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A Review of Seeding Methods
Allan D’Arcy, Aengus Mac Sweeney, Alex Haber
RAMC, 2005
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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
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3 Review of Seeding Methods / RAMC 2005
Nucleation and seeding
~50% of proteins require seeding to obtain good crystals
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4 Review of Seeding Methods / RAMC 2005
Nucleation and seeding
Nucleation Different types of seedingScreening with seeding
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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
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6 Review of Seeding Methods / RAMC 2005
Nucleation and crystal growth
Many drops remain clear during screening
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7 Review of Seeding Methods / RAMC 2005
A Real Phase Diagram2 Points Interpolation2 Points Interpolation
[Protein]
[Precipitant]
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8 Review of Seeding Methods / RAMC 2005
What affects nucleation?
Sample concentration
Sample preparation (filtering, storage)
Method used: VD, MB, FID
Mixing
Surfaces
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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
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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
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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
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12 Review of Seeding Methods / RAMC 2005
Different Seeding Methods (II)
Streak seeding with needle or hair
Micro
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13 Review of Seeding Methods / RAMC 2005
“Seed anything that might be microcrystalline” (Terese Bergfors)
(Spherulites are crystalline – test diffraction)
FabD spherulites
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14 Review of Seeding Methods / RAMC 2005
Seed Beads
Microdilution
Different Seeding Methods (III)
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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
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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
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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
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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
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19 Review of Seeding Methods / RAMC 2005
Influencing nucleation using growth surfaces
“Built-in” Heterogeneous Seeding
Parallel Synthesis Technologies, Inc.
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20 Review of Seeding Methods / RAMC 2005
Natural heterogeneous seeding?
Viral serine protease
Tryptase Elastase
Cysteine protease
Cysteine protease
Serine protease
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21 Review of Seeding Methods / RAMC 2005
Crystals of chicken triose phosphate isomerase (the first TIM barrel) taken in 1970
Courtesy of David Banner
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22 Review of Seeding Methods / RAMC 2005
Horse hair is used for streak seeding and has a complex surface structure
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23 Review of Seeding Methods / RAMC 2005
Nucleation on horse hair
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24 Review of Seeding Methods / RAMC 2005
Using the sonicated stock
Control: no hair added Diluted hair addedConcentrated hair added
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25 Review of Seeding Methods / RAMC 2005
Crystals growing on crushed horse hair
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26 Review of Seeding Methods / RAMC 2005
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
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27 Review of Seeding Methods / RAMC 2005
A difficult case: Fab-D
Hair has been
incorporated within
the crystal
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28 Review of Seeding Methods / RAMC 2005
Thanks to:
Frederic Villard
Daniel Mareque
Terese Bergfors
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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