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Microseed it! Introduction to microseeding Patrick Shaw Stewart

Microseed it! Introduction to microseeding Patrick Shaw Stewart Douglas Instruments Limited (near Oxford, UK): Peter Baldock, Patrick Shaw Stewart, Richard Briggs, Stefan Kolek. Crystallization breakthroughs. Jankaric and Kim (1991) – sparse matrix screening

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Microseed it! Introduction to microseeding Patrick Shaw Stewart

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

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

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

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

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

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

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

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

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

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

  14. Microseeding in screening experiments D’Arcy et al. ActaCryst. (2007). D63

  15. Microseeding in screening experiments D’Arcy et al. ActaCryst. (2007). D63

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

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

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

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

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

  21. Case study – Obmolovaet al, ActaCryst (2010) D66, 927 - 933 Conventional methods Complexes: IL-13/C836 (mouse antibody) IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) 40 residues changed 4 residues changed

  22. Case study – Obmolovaet al, ActaCryst (2010) D66, 927 - 933 Conventional methods Complexes: IL-13/C836 (mouse antibody) 192 conditions IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) No hits 40 residues changed 4 residues changed

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

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

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

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

  27. Case study – Obmolovaet al, ActaCryst (2010) D66, 927 - 933 Conventional methods Random microseeding (rMMS) Complexes: IL-13/C836 (mouse antibody) IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) No hits 40 residues changed Microseeding One hit Optimization 4 residues changed Both 1.9 Å resolution orthorhombic P212121 No hits

  28. Case study – Obmolovaet al, ActaCryst (2010) D66, 927 - 933 Conventional methods Random microseeding (rMMS) Complexes: IL-13/C836 (mouse antibody) IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) No hits 40 residues changed Microseeding One hit Optimization 4 residues changed Both 1.9 Å resolution orthorhombic P212121 No hits Cross-seeding 2.8 Å res. P212121

  29. Case study – Obmolovaet al, ActaCryst (2010) D66, 927 - 933 Conventional methods Random microseeding (rMMS) Complexes: IL-13/C836 (mouse antibody) IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) No hits 40 residues changed Microseeding One hit Cross-seeding Optimization 4 residues changed Both 1.9 Å resolution orthorhombic P212121 No hits Cross-seeding 2.8 Å res. P212121

  30. Case study – Obmolovaet al, ActaCryst (2010) D66, 927 - 933 Conventional methods Random microseeding (rMMS) Complexes: IL-13/C836 (mouse antibody) IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) 2.0 Å res. monoclinic P21 No hits Optimization 40 residues changed Microseeding One hit Cross-seeding Optimization 4 residues changed Both 1.9 Å resolution orthorhombic P212121 No hits Cross-seeding 2.8 Å res. P212121

  31. Case study – Obmolovaet al, ActaCryst (2010) D66, 927 - 933 Conventional methods Random microseeding (rMMS) Complexes: IL-13/C836 (mouse antibody) IL-13/H2L6 (humanized mAb) IL-13/M1295 (affinity-matured humanized mAb) 2.0 Å res. monoclinic P21 2.0 Å res. monoclinic P21 No hits Optimization 40 residues changed Microseeding Microseeding One hit Cross-seeding Optimization 4 residues changed Microseeding Both 1.9 Å resolution orthorhombic P212121 No hits Cross-seeding 2.8 Å res. P212121

  32. Matrix seeding volumes: • 0.3 µl protein • + 0.2 µl reservoir solution • + 0.1 µl seed stock

  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. Microseeding toolkit

  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/cg2001442

  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/cg2001442

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

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

  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. random Microseed Matrix-Screening

  44. “Receptive” conditions • Conditions where: • crystals don’t grow without seeds in four drops, but • crystals grow in at least three out of four drops with seeds.

  45. “Receptive” conditions • Conditions where: • crystals don’t grow without seeds in four drops, but • crystals grow in at least three out of four drops with seeds. • 25 receptive conditions were found

  46. “Hit Solution”

  47. Do any other precipitants work better than the Hit Solution for suspending seed crystals?

  48. Focusing on “pregnant” conditions (a) (b) (c)

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