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Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15X-ray crystallographyOverview of imagingBragg’s lawLearned two things from Bragg’s LawSlide 20Slide 21The electron density equation and the phase problemX-ray detectionSynchrotron x-ray sources1Bi 1 Lecture 3 Thursday, March 30, 2006What is a Receptor? Receptors and Ion Channels as Examples of Proteins2receptorMost drug receptors are proteins.a molecule on the cell surface or in the cell interior that has an affinity for a specific molecule (the ligand).Latin, “to tie”Greek, “first”3 side chains“peptide”oramide bondslink the“backbone”or“main chain”or “-carbons”Little Alberts Figure 2-22© Garland publishingshortest: 9longest: 550020 types4  helices  sheetshttp://www.its.caltech.edu/~lester/Bi-1/alpha-helix-alphabetical.pdbhttp://www.its.caltech.edu/~lester/Bi-1/beta-sheet-antiparallel.pdbProteins contain a few structural motifs: Hide side chainsShow H-bonds and distancesShow ribbons & arrowsShow side chainsShow Van der Waals radii(Swiss-prot viewer must be installed on your computer)5nicotinic acetylcholine receptorMost drug receptors are membrane proteinsOutside the cellInside the cell = cytosol(view in ~1995)natural ligand(agonist)nicotine, another agonistMembrane = lipid bilayer~ 100 Å= 10 nm6 Overall topology of the nicotinic acetylcholine receptor(view in ~2000)outside the cell:5 subunitseach subunit has 4 -helices in the membrane (20 membrane helices total)Little Alberts figure 12-42© Garland publishingBinding Region7 The acetylcholine binding protein (AChBP) from a snail, discovered in 2001, strongly resembles the binding region(Swiss-prot viewer must be installed on your computer)Color by chainShow 2 subunits,Chains,Ribbons5 subunitsLittle Alberts figure 12-42© Garland publishinghttp://www.its.caltech.edu/~lester/Bi-1/AChBP+Carb-5mer.pdb8http://www.its.caltech.edu/~lester/Bi-1-2004/AChBP-2004-BindingSite.pdbThe AChBP binding site occupied by an acetylcholine analog (2004)http://www.its.caltech.edu/~lester/Bi-1/AChBP-2004-BindingSite.pdb9Binding regionMembrane regionCytosolicregionColored by secondary structureColored by subunit(chain)Nearly Complete Nicotinic Acetylcholine Receptor (February, 2005)http://pdbbeta.rcsb.org/pdb/downloadFile.do?fileFormat=PDB&compression=NO&structureId=2BG9~ 2200 amino acids in 5 chains (“subunits”), MW ~ 2.5 x 10610 How the binding of agonist (acetylcholine or nicotine) might open the channel: June 2003 viewM2M1M3M4Ligand-bindingregion11Nicotinic acetylcholine receptorMost drug receptors are membrane proteinsSome drugs bind on the axisSome drugs compete with nicotine or acetylcholinemembraneregion12ProteinLecture #Ligand-gated Ion Channels3 (today)Pumps and transporters 5, 13Motors 10G protein-coupled receptors and G proteins 12Enzymes 13, 15DNA-binding proteins 18RNA polymerase, ribosome 18Cystic Fibrosis Transmembrane Regulator 20Rhodopsin 26All I really need to know about lifeI learned in Bi 11. If you want a job done right, get a protein13Protein structure prediction: An important 21st-century problemWant to test your own skill at predicting protein structure?Then enter “Critical Assessment of Techniques for Structure Prediction”or CASP 7http://predictioncenter.org/Winners earn an automatic “A+” in Bi1 (retroactively, if appropriate)14Protein Folding vs. “Inverse Folding” = Computational Protein DesignProtein Folding(no degeneracy)Inverse Folding(large degeneracy)Set of AllStructuresSet of AllSequencesIndividualamino acidsSeveral ways to make an arch15X-ray CrystallographyCrystal GrowthX-ray DataElectron DensityProtein Modelhttp://www.search.caltech.edu/CIT_People/action.lasso?-database=CIT_People&-response=Detail_Person.html&-layout=all_fields&person_id=29067&-searchBi 1 Cameo by Professor Pamela J. Bjorkman16X-ray crystallography•Why X-rays?Right wavelength to resolve atoms•Why crystals?Immobilize protein, enhance weak signal from scattering•What is a protein crystal?Large solvent pathways (20-80% solvent)Same density as cytoplasmEnzymes active in crystals•Are crystal structures valid compared with solution structures?Usually -- Compare NMR and X-ray structuresStructures correlate with biological functionMultiple crystal forms look same -- small effects of packing17Overview of imagingNo lens to refocus X-rays, so must understand reciprocal space and diffractionDiffraction:Scattering followed by interference18Bragg’s lawConsider simultaneous reflection of a large number of x-rays. See diffraction maximum in direction only if diffracted waves are in phase. Path difference (2dsin) must represent an integral number of wavelengths to get constructive interference.19Learned two things from Bragg’s Law•sin = n/2 x 1/dLow angle: large interplanar spacingHigh angle: small interplanar spacingSince sin  1/d, structures with large interplanar spacings (d) will have diffraction patterns with small spacings and vice-versa.•Repeating unit in real space (crystal) --> diffraction maxima and minima20Same molecular transform sampled by different latticesModified from Lipson & Taylor, 1964a) Molecular transform b) Latticed - f ) Same molecular transform sampled by different latticesc) Convolution of lattice and transform21ResolutionFrom Harburn, Taylor, Wellbery, An Atlas of Optical TransformsAn inverse Fourier transform (FT) including all of the high angle information gives back the original image.An inverse FT including only the low angle information gives back a low resolution view of Mickey.22The electron density equation and the phase problem•There are experimental methods for determining the phase for each reflection hkl.(xyz) 1Vhk| F(hkl) | exp[(  2i(hk  ky  lz )  ihkl]lCan measure this|F(hkl)|=I1/2Can’t measure this23X-ray detection•Film (relic of the past)•Diffractometers (almost relic of past, but used for small molecules)•Multiwire detectors (almost relic of past)•Phosphorimager detectors (R-AXIS, MAR)•CCD detectors24Synchrotron x-ray sources•High-intensity x-ray emitted by charged particles accelerated in a curved path•X-ray wavelength in range of 0.5 - 2 Å (from E=h=hc/)•Is tunable!!Radiation emitted by accelerating charged particle tangent to path of


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