1. Crystal Manipulation for Data Collection at Low Temperature Sean Parkin - Department of Chemistry, University of Kentucky

2. Benefits of low temperature Crystal treatment prior to cooling Cooling methods and cryogens Crystal mounting etc. Potential problems Annealing methods When nothing seems to work …

3. Benefits of Low Temperature Reduced radiation damage i) Primary – dose dependent ii) Secondary – time dependent iii) Thermal damage Decreased thermal motion (& disorder) iv)Improved resolution limit v) Possibility of disorder resolution vi) Sharper electron density Increased crystal lifetime Full data collection for most structures can be done on one single crystal.

4. Haas & Rossmann (1970) Acta Cryst. B26, 998–1004 Intensity decay from radiation damage

5. Teng & Moffat (2000) J. Synch. Rad. 7, 313-317 Visible damage and reduced resolution Lysozyme diffraction at 100K before and after ~22 minutes on beamline 14-BM-C at APS

6. Primary damage is dose dependent Some molecules become chemically altered so the average electron density gets smeared. Primary radiation damage to H 2 O produces OH radicals that can initiate further damage via secondary events Low temperature has little or no effect on primary damage.  ––––>      x-rays     ––––>   ––––> 

7. Secondary damage is time (and dose) dependent A cascade of free-radical initiated reactions destroys long-range order even more, further smearing the electron density which increasingly destroys the high resolution data Low temperature can inhibit secondary damage  ––––>     x-rays           time       ––––>       ––––>     

8. Thermal damage from very intense sources* X-ray absorption dumps energy into the crystal Heating accelerates secondary damage Non-uniform heating causes temperature gradients and stress-induced damage A consideration on insertion device beamlines at third generation sources. All modes of damage will compound each other    ––––>         ––––>             intense        x-rays      ––––>    ––––>   *e.g. Teng & Moffat (2000) J. Synch. Rad. 7, 313-317

9. Improvement of resolution I  I o exp{-2B(sin  / ) 2 } T 1 = 300 K T 2 = 100 K, B o = 5 Å 2 b = 0.05 Å 2 K -1 assume B(T) ≈ B o + bT (B o + bT 1 )/r 1 2 = (B o + bT 2 )/r 2 2 r 2 = r 1 {(B o + bT 2 )/(B o + bT 1 )} 1/2 Roughly three times as much data at 100K vs 300K Hope, H. (1988) Acta Cryst. B44, 22-26.

10. Reduced thermal motion General reduction in refined B values. Dramatic reduction may indicate resolved disorder. BPTI, main chainBPTI, side chains Parkin & Hope (1996) Acta Cryst. D52, 18-29.

11. Resolved disorder in favourable cases BPTI C-terminus, 298KBPTI C-terminus, 125K Parkin & Hope (1996) Acta Cryst. D52, 18-29.

12. Other benefits: Full datasets are usually obtained from just one crystal. For MAD data especially, systematic errors are minimized. Crystals can be harvested and stored. Important if crystals degrade, e.g. oxidization of Se-Met. Crystal mounting can be much less damaging to crystals … because of reduced amount of manipulation.

13. Benefits of low temperature Crystal treatment prior to cooling Cooling methods and cryogens Crystal mounting etc. Potential problems Annealing methods When nothing seems to work …

14. Small organics can bind at active sites - not good ! Prior to cooling the crystal i)Find a suitable cryoprotectant Paratone oil, mineral oil, polyfluoroethers ~60% success rate Remove surface water oils antifreezes PEG < 4K : increase PEG, add other small PEGs PEG > 4K : add small PEGs MPD : increase MPD concentration Salt: add MPD/glycerol or even more salt ? exchange salt e.g. to sodium formate n.b. low salt requires more help than high salt Modify surface water

15. Mitchell & Garman (1996) J. Appl. Cryst. 29, 584–585 Elspeth Garman’s table of minimum amounts of glycerol needed to prevent ice formation in Hampton Screen I. These were arrived at by dilution rather than by replacement of water, so the numbers should be used with caution. Finding a suitable cryoprotectant

16. Garman & Doublié (2003) Meth. Enzymol. 368, 188-216. Structures in Acta Cryst. D57, 2001.

17. Finding a suitable cryoprotectant Structures in Acta Cryst D56-57, 2000-2001. Garman & Doublié (2003) Meth. Enzymol. 368, 188-216.

18. Garman (1999) Acta Cryst. D55, 1641–1653 Finding a suitable cryoprotectant

19. ii)Optimize concentration of antifreeze Mitchell & Garman (1994) J. Appl. Cryst. 27, 1070–1074 Prior to cooling the crystal The minimum required to suppress ice is not necessarily the optimum amount

20. iii)Introduction of antifreeze Single or multi-step soak / wash Sequential partial exchange of mother liquor Dialysis iv)Length of soak / wash Seconds to wash, minutes (hours ?) to soak The goal has been to minimize the shock to the crystal Generally little optimization done unless there are real problems Garman (1999) Acta Cryst D55, 1641-1653 Prior to cooling the crystal a) Move crystal between solutions 0 10 20 30 40 012345 Time (minutes) % cryo-solution b) Solution pipetted onto crystal 0 10 20 30 40 012345 Time (minutes) % cryo-solution

21. Benefits of low temperature Crystal treatment prior to cooling Cooling methods and cryogens Crystal mounting etc. Potential problems Annealing methods When nothing seems to work …

22. Cooling methods - cryogen characteristics Stream cool Variable temperature is easy Very controllable Single step EASY Fixed temp., 77 K Very controllable Two steps EASY Harder Several steps Variable temp. possible but tricky Not easy to control without practice LN 2 dunk Propane dunk

23. Cooling methods - cryogen cooling rates Walker, Moreno & Hope (1998) J. Appl. Cryst. 31, 954–956Teng & Moffat (1998) J. Appl. Cryst. 31, 252–257 N 2 streamliquid N 2 liquid propane N 2 streamliquid N 2 liquid propane

24. Cooling methods – points to consider i) Cooling rate is proportional to  T ii)Liquid propane can be dangerous around potential sources of ignition iii)Due to inherent complexity, liquid propane methods are the hardest to make reproducible iv)Leidenfrost gas layer insulation of large objects is insignificant with ordinary-sized crystals v)Liquid propane has a large liquid range - constant stirring is required for reproducibility vi) Make the process as simple as possible

25. Benefits of low temperature Crystal treatment prior to cooling Cooling methods and cryogens Crystal mounting etc. Potential problems Annealing methods When nothing seems to work …

26. Crystal mounting – tools Pictures: Hampton Research; Bruker-Nonius; Sean Parkin loops - homemade loops - bought arcstongs special vialsvials and holders

27. Crystal mounting – aqueous film removal under oil Aqueous films clinging to the crystal can often be teased away with a needle point. Or they may be wicked away with a wedge of pre- moistened filter paper. Most dry oils will accept a little water so small amounts will diffuse into the oil. This may be good or it may be bad.

28. Crystal mounting – crystal pick up

29. Crystal mounting – manipulations in the dewar Parkin & Hope (1998) J. Appl. Cryst. 31, 945-953. 1) Pre-cool tongs, plunge crystal. 2) Clasp the mounting pin. 3) Remove the pin holder. 4) Carry it to diffractometer.

30. Crystal mounting - Tongs … transfer to diffractometer takes a couple of seconds … … open the tongs so that the cold stream blows in the gap.

31. Crystal mounting - Cryovials pictures courtesy of MSC Note the inverted  axis … … means no cryogen spillage.

32. Crystal mounting - robots pictures courtesy of MSC, SSRL, Bruker-AXS Automated Fast Reproducible Expensive MSC - Actor BruNo SSRL

33. Benefits of low temperature Crystal treatment prior to cooling Cooling methods and cryogens Crystal mounting etc. Potential problems Annealing methods When nothing seems to work …

34. Potential problems: Control and reproducibility Parkin & Hope (1998) J. Appl. Cryst. 31, 945-953 Temperature versus time for a "crystal" held in stainless steel block tongs. Warming rate is about 0.5° per second (depends on tongs) Crystal environment should be controlled so that it is reproducible.

35. Potential problems: Control and reproducibility Parkin & Hope (1998) J. Appl. Cryst. 31, 945-953 Temperature vs time during mount / dismount Ensure the crystal temperature is controlled throughout mounting and that it is reproducible. mount temp. dismount temp.

36. Pictures: Elspeth Garman (Oxford University) Ice caused by inadequate cryoprotectant. Solution: optimize concentration Ice caused by snow from slushy liquid nitrogen sticking to the drop. Solution: carefully remount from fresh cryogen, gently tease off the snow etc. Potential problems: Ice

37. Pictures: (1) Sean Parkin; (2,3) Elspeth Garman (Oxford University) More ice problems A poorly positioned nozzle or draughts will cause snow to grow on the pin end. This can get serious if left too long. 12 3

38. Potential problems: Ice prevention Even in humid environments ice can be prevented without elaborate contraptions. The important point is a well- defined geometric relationship between cold stream, mounting pin and goniometer head and to rigorously exclude draughts Pin design Cold stream geometry Turbulence Exclude draughts Parkin & Hope (1998) J. Appl. Cryst. 31, 945-953.

39. Potential problems: Mosaic spread Garman, E. (1999) Acta. Cryst. D55, 1641-1653.Dauter, Z. (1999) Acta. Cryst. D55, 1703-1717. Minimize mosaic spread to optimise data quality. It should prove possible to approximate the mosaicity of crystals at room temperature. Therefore it helps to know what this is !

40. Potential problems: maximize cooling rate Thus a plate should cool faster than a rod or a block. 1) Keep cryogen close at hand 2) Go for a large surface area to volume ratio - so small crystals have an advantage. Generally, S/V > 12mm -1 e.g.0.4mm x 0.4mm x 0.4mm block, S/V = 15mm -1 0.5mm x 0.5mm x 0.5mm block, S/V = 10mm -1 0.4mm x 0.5mm x 0.2mm block, S/V = 19mm -1

41. Things to consider Be in control throughout the experiment The bare minimum antifreeze concentration needed to suppress ice formation is probably not the optimum amount for minimizing mosaic spread and maximizing resolution. Minimize crystal handling. Smaller crystals are easier to cool evenly. Attempt some sort of annealing (next). Make the mounting and retrieval process as simple as possible, but not simpler Simplicity leads to reproducibility

42. Benefits of low temperature Crystal treatment prior to cooling Cooling methods and cryogens Crystal mounting etc. Potential problems Annealing methods When nothing seems to work …

43. Annealing of macromolecular crystals Quick and (hopefully not so) dirty approaches Macromolecular Crystal Annealing Flash Annealing Systematic approaches Controlled slow annealing Controlled flash annealing

44. 1) "Macromolecular Crystal Annealing" - procedure Crystal is quickly removed … … placed in cryoprotectant … … for ~ three minutes … … and then re-flash cooled. Harp, Timm, & Bunick, (1998) Acta Cryst. D54 622-628

45. Increased resolution and better mosaicity for nucleosome core particle crystal after 3 minute anneal in antifreeze or oil Harp, Timm, & Bunick, (1998) Acta Cryst. D54 622-628 Macromolecular Crystal Annealing

46. Hansen, Harp, & Bunick, (2003) Meth. Enzymol. 368, 217-235 A flash-cooled crystal of Patatin gave diffraction to 3.7 Å clearly showing multiple lattices. On annealing it broke into two pieces. On remounting, the larger piece diffracted to ~2.3 Å. Macromolecular Crystal Annealing

47. An otherwise trashed nucleosome core particle crystal resurrected after a 3 minute anneal Macromolecular Crystal Annealing Harp, Timm, & Bunick, (1998) Acta Cryst. D54 622-628

48. 2) "Flash Annealing" - procedure Yeh & Hol (1998) Acta Cryst. D54, 479-480 The cold stream blowing over the flash cooled crystal is blocked for a short period of time (seconds) until it has thawed. Then the obstruction is released to re-cool the crystal. Flash cooled …… stream diverted …… re-flash cooled.

49. Flash annealing - results Glycerol kinase, resolution limit ~4 Å, poor mosaic spread. Flash annealing by blocking the cold stream for 1.5 - 2 seconds three times gives ~2.8 Å resolution and better mosaic spread. Yeh & Hol (1998) Acta Cryst. D54, 479-480

50. Accomplished in two ways: Slow warming using a controllable stream heater. Rapid warming to some pre-determined temperature by either dynamic mixing of cold and warm streams or by rapid switching of two cold gas streams. Questions to be answered: Is it a protein or a bulk water phenomenon ? How does annealing work ? Why does annealing work ? What are the mechanisms of protein crystal annealing ? Controlled annealing without thawing

51. 3) Slow annealing: Cell volume vs temperature  V > 1200 Å 3 Concanavalin A 1 2) Weik et al. (2001) Acta Cryst. D57, 566-573 TcAChE (trigonal) 2 TcAChE (orthorhombic) 2 Crystals with channels show an abrupt volume jump at some well defined temperature. Implies that effect is in the bulk water and that there is a surface effect via the connection to the crystal surface. 1) Parkin (1993) Ph.D. Thesis, UC Davis

52. Cell dimension changes on annealing temperature (K) change relative to 95 K a axisb axisc axis There is an abrupt jump in both the b and c axes in concanavalin A on warming from 160 to 165 K. There is no corresponding jump in a. Parkin & Hope (2003) Acta Cryst. D59, 2228-2236

53. (view down c ) c axis expands protein moves waters move (view down b ) b axis expands protein moves waters move (view down a ) a axis constant - waters move Origin of annealing effects c a b c a b Parkin & Hope (2003) Acta Cryst. D59, 2228-2236

54. Annealing affects diffuse solvent diffraction Overall background for concanavalin A diffraction is reduced after annealing. It is also a bit smoother. Still don’t know what the likely mechanism of annealing is. Parkin & Hope (2003) Acta Cryst. D59, 2228-2236

55. 4) Flash annealing without thawing Rapid adjustment of warm and cold gas flows onto the crystal. Can be tricky With two low-temperature machines we can rapidly switch cold streams. Much easier Kriminski, Caylor, Nonato, Finkelstein, & Thorne Acta Cryst. (2002), D58, 459-471 Parkin (2002) unpublished

56. Flash annealing seen by in-situ X-ray imaging At room temp. the whole crystal is in the diffracting position over a very small angular range. After flash cooling, the mosaic spread is much worse and the resolution limit was severely degraded to 4.3 Å. After 25 s controlled anneal at 250 K, resolution limit is 2.4 Å, mosaic spread generally not as good as pre-cool value. Kriminski, Caylor, Nonato, Finkelstein, & Thorne Acta Cryst. (2002), D58, 459-471

57. What happens to the mosaic structure ? On flash cooling, individual domains have a mosaic spread similar to that of the whole crystal. After annealing, small domains have much narrower mosaic spread, but are themselves somewhat mis-aligned. Kriminski, Caylor, Nonato, Finkelstein, & Thorne Acta Cryst. (2002), D58, 459-471

58. What happens to the water ? a)The distribution of water is fairly uniform in solvent regions of fresh crystals at room temperature. b)During flash cooling, water is squeezed out of small domains and collects in the regions surrounding the domains. c)Which leaves the domains somewhat more mis-aligned. Annealing likely gives a partial fix by increasing order within domains and by reducing the spread of lattice spacings within a crystal. Kriminski, Caylor, Nonato, Finkelstein, & Thorne Acta Cryst. (2002), D58, 459-471

59. i) Consider annealing if diffraction is uncharacteristically poor after flash cooling. ii)Try the quick methods first. MCA appears to be more general. iii)For MCA, the crystal must be stable in its cryoprotectant. iv)Size may be a less important factor than shape. Thin crystals may be better suited to flash annealing. v)In MCA, a full three minutes may not be needed. Longer times appear less likely to yield optimum results. vi) Multiple cycles of MCA are not necessary and may be undesirable. For flash annealing, multiple cycles may be required. Annealing - General comments Hansen, Harp, & Bunick, (2003) Meth. Enzymol. 368, 217-235

60. Benefits of low temperature Crystal treatment prior to cooling Cooling methods and cryogens Crystal mounting etc. Potential problems Annealing methods When nothing seems to work …

61. When nothing seems to work (in no particular order): Does it diffract at room temperature ? Try other cryoprotectants Try more than once. Vary time and temperature of crystal handling steps Match antifreeze to the system Exchange buffers etc. Attempt annealing.

62. Does the crystal diffract at room temperature ? Photograph: Elspeth Garman (Oxford University) Capillary scheme lifted from “Practical Protein Crystallography” by Duncan McRee

63. Try more than once. Picture courtesy Elspeth Garman, after Schneider, Bravo & Hansen

64. Match antifreeze to the system - Osmotic Pressures: 1) Find osmotic pressure of mother liquor in the CRC Handbook of Chemistry and Physics (section D232) 11th column, O (Os/Kg) 2) Find osmotic pressure of your antifreeze. 3) Modify the concentration in mother liquor to minimize the change in osmotic pressure. Osmotic shock: Water will either be pumped into the crystal or sucked out of the crystal. Either one may cause cracks, increase mosaicity, lower resolution etc. All bad. Rapid transfers between solutions will give greatest shock but will minimize the time over which damage could occur. Experiment !

65. Osmotic pressure matching - Elspeth’s worked example. Mother Liquor:Osmolarity (Os/Kg) 2.0 M NaCl 50mM pH 7.8 Tris HCl3.95 Cryoprotectant: 20% glycerol2.90 Difference here is 1.05, so the plan is to alter the cryoprotectant so that it matches. From the CRC Handbook, 0.55M NaCl exerts an osmotic pressure of 1.05 Os/Kg. Hence try 0.55M NaCl, 20% glycerol in 50mM pH 7.8 Tris HCl

66. Advantages and disadvantages of low temperature work. FOR: Reduced radiation damage Gentler mounting Lower backgrounds Higher resolution Fewer crystals Transportation is easy Harvest crystals at their peak. AGAINST: Expense (money) Expense (time) Mosaic spread increase