1. Douglas Instruments Microseeding slide 1 Microseed it! Introduction to microseeding Patrick Shaw Stewart Douglas Instruments Limited (near Oxford, UK): Peter Baldock, Patrick Shaw Stewart, Richard Briggs, Stefan Kolek

2. Douglas Instruments Microseeding slide 2 Crystallization breakthroughs Jankaric and Kim (1991) – sparse matrix screening Santarsiero et al. (2001). ‘Methods and apparatus for performing array microcrystallizations.’ US patent 6,296,673 (B1). (Nanovolume crystallization) Next-generation screens from data mining e.g. JCSG+

3. Douglas Instruments Microseeding slide 3 Crystallization breakthroughs Jankaric and Kim (1991) – sparse matrix screening Santarsiero et al. (2001). ‘Methods and apparatus for performing array microcrystallizations.’ US patent 6,296,673 (B1). (Nanovolume crystallization) Next-generation screens from data mining e.g. JCSG+ D’Arcy et al. Acta Cryst. (2007). D63. 'An automated microseed matrix-screening method for protein crystallization’

4. Douglas Instruments Microseeding slide 4 random Microseed Matrix-Screening D’Arcy et al. Acta Cryst. (2007). D63. 'An automated microseed matrix-screening method for protein crystallization’

5. Douglas Instruments Microseeding slide 5 1.Add seed crystals to a random screen random Microseed Matrix-Screening D’Arcy et al. Acta Cryst. (2007). D63. 'An automated microseed matrix-screening method for protein crystallization’

6. Douglas Instruments Microseeding slide 6 1.Add seed crystals to a random screen 2.Suspend crushed crystals in the reservoir solution that gave the hits used (“hit solution”) random Microseed Matrix-Screening D’Arcy et al. Acta Cryst. (2007). D63. 'An automated microseed matrix-screening method for protein crystallization’

7. Douglas Instruments Microseeding slide 7 1.Add seed crystals to a random screen 2.Suspend crushed crystals in the reservoir solution that gave the hits used (“hit solution”) 3.Automate! random Microseed Matrix-Screening D’Arcy et al. Acta Cryst. (2007). D63. 'An automated microseed matrix-screening method for protein crystallization’

8. Douglas Instruments Microseeding slide 8 random Microseed Matrix-Screening To get: D’Arcy et al. Acta Cryst. (2007). D63. 'An automated microseed matrix-screening method for protein crystallization’ 1.Add seed crystals to a random screen 2.Suspend crushed crystals in the reservoir solution that gave the hits used (“hit solution”) 3.Automate!

9. Douglas Instruments Microseeding slide 9 random Microseed Matrix-Screening To get: (1) more hits D’Arcy et al. Acta Cryst. (2007). D63. 'An automated microseed matrix-screening method for protein crystallization’ 1.Add seed crystals to a random screen 2.Suspend crushed crystals in the reservoir solution that gave the hits used (“hit solution”) 3.Automate!

10. Douglas Instruments Microseeding slide 10 1.Add seed crystals to a random screen 2.Suspend crushed crystals in the reservoir solution that gave the hits used (“hit solution”) 3.Automate! random Microseed Matrix-Screening To get: (1) more hits (2) better crystals D’Arcy et al. Acta Cryst. (2007). D63. 'An automated microseed matrix-screening method for protein crystallization’

11. Douglas Instruments Microseeding slide 11 Microseeding in screening experiments Allan D’Arcy Novartis, Basle 2006 ‘Matrix-seeding script’ 1.protein 2.reservoir solution 3-bore tip

12. Douglas Instruments Microseeding slide 12 Microseeding in screening experiments Allan D’Arcy Novartis, Basle 2006 ‘Matrix-seeding script’ 1.protein 2.reservoir solution 3.seeds 3-bore tip

13. Douglas Instruments Microseeding slide 13 Microseeding in screening experiments Allan D’Arcy, Novartis, Basle. 2006 ‘Matrix-seeding script’

14. Douglas Instruments Microseeding slide 14 Microseeding in screening experiments D’Arcy et al. Acta Cryst. (2007). D63 MMP12 BVP USP7 Trypsin PPE Regular screenScreen with seeds

15. Douglas Instruments Microseeding slide 15 Microseeding in screening experiments D’Arcy et al. Acta Cryst. (2007). D63 MMP12 BVP USP7 Trypsin PPE Regular screenScreen with seeds

16. Douglas Instruments Microseeding slide 16 Microseeding in screening experiments USP7 crystals used for seeds grown in 30% PEG 3350, 100 mM HEPES pH 7.0 USP7 crystals after seeding in 20% PEG 3350, 200 mM magnesium hexahydrate D’Arcy et al. Acta Cryst. (2007). D63

17. Douglas Instruments Microseeding slide 17 random Microseed Matrix-Screening (rMMS) “MMS” Ireton and Stoddard. 'Microseed matrix screening to improve crystals of yeast cytosine deaminase'. Acta Crystallographica section D60 (2004), 601–605. “rMMS” D’Arcy et al. Acta Cryst. (2007). D63. 'An automated microseed matrix-screening method for protein crystallization’

18. Douglas Instruments Microseeding slide 18 See www.douglas.co.uk/mms.com 1.Break crystals 2.Place contents of well in 50 μl of reservoir solution 3.Vortex with Hampton “Seed Bead” 4.Make a dilution series immediately 5.Freeze! Look after your seeds! How to make the seed stock

19. Douglas Instruments Microseeding slide 19 Phase diagram of a protein [Protein] [Precipitant] clear precipitate nucleation metastable zone

20. Douglas Instruments Microseeding slide 20 Case study – Galina Obmolova, Tom Malia et al, Acta Cryst (2010) D66, 927 - 933 IL-13/C836 mouse antibody IL-13/H2L6 humanized IL-13/M1295 affinity-matured variant

21. Douglas Instruments Microseeding slide 21 Complexes: IL-13/C836 (mouse antibody) IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) Conventional methods 40 residues changed 4 residues changed Case study – Obmolova et al, Acta Cryst (2010) D66, 927 - 933

22. Douglas Instruments Microseeding slide 22 Complexes: IL-13/C836 (mouse antibody) 192 conditions IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) Conventional methods 40 residues changed 4 residues changed No hits Case study – Obmolova et al, Acta Cryst (2010) D66, 927 - 933

23. Douglas Instruments Microseeding slide 23 Complexes: IL-13/C836 (mouse antibody) 192 conditions IL-13/H2L6 (humanized mAb) 192 conditions IL-13/M1295 (affinity-matured humanized mAb) Conventional methods 40 residues changed 4 residues changed No hits One hit Case study – Obmolova et al, Acta Cryst (2010) D66, 927 - 933

24. Douglas Instruments Microseeding slide 24 Complexes: IL-13/C836 (mouse antibody) 192 conditions IL-13/H2L6 (humanized mAb) 192 conditions IL-13/M1295 (affinity-matured humanized mAb) 192 conditions Conventional methods 40 residues changed 4 residues changed No hits One hit Case study – Obmolova et al, Acta Cryst (2010) D66, 927 - 933

25. Douglas Instruments Microseeding slide 25 Complexes: IL-13/C836 (mouse antibody) 192 conditions IL-13/H2L6 (humanized mAb) 192 conditions IL-13/M1295 (affinity-matured humanized mAb) 192 conditions Conventional methods 40 residues changed 4 residues changed No hits One hit Case study – Obmolova et al, Acta Cryst (2010) D66, 927 - 933 Could not be optimized

26. Douglas Instruments Microseeding slide 26 Complexes: IL-13/C836 (mouse antibody) IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) Conventional methods Random microseeding (rMMS) 40 residues changed 4 residues changed No hits One hit Case study – Obmolova et al, Acta Cryst (2010) D66, 927 - 933 Microseeding

27. Douglas Instruments Microseeding slide 27 Complexes: IL-13/C836 (mouse antibody) IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) Both 1.9 Å resolution orthorhombic P2 1 2 1 2 1 Conventional methods Random microseeding (rMMS) 40 residues changed 4 residues changed No hits One hit Case study – Obmolova et al, Acta Cryst (2010) D66, 927 - 933 Microseeding Optimization

28. Douglas Instruments Microseeding slide 28 Complexes: IL-13/C836 (mouse antibody) IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) Both 1.9 Å resolution orthorhombic P2 1 2 1 2 1 2.8 Å res. P2 1 2 1 2 1 Cross-seeding Conventional methods Random microseeding (rMMS) 40 residues changed 4 residues changed No hits One hit Case study – Obmolova et al, Acta Cryst (2010) D66, 927 - 933 Microseeding Optimization

29. Douglas Instruments Microseeding slide 29 Complexes: IL-13/C836 (mouse antibody) IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) Both 1.9 Å resolution orthorhombic P2 1 2 1 2 1 2.8 Å res. P2 1 2 1 2 1 Cross-seeding Conventional methods Random microseeding (rMMS) 40 residues changed 4 residues changed No hits One hit Case study – Obmolova et al, Acta Cryst (2010) D66, 927 - 933 Microseeding Optimization

30. Douglas Instruments Microseeding slide 30 Complexes: IL-13/C836 (mouse antibody) IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) Both 1.9 Å resolution orthorhombic P2 1 2 1 2 1 2.8 Å res. P2 1 2 1 2 1 2.0 Å res. monoclinic P2 1 Cross-seeding Conventional methods Random microseeding (rMMS) 40 residues changed 4 residues changed No hits One hit Case study – Obmolova et al, Acta Cryst (2010) D66, 927 - 933 Optimization Microseeding Optimization

31. Douglas Instruments Microseeding slide 31 Complexes: IL-13/C836 (mouse antibody) IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) Both 1.9 Å resolution orthorhombic P2 1 2 1 2 1 2.8 Å res. P2 1 2 1 2 1 2.0 Å res. monoclinic P2 1 2.0 Å res. monoclinic P2 1 Cross-seeding Conventional methods Random microseeding (rMMS) 40 residues changed 4 residues changed No hits One hit Case study – Obmolova et al, Acta Cryst (2010) D66, 927 - 933 Optimization Microseeding Optimization

32. Douglas Instruments Microseeding slide 32 Matrix seeding volumes: 0.3 µl protein + 0.2 µl reservoir solution + 0.1 µl seed stock

33. Douglas Instruments Microseeding slide 33 Villasenor et al. (2010) Acta Cryst. D66, 568-576. ‘Acoustic matrix microseeding: improving protein crystal growth with minimal chemical bias’ Yellow: normal screen Orange: additive screen with Hit Solution Blue: random rMMS (screen with seeds in Hit Solution)

34. Douglas Instruments Microseeding slide 34 Microseeding toolkit

35. Douglas Instruments Microseeding slide 35 If you want to know more: Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen and Peter F.M. Baldock. 'Getting the most out of the random microseed matrix- screening method in protein crystallization'. Cryst. Growth Des., 2011, 11 (8), p3432. On-line at http://pubs.acs.org/doi/abs/10.1021/cg2001442http://pubs.acs.org/doi/abs/10.1021/cg2001442

36. Douglas Instruments Microseeding slide 36 If you want to know more: Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen and Peter F.M. Baldock. 'Getting the most out of the random microseed matrix- screening method in protein crystallization'. Cryst. Growth Des., 2011, 11 (8), pp 3432–3441. On-line at http://pubs.acs.org/doi/abs/10.1021/cg2001442http://pubs.acs.org/doi/abs/10.1021/cg2001442

37. Douglas Instruments Microseeding slide 37 rMMS with membrane proteins Crystals of membrane proteins are often unstable Remember that the reservoir normally has no detergent! Harvest several large drops without dilution 1.5 microlitres are enough! (20 nl per well)

38. Douglas Instruments Microseeding slide 38 rMMS with membrane proteins Crystals of membrane proteins are often unstable Remember that the reservoir normally has no detergent! Harvest several large drops without dilution 1.5 microlitres are enough! (20 nl per well) Does it work? - ask Isabel! Extra hits in 5 / 7 cases Moved on at least one project Will not work if sample is not crystallizable

39. Douglas Instruments Microseeding slide 39 Microseeding Opticryst – a consortium of European institutions and companies aiming to improve crystal optimization. 2007 – 2010.

40. Douglas Instruments Microseeding slide 40 Microseeding Opticryst – a consortium of European institutions and companies aiming to improve crystal optimization. 2007 – 2010. We decided to look into microseeding, especially the stability of seeds.

41. Douglas Instruments Microseeding slide 41 Microseeding Opticryst – a consortium of European institutions and companies aiming to improve crystal optimization. 2007 – 2010.

42. Douglas Instruments Microseeding slide 42 Microseeding Opticryst – a consortium of European institutions and companies aiming to improve crystal optimization. 2007 – 2010. Stefan set up 30,000 drops and estimated the number of crystals In 15,000 drops!

43. Douglas Instruments Microseeding slide 43 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? (2) How stable are the seed stocks? (3) Is “preseeding” the protein stock helpful? (4) How can we avoid salt crystals? (5) How can we get more diverse crystals? (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

44. Douglas Instruments Microseeding slide 44 Protein SourceConcentration Glucose IsomeraseHampton Research33 mg/ml HemoglobinSigma Aldrich60 mg/ml ThaumatinSigma Aldrich30 mg/ml ThermolysinSigma Aldrich15 mg/ml TrypsinSigma Aldrich30 mg/ml XylanaseMacro Crystal36 mg/ml

45. Douglas Instruments Microseeding slide 45 “Receptive” conditions Conditions where: (1)crystals don’t grow without seeds in four drops, but (2)crystals grow in at least three out of four drops with seeds.

46. Douglas Instruments Microseeding slide 46 “ Receptive” conditions Conditions where: (1)crystals don’t grow without seeds in four drops, but (2)crystals grow in at least three out of four drops with seeds. 25 receptive conditions were found

47. Douglas Instruments Microseeding slide 47 1Glucose IsomeraseJCSG+2-22 M (NH4)2SO4, 0.2 M NaCl, 0.1 M Na MES, PH 6.5 2Glucose IsomeraseJCSG+2-4325%(w/v) PEG 3350, 0.2 M (NH4)2SO4, 0.1 M bis-tris 3HemoglobinJCSG+2-2530%(w/v) Jeffamine ED-2001, 0.1 M Na HEPES, PH 7.0 4HemoglobinJCSG+2-3330%(w/v) PEG 2000 MME, K thiocyanate 5HemoglobinJCSG+2-3430%(w/v) PEG 2000 MME, K bromide 6HemoglobinJCSG+2-4425%(w/v) PEG 3350, 0.2 M NaCl, 0.1 M bis-tris, PH 5.5 7ThaumatinStructure screen 1730%(w/v) PEG 4K, 0.2 M ammonium acetate, 0.1M Na citrate, PH 5.6 8ThaumatinStructure screen 1920%(v/v) IPA, 20%(w/v) PEG 4K, 0.1 M Na citrate, PH 5.6 9ThaumatinStructure screen 11430%(w/v) PEG 8K, 0.2 M (NH4)2SO4, 0.1 M Na cacodylate, PH 6.5 10ThaumatinStructure screen 11520%(w/v) PEG 8K, 0.2M magnesium acetate, 0.1 M Na cacodylate, PH6.5 11ThaumatinStructure screen 1322 M (NH4)2SO4, 0.1 M tris, PH 8.5 12ThaumatinJena Bioscience Membrane screen3D51.5 M Li2SO4, 0.1 M Na HEPES, PH 7.5 13ThermolysinJCSG+ (2:1 water)1-220%(w/v) PEG 3K, 0.1 M Na citrate, PH 5.5 14ThermolysinJCSG+ (2:1 water)1-2120%(w/v) PEG 6k, 0.1 M citric acid, PH 5.0 15ThermolysinJCSG+ (2:1 water)2-1810%(v/v) MPD, 0.1 M bicine, PH 9.0 16ThermolysinJCSG+ (2:1 water)2-190.8 M succinic acid, PH 7.0 17ThermolysinJCSG+ (2:1 water)2-212.4 M Na malonate, PH 7.0 18ThermolysinJCSG+ (2:1 water)2-220.5%(w/v) Jeffamine ED-2001, 1.1 M Na malonate, 0.1 M Na HEPES, PH 7.0 19TrypsinJena Bioscience Membrane screen3D31.5 M NaCl, 0.1M Na acetate, PH 4.6 20TrypsinJena Bioscience Membrane screen3D31.5 M NaCl, 0.1M Na acetate, PH 4.6 21TrypsinJena Bioscience Membrane screen3D62 M NaCl, 0.1 M Na citrate 22TrypsinJena Bioscience Membrane screen3D62 M NaCl, 0.1 M Na citrate 23XylanaseStructure screen 1322 M (NH4)2SO4, 0.1 M tris, PH 8.5 24XylanaseStructure screen 13730%(w/v) PEG 4K, 0.2 M Na acetate, 0.1 M tris, PH 8.5 25XylanaseStructure screen 1454 M Na formate 26XylanaseJena Bioscience Membrane screen3B53.5 M (NH4)2SO4, 0.25M NaCl, 50mM Na/K phosphate, PH 7.5 27XylanaseJena Bioscience Membrane screen3D41.5 M K phosphate, PH 7.0

48. Douglas Instruments Microseeding slide 48 1Glucose IsomeraseJCSG+2-22 M (NH4)2SO4, 0.2 M NaCl, 0.1 M Na MES, PH 6.5 2Glucose IsomeraseJCSG+2-4325%(w/v) PEG 3350, 0.2 M (NH4)2SO4, 0.1 M bis-tris 3HemoglobinJCSG+2-2530%(w/v) Jeffamine ED-2001, 0.1 M Na HEPES, PH 7.0 4HemoglobinJCSG+2-3330%(w/v) PEG 2000 MME, K thiocyanate 5HemoglobinJCSG+2-3430%(w/v) PEG 2000 MME, K bromide 6HemoglobinJCSG+2-4425%(w/v) PEG 3350, 0.2 M NaCl, 0.1 M bis-tris, PH 5.5 7ThaumatinStructure screen 1730%(w/v) PEG 4K, 0.2 M ammonium acetate, 0.1M Na citrate, PH 5.6 8ThaumatinStructure screen 1920%(v/v) IPA, 20%(w/v) PEG 4K, 0.1 M Na citrate, PH 5.6 9ThaumatinStructure screen 11430%(w/v) PEG 8K, 0.2 M (NH4)2SO4, 0.1 M Na cacodylate, PH 6.5 10ThaumatinStructure screen 11520%(w/v) PEG 8K, 0.2M magnesium acetate, 0.1 M Na cacodylate, PH6.5 11ThaumatinStructure screen 1322 M (NH4)2SO4, 0.1 M tris, PH 8.5 12ThaumatinJena Bioscience Membrane screen3D51.5 M Li2SO4, 0.1 M Na HEPES, PH 7.5 13ThermolysinJCSG+ (2:1 water)1-220%(w/v) PEG 3K, 0.1 M Na citrate, PH 5.5 14ThermolysinJCSG+ (2:1 water)1-2120%(w/v) PEG 6k, 0.1 M citric acid, PH 5.0 15ThermolysinJCSG+ (2:1 water)2-1810%(v/v) MPD, 0.1 M bicine, PH 9.0 16ThermolysinJCSG+ (2:1 water)2-190.8 M succinic acid, PH 7.0 17ThermolysinJCSG+ (2:1 water)2-212.4 M Na malonate, PH 7.0 18ThermolysinJCSG+ (2:1 water)2-220.5%(w/v) Jeffamine ED-2001, 1.1 M Na malonate, 0.1 M Na HEPES, PH 7.0 19TrypsinJena Bioscience Membrane screen3D31.5 M NaCl, 0.1M Na acetate, PH 4.6 20TrypsinJena Bioscience Membrane screen3D31.5 M NaCl, 0.1M Na acetate, PH 4.6 21TrypsinJena Bioscience Membrane screen3D62 M NaCl, 0.1 M Na citrate 22TrypsinJena Bioscience Membrane screen3D62 M NaCl, 0.1 M Na citrate 23XylanaseStructure screen 1322 M (NH4)2SO4, 0.1 M tris, PH 8.5 24XylanaseStructure screen 13730%(w/v) PEG 4K, 0.2 M Na acetate, 0.1 M tris, PH 8.5 25XylanaseStructure screen 1454 M Na formate 26XylanaseJena Bioscience Membrane screen3B53.5 M (NH4)2SO4, 0.25M NaCl, 50mM Na/K phosphate, PH 7.5 27XylanaseJena Bioscience Membrane screen3D41.5 M K phosphate, PH 7.0 “Hit Solution”

49. Douglas Instruments Microseeding slide 49 Do any other precipitants work better than the Hit Solution for suspending seed crystals?

50. Douglas Instruments Microseeding slide 50 (a)(b)(c) Focusing on “pregnant” conditions

51. Douglas Instruments Microseeding slide 51 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? (3) Is “preseeding” the protein stock helpful? (4) How can we avoid salt crystals? (5) How can we get more diverse crystals? (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

52. Douglas Instruments Microseeding slide 52 Phase diagram of a protein [Protein] [Precipitant] clear precipitate nucleation metastable zone Reservoir stock Seed stock Protein stock

53. Douglas Instruments Microseeding slide 53

54. Douglas Instruments Microseeding slide 54 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful? (4) How can we avoid salt crystals? (5) How can we get more diverse crystals? (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

55. Douglas Instruments Microseeding slide 55 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful? (4) How can we avoid salt crystals? (5) How can we get more diverse crystals? (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

56. Douglas Instruments Microseeding slide 56 Phase diagram of a protein [Protein] [Precipitant] clear precipitate nucleation metastable zone Reservoir stock Seed stock Protein stock “Preseeded” protein stock

57. Douglas Instruments Microseeding slide 57 (a)(b)(c) Preseeding the protein

58. Douglas Instruments Microseeding slide 58 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful?Not so good! (4) How can we avoid salt crystals? (5) How can we get more diverse crystals? (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

59. Douglas Instruments Microseeding slide 59 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful?Not so good! (Better than nothing. It’s free!) (4) How can we avoid salt crystals? (5) How can we get more diverse crystals? (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

60. Douglas Instruments Microseeding slide 60 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful?Not so good! (Better than nothing. It’s free!) (4) How can we avoid salt crystals? (5) How can we get more diverse crystals? (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

61. Douglas Instruments Microseeding slide 61 Why would you want to avoid the Hit Solution? 1.To avoid salt crystals Sulfate + Ca 2+ = gypsum (plaster-of-Paris) Phosphate + Mg 2+, Ca 2+, Zn 2+, or Cd 2+ = salt crystals Matrix seeding volumes: 0.3 µl protein + 0.2 µl reservoir solution + 0.1 µl seed stock

62. Douglas Instruments Microseeding slide 62 (a)(b)(c) What can we replace the Hit Solution with?

63. Douglas Instruments Microseeding slide 63 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful?Not so good! (Better than nothing. It’s free!) (4) How can we avoid salt crystals?Suspend the seed crystals in PEG or NaCl (5) How can we get more diverse crystals? (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

64. Douglas Instruments Microseeding slide 64 Why would you want to avoid the Hit Solution? 1.To avoid salt crystals Sulfate + Mg 2+ = gypsum (plaster-of-Paris) Phosphate + Mg 2+, Ca 2+, Zn 2+, or Cd 2+ = salt crystals 2.To avoid the additive effect, thereby increasing the diversity of crystals

65. Douglas Instruments Microseeding slide 65 Increasing the diversity of crystals PDB code 2VU6: thaumatin with tartrate. P4 1 2 1 2 bipyramid crystal form. Thaumatin crystallized in the absence of tartrate. P6 1 hexagonal crystal form.

66. Douglas Instruments Microseeding slide 66 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful?Not so good! (Better than nothing. It’s free!) (4) How can we avoid salt crystals?Suspend the seed crystals in PEG or NaCl (5) How can we get more diverse crystals? Remove ingredients that you suspect may be interacting with your protein from the seed stock (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

67. Douglas Instruments Microseeding slide 67 Why would you want to avoid the Hit Solution? 1.To avoid salt crystals Sulfate + Mg 2+ = gypsum (plaster-of-Paris) Phosphate + Mg 2+, Ca 2+, Zn 2+, or Cd 2+ = salt crystals 2.To avoid the additive effect, thereby increasing the diversity of crystals 3.For crystallizing complexes, including heavy atom, small molecule and peptide derivatives

68. Douglas Instruments Microseeding slide 68 Suggested by Lesley Haire

69. Douglas Instruments Microseeding slide 69 Cross-seeding A natural approach, especially when you are adding something small e.g. a peptide Complex Uncomplexed protein crystals

70. Douglas Instruments Microseeding slide 70 Cross-seeding You don’t have to match the unit cell, only one of the structural planes of the crystals

71. Douglas Instruments Microseeding slide 71 Cross-seeding You don’t have to match the unit cell, only one of the structural planes of the crystals

72. Douglas Instruments Microseeding slide 72 Crystallizing complexes Radaev and Sun. Crystallization of protein-protein complexes. J. Appl. Cryst. (2002). 35, 674-676 PEG / (NH4)2SO4 / other salts / organic solvents (including 2-propanol, MPD, ethanol) Random samples, all protein-protein complexes included in this survey, immune complexes, antibody- antigen complexes, signal transduction complexes, receptor and ligand complexes, miscellaneous protein- protein complexes, enzyme related complexes, oligomeric protein complexes

73. Douglas Instruments Microseeding slide 73 (a)(b)(c) What can we replace the Hit Solution with?

74. Douglas Instruments Microseeding slide 74 Investigate stability of complex with isothermal calorimetry, fluorescence anisotropy, thermal shift assay etc. Test stability of seed crystals by incubation of uncrushed crystals in the suggested solution for 1 day Try to find a solution that both the seed crystals and the complex are stable in

75. Douglas Instruments Microseeding slide 75 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful?Not so good! (Better than nothing. It’s free!) (4) How can we avoid salt crystals?Suspend the seed crystals in PEG or NaCl (5) How can we get more diverse crystals? Remove ingredients that you suspect may be interacting with your protein from the seed stock (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? Avoid high salt in your seed stock; remove ingredients.... (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

76. Douglas Instruments Microseeding slide 76 Can we predict which solutions the seed crystals will be stable in?

77. Douglas Instruments Microseeding slide 77 (a)(b)(c) Focusing on “pregnant” conditions

78. Douglas Instruments Microseeding slide 78 Appearance of crystals after incubation for one day Protein Crystals in Hit Sol. Crystals in Isopropanol Crystals in PEG 600 Crystals in Amm.sul. Crystals in NaCl Crystals in protein stock Gluc. Isom.OKCrackedShatteredCrackedDissolved HemoglobinOKCrackedOKDissolved Thaumatin Thermolysin OK Cracked OK Shattered OK Dissolved Grew Trypsin Xylanase OK Dissolved Cracked OK Dissolved

79. Douglas Instruments Microseeding slide 79 Appearance of crystals after incubation for one day Protein Crystals in Hit Sol. Crystals in Isopropanol Crystals in PEG 600 Crystals in Amm.sul. Crystals in NaCl Crystals in protein stock Gluc. Isom.OKCrackedShatteredCrackedDissolved HemoglobinOKCrackedOKDissolved Thaumatin Thermolysin OK Cracked OK Shattered OK Dissolved Grew Trypsin Xylanase OK Dissolved Cracked OK Dissolved Glucose isomeraseJCSG+2-43 25%(w/v) PEG 3350, 0.2 M (NH4)2SO4, 0.1 M bis-tris HemoglobinJCSG+2-33 30%(w/v) PEG 2000 MME, K thiocyanate

80. Douglas Instruments Microseeding slide 80 (a)(b)(c) Preseeding the protein

81. Douglas Instruments Microseeding slide 81 Appearance of crystals after incubation for one day Protein Crystals in Hit Sol. Crystals in Isopropanol Crystals in PEG 600 Crystals in Amm.sul. Crystals in NaCl Crystals in protein stock Gluc. Isom.OKCrackedShatteredCrackedDissolved HemoglobinOKCrackedOKDissolved Thaumatin Thermolysin OK Cracked OK Shattered OK Dissolved Grew Trypsin Xylanase OK Dissolved Cracked OK Dissolved

82. Douglas Instruments Microseeding slide 82 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful?Not so good! (Better than nothing. It’s free!) (4) How can we avoid salt crystals?Suspend the seed crystals in PEG or NaCl (5) How can we get more diverse crystals? Remove ingredients that you suspect may be interacting with your protein from the seed stock (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? Avoid high salt in your seed stock; remove ingredients (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

83. Douglas Instruments Microseeding slide 83 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful?Not so good! (Better than nothing. It’s free!) (4) How can we avoid salt crystals? Suspend the seed crystals in PEG or NaCl... test by incubation for 1 day (5) How can we get more diverse crystals? Remove ingredients that you suspect may be interacting with your protein from the seed stock (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? Avoid high salt in your seed stock; remove ingredients.... test by incubation for 1 day (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

84. Douglas Instruments Microseeding slide 84 rMMS with membrane proteins Crystals of membrane proteins are often unstable Remember that the reservoir normally has no detergent! Harvest several large drops without dilution 1.5 microlitres are enough! MPL (Diamond Light Source) / Douglas Instruments: 2 of 5 projects worked very well

85. Douglas Instruments Microseeding slide 85 random Microseed Matrix-Screening Our questions:Take-home practical suggestions: (1) How can we get as many hits as possible? Stick to the ‘hit solution’ for suspending seed crystals for routine rMMS (2) How stable are the seed stocks? Not completely stable so use your seed stock quickly, then freeze. Or cross-link. (3) Is “preseeding” the protein stock helpful?Not so good! (Better than nothing. It’s free!) (4) How can we avoid salt crystals? Suspend the seed crystals in PEG or NaCl... test by incubation for 1 day (5) How can we get more diverse crystals? Remove ingredients that you suspect may be interacting with your protein from the seed stock (6) How can we stabilize protein complexes, including heavy atom, small molecule and peptide derivatives ? Avoid high salt in your seed stock; remove ingredients.... test by incubation for 1 day (7) Can we harvest seed crystals from microfluidic devices? (8) What can you do if you have no crystals?

86. Douglas Instruments Microseeding slide 86

87. Douglas Instruments Microseeding slide 87 Thank you for listening!

88. Douglas Instruments Microseeding slide 88 Thank you for listening! Shaw Stewart et al., Cryst. Growth Des., 2011, 11 (8), p3432.

89. Douglas Instruments Microseeding slide 89 Crosslinking seed crystals after crushing Protein Seeds in Hit Sol Seeds in PEG 600 X-linked seeds in PEG, used immediately X-linked seeds in PEG, 1wk 20°C Seeds in NaCl X-linked seeds in NaCl, used immediately X-linked seeds in NaCl, 1wk 20°C Gluc. isom.18199099 Hemoglobin10 70000 Trypsin36 13333625