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

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

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

5. Frying Thaumatin Thaumatin: 207 Residues, 8 disulfide bridges Thaumatin: 207 Residues, 8 disulfide bridges P4(1)2(1)2: Easy to get a complete data set fairly quickly 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 Collect the same angular range several times to investigate radiation damage Data collected at NSLS X9B Data collected at NSLS X9B Resolution 1.45 Å Wavelength 0.979 Å SpacegroupP4(1)2(1)2 Osc. Range 90° ; 1.0 ° Redundancy / comp 7 / 99% # Data sets 20 I/sigma (HR) 7.4 -> 2.3 R merge 4.3 % -> 5.8 %

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

7. F Heavy becomes smaller due to damage resulting in F Tot to decrease F Heavy becomes smaller due to damage resulting in F Tot to decrease

8. F Heavy becomes smaller due to damage resulting in F Tot to increase F Heavy becomes smaller due to damage resulting in F Tot to increase

9. F Heavy becomes smaller due to damage resulting in F + and F - to increase, while anomalous difference becomes smaller F Heavy 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 Say we have two main processes over the course of our data collection Major change (disulphide breakage + carboxyl diss.+ etc) Major change (disulphide breakage + carboxyl diss.+ etc) Involves lots of electrons Involves lots of electrons Minor change (carboxyl dissociation+etc) Minor change (carboxyl dissociation+etc) Imagine the following scenario: Imagine the following scenario: State 0State 1State 2 Major changeMinor change

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

12. Signs of radiation damage Roughly spoken: Roughly spoken:  2  (I(t) - ) 2 R  |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) 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 ‘Late’ radiation damage State 1 @ frame 100 State 1 @ frame 100 ‘Early’ radiation damage ‘Early’ radiation damage State 1 @ frame 40 State 1 @ frame 40 ‘Very Early’ radiation damage ‘Very Early’ radiation damage State 1 @ frame 20 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 Visualise structural changes by isomorphous difference maps Refine model against first data set Refine model against first data set Use phases and isomorphous differences to compute map Use phases and isomorphous differences to compute map {|F 1 |-|F X>1 |,  1 } synthesis {|F 1 |-|F X>1 |,  1 } synthesis Positive peaks: disappearing electrons (red) Positive peaks: disappearing electrons (red) Negative peaks: appearing electrons (green) 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. Radiation-damage induced phasing (RIP) Disulfide breakage involves large rearrangement of electrons: X-ray induced derivative Disulfide breakage involves large rearrangement of electrons: X-ray induced derivative |F| |F mod | Substructure solution Phasing Density modification Model building

21. Radiation-damage induced phasing (RIP) Map resulting from (1,15) iso differences after shelxd, sharp and DM Map resulting from (1,15) iso differences after shelxd, sharp and DM (1,5) iso was enough as well (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? What would be the effect of anomalous dispersion in substructure solution and phasing? RIP: SIR like; RIPAS: SIRAS like RIP: SIR like; RIPAS: SIRAS like Both anomalous difference in ‘native’ and ‘derivative’ though Both anomalous difference in ‘native’ and ‘derivative’ though Most damaged data is treated as ‘native’ 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 Iodinate tyrosine by treating protein with N-Iodo-succinamide Either prior to or after crystallisation Either prior to or after crystallisation Iodinated tyrosines are sensitive to radiation damage Iodinated tyrosines are sensitive to radiation damage Se-Met not extremely sensitive to RD Se-Met not extremely sensitive to RD Collect 4 data sets on two derivatives Collect 4 data sets on two derivatives Thaumatin Thaumatin Iodinated prior to crystallization: IC (co-crystal) Iodinated prior to crystallization: IC (co-crystal) Iodinated after crystallization: CS (soak) Iodinated after crystallization: CS (soak)

24. Radiation-damage induced phasing with anomalous scattering (RIPAS) CSIC Resolution 2.5 Å 2.0 Å Wavelength 1.54 Å SpacegroupP4(1)2(1)2P4(1)2(1)2 Osc. Range 60° ; 1.0° 90° ; 0.5 ° Ano red. / comp. 2.4 / 97% 3.8 / 97% # Data sets 44 I/sigma (HR) 2.4 -> 1.9 4.0 -> 1.4 R merge 5.6% -> 5.2% 5.9 % -> 7.2 %

25. Radiation-damage induced phasing with anomalous scattering (RIPAS) Radiation damage was apparent within a single data set 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) Anom diffs indicate presence of iodine (green) (1,2) iso diffs indicate loss of iodines (red) (1,2) iso diffs indicate loss of iodines (red) Blue: 2Fo-FC 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) 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 Substructure solution success rate increases compared to SAD/RIP Iso and Ano diffs can be used simultaneously in SHARP for phasing Iso and Ano diffs can be used simultaneously in SHARP for phasing RIP/SAD phase ambiguity is broken 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 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 SAD RIP RIPAS

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

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

32. Other Possible derivatives for RIP(AS) Osmium Chloride Osmium Chloride Over the course of 5 datasets, Osmium Chloride clusters disappear 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 Anomalous signal so strong and Osmium so large, that combining with RIP signal does not improve phasing Cl Os 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 The presence of radiation damage can be spotted in several ways Disulfides break and push / release surrounding main and side chains 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 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 Iodinated tyrosines are susceptible to radiation damage Other RIPAS type derivatives are available Other RIPAS type derivatives are available

34. Acknowledgements Banumathi Sankaran Mirka Dauter Zbigniew Dauter

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