A Review of Seeding Methods

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

2. Optimisation of crystal quality • 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

3. Nucleation and seeding • ~50% of proteins require seeding to obtain good crystals

4. Nucleation and seeding Nucleation Different types of seeding Screening with seeding

5. Nucleation • The formation of a core that allows further crystal growth 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.

6. Nucleation and crystal growth Many drops remain clear during screening

7. A Real Phase Diagram

8. What affects nucleation? • Sample concentration • Sample preparation (filtering, storage) • Method used: VD, MB, FID • Mixing • Surfaces

9. Mixing drops on set up • Unmixed drops sample a greater range of protein and precipitant concentrations – nucleation is more likely

10. Uses for seeding • Reproducing difficult crystallizations • Obtaining favoured crystal form • Cocrystallizing with inhibitors • Improve screening success • Screening for “crystal growth” conditions

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

12. Different Seeding Methods (II) Micro Streak seeding with needle or hair

13. “Seed anything that might be microcrystalline” (Terese Bergfors) • FabD spherulites • (Spherulites are crystalline – test diffraction)

14. Different Seeding Methods (III) Microdilution Seed Beads

15. Microseeding Dilution series of stock made with seed bead Concentrated seed stock 1:5 diluted seed stock 1:25 diluted seed stock • Same protein streak and micro (bead) seeded

16. Microseed matrix screening 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.

17. Influencing nucleation at the start • Problems: • We need seeds • The seeds must be stable in the initial conditions

18. 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. Influencing nucleation using growth surfaces • “Built-in” Heterogeneous Seeding Parallel Synthesis Technologies, Inc.

20. Natural heterogeneous seeding? Serine protease • Cysteine protease • Viral serine protease Elastase • Cysteine protease Tryptase

21. Crystals of chicken triose phosphate isomerase (the first TIM barrel) taken in 1970 Courtesy of David Banner

22. Horse hair is used for streak seeding and has a complex surface structure

23. Nucleation on horse hair

24. Using the sonicated stock Control: no hair added Concentrated hair added Diluted hair added

25. Crystals growing on crushed horse hair

26. Testing on different proteins • Protein Control Seed dilution • (0 seeds) 1:5 1:25 • Trypsin 0 75 8 • Glucose Isom. 1 125 64 • Lysozyme 1 9 6 • FabD 0 8 2

27. A difficult case: Fab-D • Hair has been • incorporated within • the crystal

28. Thanks to: • Frederic Villard • Daniel Mareque • Terese Bergfors

29. Workshop • Lysozyme 30 mg/ml • 500ul reservoir solution • 1 crystal drop each for seeding • Streak seeding • Edge seeding • Solution Microseeding • Heterogeneous seeding