Structural Genomics aims in identifying as many new folds as possible.

1. Macromolecular Crystallography and Structural Genomics – Recent TrendsProf. D. VelmuruganDepartment of Crystallography and BiophysicsUniversity of MadrasGuindy Campus, Chennai – 25.

2. Structural Genomics aims in identifying as many new folds as possible. • This eventually requires faster ways of determining the three dimensional structures as there are many sequences before us for which structural information is not yet available. • Although Molecular Replacement technique is still used in Crystallography for solving homologous structures, this method fails if there is not sufficient percentage of homology. • The Multiwavelength Anomalous Diffraction (MAD) techniques have taken over the conventional Multiple Isomorphous Replacement (MIR) technique.

3. With the advent of high energy synchrotron sources and powerful detectors for the diffracted intensities, developments in methodologies of macromolecular structure determination, there is a steep increase in the number of macromolecular structures determined and on an average eight new structures are deposited in the PDB every day and the total entries in the PDB is now around 29,000. • Instead of using the three wavelength strategies in MAD experiments, the use of single wavelength anomalous diffraction using Sulphur anomalous scattering is recently proposed. This will reduce the data collection time to 1/3rd. • Also, the judicious use of the radiation damage during redundant data measurements in second generation synchrotron source and also during regular data collection in the third generation synchrotron source has been pointed out recently (RIP & RIPAS).

4. Protein Structure Determination • X-ray crystallography • NMR spectroscopy • Neutron diffraction • Electron microscopy • Atomic force microscopy

5. As the number of available amino acid sequences exceeds far in number than the number of available three-dimensional structures, high-throughput is essential in every aspect of X-ray crystallography.

7. Procedure Protein Crystal

9. The 14 Bravais lattices 2: Monoclinic 1: Triclinic (Blue numbers correspond o the crystal system)

10. The 14 Bravais lattices 3: Orthorhombic (Blue numbers correspond to the crystal system)

11. The 14 Bravais lattices 4: Rhombohedral 5: Tetragonal 6: Hexagonal (Blue numbers correspond to the crystal system)

12. The 14 Bravais lattices 7: Cubic (Blue numbers correspond to the crystal system)

15. Synchrotron radiation More intense X-rays at shorter wavelengths mean higher resolution & much quicker data collection

18. Diffraction Apparatus

19. Diffraction Principles nl = 2dsinq

20. The diffraction experiment

21. The amplitudes of the waves scattered by an atom to that of an single electron– atomic scattering factor The amplitude of the waves scattered by all the atoms in a unit cell to that of a single electron (The vector (amplitude and phase) representing the overall scattering from a particular set of Bragg planes) | Fhkl | – structure factor The structure factor magnitude F(hk/) is represented by the length of a vector in the complex plane. The phase angle a(hk/) is given by the angle. measured counterclockwise, between the positive real axis and the vector F.

22. unit cell F (h,k,l) = Vx=0 y=0 z=0 (x,y,z).exp[2I(hx + ky + lz)].dxdydz A reflection electron density V = the volume of the unit cell |Fhkl| = the structure-factor amplitude (proportional to the square-root of reflection intensities) ahkl = the phase associated with the structure-factor amplitude |Fhkl|We can measure the amplitudes, but the phases are lost in the experiment. This is the phase problem.

24. Fourier Transform requires both structure factors and phases Electron density calculation Σ Σ Σ ρ α π Unknown

28. Patterson function • Patterson space has the same dimension as the real-space unit cell • The peaks in the Patterson map are expressed in fraction coordinates • To avoid confusion, the x, z and z dimensions of Patterson vector-space are called (u, v, w).

29. What does Patterson function represent? • It represents a density map of the vectors between scattering atoms in the cell • Patterson density is proportional to the squared term of scattering atoms, therefore, the electron rich, i.e., heavy atoms, contribute more to the patterson map than the light atoms.

30. Patterson function – no phase info required Consider phaseless term (h, k, l, F2) Σ Σ Σ P No phase term

31. Patterson map Direct space Density and position Patterson map Fourier transformation Fourier transformation Amplitudes and phases Intensities Reciprocal space

32. Patterson map symmetry Patterson map with symmetry Harker vectors u, v, w 2x, 1/2, 2z P21 x, y, z -x, y+1/2, -z

35. Diffracting a Cat Diffraction data with phase information Real Diffraction Data

36. Reconstructing a Cat FT Easy FT Hard

37. The importance of phases

38. Phasing Methodsall assume some prior knowledge of the electron density or structure

39. The Phase Problem • Diffraction data only records intensity, not phase information (half the information is missing) • To reconstruct the image properly you need to have the phases (even approx.) • Guess the phases (molecular replacement) • Search phase space (direct methods) • Bootstrap phases (isomorphous replacement) • Uses differing wavelengths (anomolous disp.)

40. Acronyms for phasing techniques • MR • SIR • MIR • SIRAS • MIRAS • MAD • SAD

41. Direct methods • Based on the positivity and atomicity of electron density that leads to phase relationships between the (normalized) structure factors (E). • Used to solve small molecules structures • Proteins upto ~1000 atoms, resolution better than 1.2 Å • Used in computer programs (SnB, SHELXD SHARP) to find heavy-atom substructure. Jerome Karle and Herbert A. Hauptman Nobel prize 1985 (chemistry)

42. Dm cycle Density modification procedures (e.g. solvent flattening and averaging) can be carried out as part of a cyclic process

43. Molecular Replacement (MR) Used when there is a homology model available (sequence identity > 25%). 1. Orientation of the model in the new unit cell (rotation function) 2. Translation

44. Molecular Replacement (MR) New Protein Coordinates in PDB • MR works because the Fourier transform works in both directions. • Reflections model (density) • Have to be careful of model bias MR solution

45. Isomorphous replacement • Why isomorphous replacement, making heavy atom derivatives? • Phase determination • Calculating FH FH= FPH-FP If HA position is known, FH can be calculated from ρ(xH, yH, zH) by inverse FT • HA position determination – Patterson function

49. HA shifts FP by FH

50. Isomorphous Replacement (SIR, MIR) • Collect data on native crystals (no metals) • Soak in heavy metal compounds into crystals, go to specific sites in the unit cell. • e.g. Hg, Pt, Au compounds • The unit cell must remain isomorphous • Collect data on the derivatives • As a result, only the intensity of the reflections changes but not the indices • Measure the reflection intensity differences between native and derivative data sets. • Find the position of the heavy atoms in the unit cell from the intensity differences. • generate vector maps (Patterson maps) • |FP + HA| – |FP| = |FHA| • Must have at least two heavy atom derivatives • The main limitations in obtaining accurate phasing from MIR is non isomorphism and incomplete incorporation (low occupancy) of the heavy atom compound. Native and heavy-atom derivative diffraction patterns superimposed and shifted vertically. Note:intensity differences for certain reflections. Note: the identical unit cell (reflection positions). This suggests isomorphism.