1. Optimizing structure determination How many are we solving? What is the limit? Are we there yet? Why not? What are the biggest problems?

2. How many are we solving? http://asdp.bnl.gov/asda/Libraries/pdb_statis/latest/bml/ALS.html

3. $$ → photons photons → data data → models models → results results → $$ Breaking it down

4. SecondsDescriptionPercent 104490 Assigned and available91% 42093 Shutter open40% 52684 Collecting (3026 images)50% 51806 Something else50% Operational Efficiency “representative” 8.3.1 user

5. SecondsDescriptionPercent 51806 Something else100% 247s  45 Mounting22% 229s  37 Centering16% 179s  109 Strategizing38% 309s  37 Prepping24% Operational Efficiency “representative” 8.3.1 user

6. NumberDescriptionPercent 446028 Images (~7 TB)33% 2346 Data sets47% 449 MAD/SAD (1:2)19% 48 Published2% 8.3.1 in 2003 Turning data into models

7. Top producing beamlines of the world http://asdp.bnl.gov/asda/Libraries/pdb_statis/latest/bml/ALL.html Structures credited

8. 28 operating US beamlines ~10 11 ph/μm 2 exposure limit ÷ 2x10 9 ph/μm 2 /s ~ 100,000 datasets/year ÷ 1324 str in 2003 ~ 2% efficient What is the limit?

9. DVD data archive

10. Elven Automation Elves examine images and set-up data processing Elves run… mosflm scala solve mlphare dm arp/warp

11. Apr 6 – 24 at ALS 8.3.1 Elven Automation 27,686images collected 148datasets (15 MAD) 31investigators 56unique cells 5 KDa – 23 MDaasymmetric unit 0.94 – 32 Åresolution (3.2 Å)

12. Apr 6 – 24 at ALS 8.3.1 Elven Automation 148datasets 117succeded ~3.5 (0.1-75)hours 31failed ~61 (0-231)hours 2 / 15MAD structures

13. Overlaps Signal to noise Radiation Damage Why do structures fail?

14. avoidable overlaps mosaicity phi detector c* b c a Ewald sphere

15. unavoidable overlaps mosaicity phi detector c* b c a Ewald sphere

16. Overlaps Signal to noise Radiation Damage Why do structures fail?

17. MAD phasing simulation Anomalous signal to noise ratio Correlation coefficient to correct model mlphare results

18. SAD phasing simulation Anomalous signal to noise ratio Correlation coefficient to correct model mlphare results

19. Minimum required signal (MAD/SAD)

20. Is it real, or is it MLFSOM ?

21. “We really need those high-resolution spots”

22. Incremental strategy incremental_strategy.com merged.mtz auto.mat

23. Overlaps Signal to noise Radiation Damage Why do structures fail?

24. Distention of cryo with dose before

25. Distention of cryo with dose after

26. Water ring shift saturated sucrose in 250mM WO4 0 MGy

27. Water ring shift saturated sucrose in 250mM WO4 184 MGy

28. Water ring shift Resolution (Ǻ) Photons/s/pixel  7.5 3.8 2.5 1.9 1.5 saturated sucrose in 250mM WO4

29. Protein crystal background

30. Water ring shift Absorbed dose (MGy) Water ring position (Ǻ) GCN4-p1-N16A trigonal crystal crystal background saturated sucrose

31. Water ring shift http://www.lsbu.ac.uk/water/amorph.html

32. Water ring shift bubbles? Richard D. Leapman, Songquan Sun, Ultramicroscopy (1995)

33. Water ring shift Hydrogen bubbles? Richard D. Leapman, Songquan Sun, Ultramicroscopy (1995)

34. Water ring shift Hydrogen bubbles? http://www.rcdc.nd.edu/compilations/Rxn.pdf “The hydrogen atom reacts with organic compounds by abstracting H from saturated molecules and by adding to centers of unsaturation, for example,

35. Damage model system

36. Data quality vs phasing quality Exposure time (min) Correlation coefficient

37. Individual atoms decay at different rates Exposure time (min) Correlation coefficient to observed data

38. Damage changes fluorescence spectrum Photon energy (eV) counts

39. Damage changes fluorescence spectrum fluence (10 3 photons/mm 2 ) Fraction unconverted 25mM SeMet in 25% glycerol 0.0 0.2 0.4 0.6 0.8 1.0 0 20 40 60 80 100 120 Exposing at 12680 eV Se cross-section at 12680 eV

40. fluorescence probe for damage Absorbed Dose (MGy) Fraction unconverted Wide range of decay rates seen 0.0 0.2 0.4 0.6 0.8 1.0 0 50 100 150 200 Half-dose = 41.7 ± 4 MGy “GCN4” in crystal Half-dose = 5.5 ± 0.6 MGy 8 mM SeMet in NaOH Protection factor: 660% ± 94%

41. “Can we do more with what we’ve got?”

42. Interleaved Scheduling experiment queuebeamline Minor 30s Choe 120s Alberta 60s Choe 30s Minor 30s

43. SuperTong

45. “infinite capacity” sample carousel

46. Carousel open

47. CHL idlepos