Retinal
Water
1798
1752
20
26
Rcrys (%) 22.4
Rfree (%) 24.5Belrhali et al. 1999
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Electron-density map to 1.9Å
Overall view of bR
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Electron density maps assigned to lipid molecules.