1. A Review of Seeding Methods Allan D’Arcy, Aengus Mac Sweeney, Alex Haber RAMC, 2005

2. 2 Review of Seeding Methods / RAMC 2005 Obvious First Steps Vary initial conditions: Protein concentration, precipitating agent, salt, pH, temperature Additives: Detergent, glycerol, ethylene glycol, organic solvents etc. Setup type: Microbatch, hanging drop, sitting drop, oil in reservoir Seeding: Microseeding, macroseeding Optimisation of crystal quality

3. 3 Review of Seeding Methods / RAMC 2005 Nucleation and seeding ~50% of proteins require seeding to obtain good crystals

4. 4 Review of Seeding Methods / RAMC 2005 Nucleation and seeding Nucleation Different types of seeding Screening with seeding

5. 5 Review of Seeding Methods / RAMC 2005 Nucleation The nucleation event is a parameter that can be difficult to control. A sufficiently high level of saturation is often not reached and the critical nucleation event cannot occur. Local variations in protein and precipitant concentration favour nucleation. The formation of a core that allows further crystal growth

6. 6 Review of Seeding Methods / RAMC 2005 Nucleation and crystal growth Many drops remain clear during screening

7. 7 Review of Seeding Methods / RAMC 2005 A Real Phase Diagram

8. 8 Review of Seeding Methods / RAMC 2005 What affects nucleation? Sample concentration Sample preparation (filtering, storage) Method used: VD, MB, FID Mixing Surfaces

9. 9 Review of Seeding Methods / RAMC 2005 Mixing drops on set up Unmixed drops sample a greater range of protein and precipitant concentrations – nucleation is more likely

10. 10 Review of Seeding Methods / RAMC 2005 Uses for seeding Reproducing difficult crystallizations Obtaining favoured crystal form Cocrystallizing with inhibitors Improve screening success Screening for “crystal growth” conditions

11. 11 Review of Seeding Methods / RAMC 2005 Macro Different Seeding Methods (I) With fresh crystals the “crystal washing” step is often unnecessary More focused X-ray sources reduce need for macroseeding

12. 12 Review of Seeding Methods / RAMC 2005 Different Seeding Methods (II) Streak seeding with needle or hair Micro

13. 13 Review of Seeding Methods / RAMC 2005 “Seed anything that might be microcrystalline” (Terese Bergfors) (Spherulites are crystalline – test diffraction) FabD spherulites

14. 14 Review of Seeding Methods / RAMC 2005 Seed Beads Microdilution Different Seeding Methods (III)

15. 15 Review of Seeding Methods / RAMC 2005 Concentrated seed stock 1:5 diluted seed stock 1:25 diluted seed stock Dilution series of stock made with seed bead Same protein streak and micro (bead) seeded Microseeding

16. 16 Review of Seeding Methods / RAMC 2005 Microseed matrix screening to improve crystals of yeast cytosine deaminase. Ireton, G.C. & Stoddard, B.L. Acta Crystallogr D (2004), 60(3), 601-5. A crystallization strategy termed “microseed matrix screening” is described where the optimal conditions for nucleation versus extended lattice growth are not compatible. Microseed matrix screening

17. 17 Review of Seeding Methods / RAMC 2005 Influencing nucleation at the start Problems: We need seeds The seeds must be stable in the initial conditions

18. 18 Review of Seeding Methods / RAMC 2005 McPherson & Schlichta 1987: Crushed mineral materials Punzi et al. 1991: Polyvinylidene difluoride Chayen et al. 2001: Porous silicon Fermani et al. 2002: Modified mica Haushalter & McPherson 2002: Nanoengineered Surfaces Rong et al. 2004: Porous glass The nucleation problem

19. 19 Review of Seeding Methods / RAMC 2005 Influencing nucleation using growth surfaces “Built-in” Heterogeneous Seeding Parallel Synthesis Technologies, Inc.

20. 20 Review of Seeding Methods / RAMC 2005 Natural heterogeneous seeding? Viral serine protease Tryptase Elastase Cysteine protease Serine protease

21. 21 Review of Seeding Methods / RAMC 2005 Crystals of chicken triose phosphate isomerase (the first TIM barrel) taken in 1970 Courtesy of David Banner

22. 22 Review of Seeding Methods / RAMC 2005 Horse hair is used for streak seeding and has a complex surface structure

23. 23 Review of Seeding Methods / RAMC 2005 Nucleation on horse hair

24. 24 Review of Seeding Methods / RAMC 2005 Using the sonicated stock Control: no hair addedDiluted hair addedConcentrated hair added

25. 25 Review of Seeding Methods / RAMC 2005 Crystals growing on crushed horse hair

26. 26 Review of Seeding Methods / RAMC 2005 Testing on different proteins ProteinControl Seed dilution (0 seeds)1:51:25 Trypsin0758 Glucose Isom.112564 Lysozyme196 FabD082

27. 27 Review of Seeding Methods / RAMC 2005 A difficult case: Fab-D Hair has been incorporated within the crystal

28. 28 Review of Seeding Methods / RAMC 2005 Thanks to: Frederic Villard Daniel Mareque Terese Bergfors

29. 29 Review of Seeding Methods / RAMC 2005 Workshop Streak seeding Solution Microseeding Heterogeneous seeding Edge seeding Lysozyme 30 mg/ml 500ul reservoir solution 1 crystal drop each for seeding