A Review of Seeding Methods

Allan D’Arcy, Aengus Mac Sweeney, Alex Haber

RAMC, 2005

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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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Nucleation and seeding

~50% of proteins require seeding to obtain good crystals

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Nucleation and seeding

Nucleation Different types of seedingScreening with seeding

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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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Nucleation and crystal growth

Many drops remain clear during screening

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A Real Phase Diagram2 Points Interpolation2 Points Interpolation

[Protein]

[Precipitant]

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What affects nucleation?

Sample concentration

Sample preparation (filtering, storage)

Method used: VD, MB, FID

Mixing

Surfaces

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Mixing drops on set up

Unmixed drops sample a greater range of protein and precipitant concentrations – nucleation is more likely

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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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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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Different Seeding Methods (II)

Streak seeding with needle or hair

Micro

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“Seed anything that might be microcrystalline” (Terese Bergfors)

(Spherulites are crystalline – test diffraction)

FabD spherulites

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Seed Beads

Microdilution

Different Seeding Methods (III)

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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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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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Influencing nucleation at the start

Problems:

We need seeds

The seeds must be stable in the initial conditions

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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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Influencing nucleation using growth surfaces

“Built-in” Heterogeneous Seeding

Parallel Synthesis Technologies, Inc.

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Natural heterogeneous seeding?

Viral serine protease

Tryptase Elastase

Cysteine protease

Cysteine protease

Serine protease

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Crystals of chicken triose phosphate isomerase (the first TIM barrel) taken in 1970

Courtesy of David Banner

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

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Nucleation on horse hair

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Using the sonicated stock

Control: no hair added Diluted hair addedConcentrated hair added

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Crystals growing on crushed horse hair

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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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A difficult case: Fab-D

Hair has been

incorporated within

the crystal

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Thanks to:

Frederic Villard

Daniel Mareque

Terese Bergfors

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Workshop

Streak seeding

Solution Microseeding

Heterogeneous seeding

Edge seeding

Lysozyme 30 mg/ml

500ul reservoir solution

1 crystal drop each for seeding