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X-ray crystallography – an overview (based on Bernie Brown’s talk, Dept. of Chemistry, WFU)

X-ray crystallography – an overview (based on Bernie Brown’s talk, Dept. of Chemistry, WFU). Protein is crystallized (sometimes low-gravity atmosphere is helpful e.g. NASA) X-Rays are scattered by electrons in molecule

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X-ray crystallography – an overview (based on Bernie Brown’s talk, Dept. of Chemistry, WFU)

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  1. X-ray crystallography – an overview(based on Bernie Brown’s talk, Dept. of Chemistry, WFU) • Protein is crystallized (sometimes low-gravity atmosphere is helpful e.g. NASA) • X-Rays are scattered by electrons in molecule • Diffraction produces a pattern of spots on a film that must be mathematically deconstructed • Result is electron density (contour map) – need to know protein sequence and match it to density • Hydrogen atoms not typically visible (except at very high resolution)

  2. X-ray Crystallography – in a nutshell REFLECTIONS h k l I σ(I) 0 0 2 3523.1 91.3 0 0 3 -1.4 2.8 0 0 4 306.5 9.6 0 0 5 -0.1 4.7 0 0 6 10378.4 179.8 . . . Bragg’s law Fourier transform ? Phase Problem ? MIR MAD MR Electron density: r(x y z) = 1/V SSS |F(h k l)| exp[–2pi (hx + hy + lz) + ia(h k l)]

  3. Crystal formation • Start with supersaturated solution of protein • Slowly eliminate water from the protein • Add molecules that compete with the protein for water (3 types: salts, organic solvents, PEGs) • Trial and error • Most crystals ~50% solvent • Crystals may be very fragile

  4. Visible light vs. X-raysWhy don’t we just use a microscope to look at proteins? • Size of objects imaged limited by wavelength. Resolution ~ l/2 • Visible light – 4000-7000 Å (400-700 nm) • X-rays – 0.7-1.5 Å (0.07-0.15 nm) • It is very difficult to focus X-rays (Fresnel lenses) • Getting around the problem • Defined beam • Regular structure of object (crystal) • Result – diffraction pattern (not a focused image).

  5. Diffraction pattern – lots of spots Bragg’s Law: 2d sinq = nl X-ray beam crystal ~1015 molecules/crystal Diffraction pattern is amplified Film/Image plate/CCD camera

  6. End result – really!Fourier transform of diffraction spots  electron density  fit a.a. sequence DNA pieces Protein (Dimer of dimers)

  7. Interference of waves • In crystallography, get intensity information only, not phase information • Need to deconvolute and obtain phase information: • THE PHASE PROBLEM

  8. How to get from spots to structure? • Fourier synthesis • Getting around phase problem • Trial and error • Previous structures • Heavy atom replacement – make a landmark • Ex: Selenomethionine • Plenty of computer algorithms now

  9. Electron density with incorrect phases • Red is true structure

  10. The effect of resolutionMore extensive diffraction pattern gives more structural information = higher resolution • 6.0-4.5 Å – secondary structure elements • 3.0 Å – trace polypeptide chain • 2.0 Å – side chain, bound water identification • 1.8 Å – alternate side chain orientations • 1.2 Å – hydrogen atoms

  11. With computational tools, spots become density Flexible regions give smeared density, often 2-3 conformations visible, more than that invisible

  12. Density becomes structure Need to know protein sequence to trace backbone

  13. Co-crystal structures • Because of relatively high solvent content, can often “soak in” substrate • Then can solve structure of protein with substrate bound • If crystal cracks, good sign that substrate binding or enzyme catalysis results in conformational change in protein • No longer has same crystal arrangement

  14. NMR vs. crystallography • Useful for different samples • Generally good agreement • E. coli thioredoxin: NMR X-ray Note missing region

  15. Known protein structures • ~17,000 protein structures since 1958 • Common depository of x,y,z coordinates: Protein data bank (http://www.rcsb.org) • Coordinates can be extracted and viewed • Comparisons of structures allows identification of structural motifs • Proteins with similar functions and sequences = homologs

  16. Growth in structure determination

  17. Function from structure • Might identify a pocket lined with negatively-charged residues • Or positively charged surface – possibly for binding a negatively charged nucleic acid • Rossmann fold – binds nucleotides • Zinc finger – may bind DNA

  18. Domain organization • Large proteins have polypeptide regions that fold in isolation • May have distinct functional roles • Example: glyceraldehyde-3-phosphate dehydrogenase

  19. Protein families • Similar function and overall structure • But amino acid sequence may or may not be highly conserved • Limited number of protein domains • Homologs versus structural motifs

  20. Class Folds Superfamilies Families All a 171 286 457 All b 119 234 418 Alpha & beta (a/b) 117 192 501 Alpha & beta (a+b) 224 330 532 Multi-domain proteins 39 39 50 Membrane /cell-surface proteins 34 64 128 Small proteins 61 87 135 Total 765 1232 2164 SCOP Classification Statistics Structural Classification of Proteins 18946 PDB Entries, 49497 Domains (1 March 2002) (excluding nucleic acids and theoretical models) http://scop.berkeley.edu/ or http://scop.mrc-lmb.cam.ac.uk/scop/

  21. Have all folds been found? Red = Old folds Blue = New folds

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