Radiation-damage-induced phasing with anomalous scattering

1. Radiation-damage-induced phasing with anomalous scattering Peter Zwart Physical biosciences division Lawrence Berkeley National Laboratories Not long ago: BNL/ANL/SAIC; Dauter group

2. Introduction • Radiation damage has been seen as a curse in macromolecular crystallography • Diffraction power is lost over the course of data collection • Cell dimensions change • Introduction of non-isomorphism over the course of data collection • Can we turn a problem into an opportunity? • What are the signs of radiation damage? • How does it affect the structure? • How can we use radiation damage?

3. Introduction • X-rays cause ionization events in unit cell • Protein is ionized and electronic rearrangements take place • Primary damage • Solvent is ionized and reacts with protein • Secondary damage • Secondary damage is limited by cryo-cooling

4. Introduction • Common rearrangements / reactions due to radiation damage include • Disulfide breakage • Dehalogenation of halogenated aromatic compounds (brominated uracil) • Decarboxylation of side chains • Associated main and side chain movement

5. Frying Thaumatin • Thaumatin: 207 Residues, 8 disulfide bridges • P4(1)2(1)2: Easy to get a complete data set fairly quickly • Collect the same angular range several times to investigate radiation damage • Data collected at NSLS X9B

6. Signs of radiation damage • Structural changes imply changes in intensities • A simple model: Protein + heavy atom • Assume only heavy atom is affected by RD • Assume RD diminishes occupancy • Make Argand diagram and see what happens

7. FHeavy becomes smaller due to damage resulting in FTot to decrease

8. FHeavy becomes smaller due to damage resulting in FTot to increase

9. FHeavy becomes smaller due to damage resulting in F+ and F- to increase, while anomalous difference becomes smaller

10. Signs of radiation damage • Say we have two main processes over the course of our data collection • Major change (disulphide breakage + carboxyl diss.+ etc) • Involves lots of electrons • Minor change (carboxyl dissociation+etc) • Imagine the following scenario: Major change Minor change State 0 State 1 State 2

11. Zero dose I(h) Time averaged I(h) Small difference Large difference Time Signs of radiation damage Structure at image X End of data collection

12. Signs of radiation damage • Roughly spoken: 2 (I(t) - <I(t)>)2 R |I(t) - <I(t)>| • This implies that the presence of radiation damage can be detected from the R values / Chi-squares vs frame number (in favorable cases)

13. Signs of radiation damage • ‘Late’ radiation damage • State 1 @ frame 100 • ‘Early’ radiation damage • State 1 @ frame 40 • ‘Very Early’ radiation damage • State 1 @ frame 20 Numbers for figure obtained via simulation techniques State 0: 207 residues, 17 Sulphurs State 1: Protein: 0.1 Å rmsd; Sulphurs: 0.8 Å rmsd State 2: Protein: 0.1+0.05 Å rmsd; Sulphurs: 0.8+0.1 Å rmsd

14. Signs of radiation damage Scaling the 20 individual data sets indicates radiation damage

15. Signs of radiation damage Increasing cell parameters indicates radiation damage

16. Signs of radiation damage Increase in Wilson B value indicates loss of diffraction power due to radiation damage

17. Structural changes • Visualise structural changes by isomorphous difference maps • Refine model against first data set • Use phases and isomorphous differences to compute map • {|F1|-|FX>1|, 1 } synthesis • Positive peaks: disappearing electrons (red) • Negative peaks: appearing electrons (green)

18. Structural changes Disulfide breakage over the course of the experiment (Elastase data)

19. Structural changes Disulfide breakage in thaumatin results in main and side chain movement

20. |F| Substructure solution Phasing Density modification Model building |Fmod| Radiation-damage induced phasing (RIP) • Disulfide breakage involves large rearrangement of electrons: X-ray induced derivative

21. Radiation-damage induced phasing (RIP) • Map resulting from (1,15) iso differences after shelxd, sharp and DM • (1,5) iso was enough as well

22. Radiation-damage induced phasing with anomalous scattering (RIPAS) • What would be the effect of anomalous dispersion in substructure solution and phasing? • RIP: SIR like; RIPAS: SIRAS like • Both anomalous difference in ‘native’ and ‘derivative’ though • Most damaged data is treated as ‘native’

23. Radiation-damage induced phasing with anomalous scattering (RIPAS) • Iodinate tyrosine by treating protein with N-Iodo-succinamide • Either prior to or after crystallisation • Iodinated tyrosines are sensitive to radiation damage • Se-Met not extremely sensitive to RD • Collect 4 data sets on two derivatives • Thaumatin • Iodinated prior to crystallization: IC (co-crystal) • Iodinated after crystallization: CS (soak)

24. Radiation-damage induced phasing with anomalous scattering (RIPAS)

25. Radiation-damage induced phasing with anomalous scattering (RIPAS) • Radiation damage was apparent within a single data set

26. Radiation-damage induced phasing with anomalous scattering (RIPAS) • Anom diffs indicate presence of iodine (green) • (1,2) iso diffs indicate loss of iodines (red) • Blue: 2Fo-FC

27. Radiation-damage induced phasing with anomalous scattering (RIPAS) • Iso and Ano diffs can be used simultaneously in substructure solution in Xprep (analogous to MAD) • Substructure solution success rate increases compared to SAD/RIP • Iso and Ano diffs can be used simultaneously in SHARP for phasing • RIP/SAD phase ambiguity is broken

28. Radiation-damage induced phasing with anomalous scattering (RIPAS) CS data (2.5 Å) directly after SHARP. No DM/solomon SAD RIP RIPAS

29. Radiation-damage induced phasing with anomalous scattering (RIPAS) IC data (2.0 Å) directly after SHARP. No DM/solomon SAD RIP RIPAS

30. Other Possible derivatives for RIP(AS) • International Tables for Crystallography Volume F., Page 752 • A Mercury derivative for Lysozyme

31. Other Possible derivatives for RIP(AS) • The Mercury-S bond is sensitive to radiation damage as well • Ramagopal et al, last year ACA meeting. • p-Iodo-Phenylanaline; • First talk of the day • Selenated ribose moiety / Brominated Uracil (DNA/RNA)?

32. Other Possible derivatives for RIP(AS) Cl • Osmium Chloride • Over the course of 5 datasets, Osmium Chloride clusters disappear • Anomalous signal so strong and Osmium so large, that combining with RIP signal does not improve phasing Cl Cl Os Cl Cl Cl Red: Isomorphous difference map (2 sigma) Blue: Anomalous difference map (15 sigma)

33. Conclusions • The presence of radiation damage can be spotted in several ways • Disulfides break and push / release surrounding main and side chains • Radiation damage induced isomorphous differences can be the sole source of information in substructure solution and phasing and AS enhances it • Iodinated tyrosines are susceptible to radiation damage • Other RIPAS type derivatives are available

34. Acknowledgements Mirka Dauter Zbigniew Dauter Banumathi Sankaran

35. Announcing the second INTERNATIONAL SYMPOSIUM ON RECENT TRENDS IN MACROMOLECULAR STRUCTURE AND FUNCTION Jan 18-20, 2006 Chennai, India For information, contact: Prof. D. Velmurugan: d_velu@yahoo.com isrtmsf@rediffmail.com