MAD/SAD: Is simpler better?

1. MAD/SAD:Is simpler better? Howard Robinson Brookhaven Biology Brookhaven Biology

2. We know that our crystallographic methods are able to provide us with remarkably large and complex structures. Brookhaven Biology

3. Often the electron density is accurate enough that one can find the precise position of atomsto give important chemical information. Brookhaven Biology

4. MAD map of Zn Alcohol Dehydrogenase (from Solve/Resolve, 2000) 9 hours 3 wavelengths 2.2Å x12B Brookhaven Biology

5. Questions? • Why have reports of MAD phasing vanished from the literature? • Where are the Sulfur SAD phasing reports?

6. How do we determine these structures? • Protein gives crystals • Crystals and x-rays give diffraction • Diffraction amplitudes plus phases give electron density

7. What’s more important, Amplitudes or Phases Jerome Karle Herbert Hauptman Herb’s amplitudes Jerry’s phases Jerry’s amplitudes Herb’s phases Randy Read, used by permission Brookhaven Biology

8. The goals of this lecture • Describe various ways of determining diffraction phase • Discuss isomorphous replacement • Show effect of anomalous dispersion • Show parallels between MAD and Isomorphous Replacement • Describe usefulness of SAD

9. Some sources of intelligence about crystallography and phasing (Go to wikipedia: x-ray crystallography) Brookhaven Biology

10. If crystal diffraction is good enough, one can determine structure directly from diffraction amplitudes. Popularized for proteins by especially Chuck Weeks, Hauptman, and Sheldrick. Direct Methods Brookhaven Biology

11. If the unknown molecule is similar enough to a structure that is already known, one can use Molecular Replacement to supply phases. Compare diffraction intensities from the crystal with calculated intensities from a model positioned within unit cell, and then use the model’s phases and the crystal’s amplitudes to calculate a map Brookhaven Biology

12. More generally, with large molecules like proteins, one must employ scattering from a few HEAVY atoms in a molecule to gain phase information. • Isomorphous Replacement – add a few heavy atoms, diffract with and without • MAD – add heavy atoms and exploit wavelength dispersion of anomalous and in-phase scattering • SAD – similar to MAD, but use only anomalous scattering at a single energy

13. We understand how the scattering from individual atoms combines to give a diffracted ray with predictable amplitude and phase. Note the precise angles and the color coding. Scattering from Bragg planes Structure factors Vector additive Randy Read, used by permission Brookhaven Biology

14. Look at what happens when one adds a single heavy atom. The two structure factors, FP and FPH can be used to solve for the phase. This is called Isomorphous Replacement. Randy Read, used by permission Brookhaven Biology

15. Perutz’s Fundamental Idea: Isomorphous Replacement FP = S Fatoms FPH = FP + FH FH We can approximate |FH| with |FPH - FP|. This often suffices for solving for the position of the heavy atom as if it were a small-molecule structure.

16. So for some particular reflection and a particular heavy atom, we can begin to find the phase: Knowing the position of the heavy atom allows us to calculate FH. Then we use FP = FPH + (-)FH to show that the phase triangles close with a two-fold ambiguity, at G and at H. There are several ways to resolve the ambiguity.

17. One way to resolve the ambiguity is to use a second isomorphous heavy-atom derivative.

18. David Blow and Francis Crick, when they were postdocs for Max Perutz, deduced a neat statistical way to determine the “best” phase when the MIR or SIR indications are ambiguous. We call it Figure-of-merit weighting. Randy Read, used by permission Brookhaven Biology

19. In principle, if we reflect x-rays from the “back” of Bragg planes, the amplitude should be the same and the phase should have opposite sign from when we reflect from the “front.” We call this Friedel’s Law: F(h) = F*(-h) Randy Read, used by permission Brookhaven Biology

20. What aboutFriedel’s Law: F(h) = F*(-h), here with a heavy atom? Randy Read, used by permission Brookhaven Biology

21. And what does this have to do with the fluorescence scan near the Se K-edge? Brookhaven Biology

22. Chooch analyzes the fluorescence scan and shows the elastic and inelastic scattering. Brookhaven Biology

23. However, a heavy atom might scatter anomalously, then Friedel’s law fails. In this case, the resonance between the electrons on the heavy atom and the x-rays causes a phase and amplitude shift. The symmetry of diffraction (from the front vs back of the Bragg planes) is broken. This can be measured and used.

24. One can see how to choose wavelengths to get large phase contrast for MAD phasing. * * * * * * * Maximum Real Signal * * Maximum Imaginary Signal * * * Spectrum from Phizackerly, Hendrickson, et al. Study of Lamprey Haemoglobin.

25. This Multiwavelength Anomalous Diffraction method often gives very strong phase information and is the source of many new structures.

26. Synchrotron radiation is very available and useful in the 8 to 14 keV range, and possible outside that range. Quite a number of elements can be incorporated into macromolecules that resonate in that range. Is only the bottom row really useful? Ethan Merritt, used by permission Brookhaven Biology

27. Brookhaven Biology

28. MAD map of Zn Alcohol Dehydrogenase (from Solve/Resolve) 9 hours 3 wavelengths 2.2Å x12B Brookhaven Biology

29. Modern times • Several things make data more accurate • Much better detectors • Well-collimated synchrotron beams • Improved software • There are powerful tools to improve approximate phases: Resolve, DM • We can use weaker signals to solve structures: Use one wavelength to acquire anomalous data and solve the structure from that: Single Anomalous Diffraction. Density modification improves phases.

30. SAD map of Zn Alcohol Dehydrogenase (from Solve/Resolve) 3 hours at peak wavelength 2.2Å Brookhaven Biology

31. 5 minutes at x29. How much here? Brookhaven Biology

32. Too Much Radiation Damage 360 1 sec. 1 degree = 6 min exposure at x29

33. Do you need to be near an edge? Fe anomalous map at 1.1A.

34. MAD or SAD? • Simplicity. • Other sources of phase information. • Managing radiation induced damage effects. • How about Sulfur phasing?

35. How about Sulfur phasing? • Damage to S and S-S. • S anomalous signal, optimal wavelength?

36. How about Identifying Metals? Anomalous map above and below the Cu edge