1. Methods for Structure Determination Chemistry and Chemical Biology Rutgers University

2. X-ray (X-ray crystallography) NMR (Nuclear Magnetic Resonance) EM (Electron Microscopy) Protein Data Bank Download How are macromolecular structures determined?

3. The Data Pipeline Genomic Based Target Selection Data Collection Structure Determination Isolation, Expression, Purification, Crystallization PDB Deposition & Release 3D Models Annotations Publications X-ray cryst NMR EM

4. Some Background Symmetry –Translation, Rotation, Reflection, Inversion Crystals –Lattice, Unit cell, Asymmetric Unit Diffraction –Light diffraction, X-ray diffraction

5. Translation M.C. Escher

6. Rotation M.C. Escher

7. Reflection M.C. Escher

8. ??? M.C. Escher

9. Crystals Mineral Protein

10. .... Lattice, Crystal and Unit cell............, lattice object,,,,,,,,,,,,,,,, Convolution Crystal structure,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, Unit Cell 1 Unit Cell 2

11. Macromolecular Crystal Lattice Alexander McPherson, Introduction to Macromolecular Crystallography Wiley-Liss, 2002

12. Unit Cell and Asymmetric Unit

13. Symmetry in Crystals 1-fold 2-fold 3-fold 4-fold 6-fold 4 1 3 2 6 5-, 7-, 8- and higher fold symmetries do not pack in a crystal

14. Crystal Systems Jenny Pickworth Glusker, Kenneth N. Trueblood, Crystal Structure Analysis: A Primer, Oxford University Press, 1985

15. The International Tables

16. Diffraction Sunrise through a screened window http://www.flickr.com/photos/fizzix/2458009067/in/photostream/

17. Light Diffraction Henry S. Lipson Crystals and X-rays Taylor & Francis 1970

18. Diffraction in Action http://mrsec.wisc.edu/Edetc/supplies/DNA_OTK/images/ABCH.mov

19. Principles of Microscopy

20. The Fourier Duck Fourier Transform Reverse Transform Reverse Transform with limited resolution data

21. Why Use X-rays? http://bccp.lbl.gov/Academy/wksp_pix_1/spectrum.gif

22. X-ray Diffraction Gale Rhodes, Crystallography Made Crystal Clear: A Guide for Users of Macromolecular Models, Academic Press, 1993

23. Miller Indices (hkl) For any plane in the unit cell with intercepts1/h, 1/k and 1/l along the x, y, and z axes the Miller indices are h,k,l If the resulting indices are fractions, multiply all to get integer numbers Intercepts : ½ a, a, ∞ Fractional intercepts : ½, 1, ∞ Miller Indices : (210) http://www.chem.qmul.ac.uk/surfaces/scc/scat1_1b.htm

24. http://www.bmsc.washington.edu/people/merritt/bc530/bragg/ try the Java Applet! n = 2d sin  2  angle between incident and reflected beams d spacing between planes wavelength n order of diffraction Bragg’s Law Constructive interference occurs from successive crystallographic planes (h, k, l) in the crystalline lattice

25. X-ray Diffraction Pattern Diffraction pattern is in reciprocal space Size and shape of unit cell determines position of diffraction peaks. Atomic positions within unit cell determines intensity of peaks. Experimental data: h,k,l and intensities (with errors) A precession photograph

26. Diffraction Patterns to Structure I hkl = constant.| F hkl | 2 Structure Factor  (x,y,z) = Σ F hkl e -2 π i (hx + ky +lz) Electron Density

27. Phase Problem Structure factor is dependent on type and location of atoms in unit cell The complete Structure Factor F for a reflection includes the phase, which cannot be measured directly. F hkl = | F hkl | e  i ϕ hkl Amplitude: from experimental measurements Amplitude: from experimental measurements Phase: must be estimated Phase: must be estimated Structure Factor

28. Electron Density Can be calculated by Fourier transform of diffraction data Provides an averaged image: –over all molecules in the crystal –over the time of the diffraction experiment Trp in a 1.3 A map Trp in a 2.25 A map Trp in a 4.3 A map

29. Microscopy vs X-ray Crystallography http://www.iucr.org/education/pamphlets/15/full-text

30. The X-ray Crystallography Pipeline Protein preparation Crystal growth Data collection Phase determination Model building and refinement

31. Protein Preparation Purify from natural sources: e.g. liver, muscle, leaf etc. Clone in appropriate vector Express in appropriate host – bacteria, yeast, mammalian cell lines, cell free extracts Purify target protein from cell lysate

32. Cover Slip Precipitant Solution Protein + Precipitant Crystal Growth: Vapor Diffusion Common precipitants: –Polyethylene glycol –Salts ammonium sulfate sodium chloride – Alcohols Isopropanol Methylpentanediol (MPD)

33. http://www-structmed.cimr.cam.ac.uk/Course/Crystals/ Theory/phase_methods.html Crystallization Conditions Crystallization Phase Diagram

34. Data Collection Rotating Anode Diffractometer Crystal mounted in nylon loop. Frozen in liquid N2 Crystal mounted in glass capillary

35. Synchrotron X-ray source http://www.nsls.bnl.gov NSLS Beamline X12C

36. Crystal Diffraction Low Resolution (small angle) Low Resolution (small angle) High Resolution (large angle) High Resolution (large angle) Beam Stop Shadow Water Ring ~3-5 Å Water Ring ~3-5 Å Jeff Dahl, Sars protease, http://en.wikipedia.org/wiki/File:X-ray_diffraction_pattern_3clpro.jpg

37. Different crystal forms of the same protein yield different diffraction patterns trp repressor, sodium phosphatetrp repressor, ammonium sulfate

38. Data Obtained Crystal unit cell dimensions Lattice type, possible space groups Resolution Limit Merged data set with index, intensity + error for each reflection a = 36.67 Å b = 79.39 Å c = 39.97 Å α = 90.0° ß = 91.25° γ = 90.0° Monoclinic lattice (P2 or P2 1 ) H K L intensity error 0 0 12 6714.3 347.2 0 0 18 -8.9 16.3 0 0 24 979.5 62.4 0 0 30 4136.4 272.5 1 0 3 3035.4 70.2 1 0 4 0.0 0.7 1 0 5 0.1 0.6 1 0 6 838.4 20.4 1 0 7 14903.0 535.6 1 0 8 2759.4 64.7 1 0 9 1403.5 31.0 1 0 10 109.4 5.6 1 0 11 31739.5 1611.5 1 0 12 231.9 7.6...etc. a = 36.67 Å b = 79.39 Å c = 39.97 Å α = 90.0° ß = 91.25° γ = 90.0° Monoclinic lattice (P2 or P2 1 ) H K L intensity error 0 0 12 6714.3 347.2 0 0 18 -8.9 16.3 0 0 24 979.5 62.4 0 0 30 4136.4 272.5 1 0 3 3035.4 70.2 1 0 4 0.0 0.7 1 0 5 0.1 0.6 1 0 6 838.4 20.4 1 0 7 14903.0 535.6 1 0 8 2759.4 64.7 1 0 9 1403.5 31.0 1 0 10 109.4 5.6 1 0 11 31739.5 1611.5 1 0 12 231.9 7.6...etc.

39. Direct methods –Estimate from probability relationships applied to most intense diffraction peaks Patterson methods –Multiple Isomorphous Replacement –Anomalous Dispersion Molecular replacement Density Improvement –Non-crystallographic symmetry averaging –Solvent flattening Phase Determination

40. Patterson Function Convolution of electron density with itself Evaluated at points u,v,w throughout unit cell Map of vectors between scattering atom in the real crystal cell (translated to Patterson origin) crystal Patterson map http://www.ruppweb.org/Xray/Patterson/Native_Patterson.htm

41. Isomorphous Replacement Derivative – native crystal = heavy atom Deriv. diffn – native diffn = heavy atom diffn Patterson synthesis > peaks based on distance between heavy atoms in structure gives initial phase. Real space Reciprocal space http://www.ruppweb.org/Xray/Phasing/Phasingt.html

42. Anomalous Dispersion Friedel’s Law: I hkl = I -h-k-l Members of a Friedel pair have equal amplitude and opposite phase In anomalous scattering crystals Friedel’s law is not obeyed http://skuld.bmsc.washington.edu/scatter/AS_wavechoice.html http://www.xtal.iqfr.csic.es/Cristalografia/parte_07_2-en.html

43. Molecular Replacement New structure expected to resemble one previously determined Use Patterson-based methods to find the orientation of known model in new crystal lattice (i.e. find rotation R and translation T) http://reference.iucr.org/dictionary/Molecular_replacement

44. Density Modification Improve map by adding additional “knowledge” Typical modifications: Molecular averaging Solvent Flattening Histogram Matching Image from C. Lawson

45. Calculate Map Edit model Refine Initial Model Experimental Data Stereochemical Knowledge Final Model Model Building-Refinement Cycle

46. Crystal Structures Lysozyme Hemoglobin Ribonuclease Myoglobin: Kendrew, Bodo, Dintzis, Parrish, Wyckoff, Phillips, Nature 181 662-666, 1958. Hemoglobin: Perutz, Proc. R. Soc. A265, 161-187,1962. Lysozyme: Blake, Koenig, Mair, North, Phillips, Sarma, Nature 206 757, 1965. Ribonuclease: Kartha, Bello, Harker, Nature 213, 862-865 1967. Wyckoff, Hardman, Allewell, Inagami, Johnson, Richards. J. Biol. Chem. 242, 3753-3757, 1967. Myoglobin

47. Structural Data PDB 3a6b -snip-

48. Types of Electron Density Maps Experimentally phased map: –F obs, Phi calc “model” map: –(2F obs – F calc ), Phi calc “difference” map –(F obs – F calc ) or (F obs – F obs ), Phi calc

49. R-factor Equation

50. R versus R free

51. Typical Statistical Table

52. Validation: Ramachandran Plot

53. Graphical Display and Model Fitting View maps and model together to: –Look at crystal contacts –assess map regions with unassigned density –assess model geometry problems –Build missing polymer residues –Add waters, ligands Image from C. Lawson

54. Some Movie Links Crystal Mounting Robot –http://www.youtube.com/watch?v=J4OD_b9XKh4 Crystal Diffraction –http://ucxray.berkeley.edu/~jamesh/movies/ Optical diffraction –http://mrsec.wisc.edu/Edetc/supplies/DNA_OTK/in dex.html Enjoy!

55. References IUCr Online dictionary of Crystallography –http://reference.iucr.org/dictionary/Main_Pagehttp://reference.iucr.org/dictionary/Main_Page Educational web sites and resources –http://www.iucr.org/education/resourceshttp://www.iucr.org/education/resources An interactive SF tutorial –http://www.ysbl.york.ac.uk/~cowtan/sfapplet/sf intro.htmlhttp://www.ysbl.york.ac.uk/~cowtan/sfapplet/sf intro.html