Lecture 2

Crystals: Theory and Practice

Dr. Susan Yates

Wednesday, February 2, 2011

Steps in Solving an X-ray Structure

What is a Crystal?

Crystal acts as an X-ray diffraction amplifier

Crystals• Crystals consist of a structural motif, repeated at regular spacings

• Unit cell

• The smallest repeating unit that can generate the entire

crystal using only translation operations

•Mathematical concept - one molecule does not need to fit neatly in this “box”

Crystal Lattice• Lattice

• The set of points in the

crystal that are equivalent to each other

• Geometric

arrangement of the points in space at which the atoms/molecules/ions of a crystal occur

Crystal Lattice

Precipitating Proteins

• In a concentrated protein solution, proteins interact with water and with other proteins

• Proteins stay in solution as long as the interactions they make with water are energetically more favourable than those they make with other proteins

• If you alter this equilibrium (e.g. by competing water away using high salt concentrations - salting out), proteins start to bind one another and precipitate

Protein Precipitation

• When a precipitation agent is added to a concentrated protein solution, protein-protein interactions become energetically more favourablethan protein-solvent interactions

• Proteins bind one another and come out of solution

• No preferred way that the proteins interact

• Result is a precipitate with no long range order

Protein Crystallization

• Crystallization differs from precipitation because each molecule in the “precipitate” interacts with its neighbours in the same way as every other molecule

• The result is a highly ordered arrangement - a crystal

Crystallizing a Protein• Growing a protein crystal requires controlledprecipitation

• Interactions between individual protein molecules in a crystal are stabilized by energetically favourable contacts

• The forces involved include hydrogen bonds, salt bridges, hydrophobic effect etc.

• Tricky to find this condition and prevent formation of non-specific aggregates

Energy Barrier to Crystallization

crystal representsthe lowest free-

energy

∆G=∆H-T∆S

unfavourablefavourable

Crystallization: Solubility• Protein solubility varies with the concentration of salts, polyethylene glycols and other substances in the protein solution

• A protein will quickly form an amorphous precipitate if the solubility is lowered drastically

• A protein might crystallize if the concentration is slightly above the solubility limit

Crystallization Phase Diagram

Crystallization

Clear

Crystallization Process• Productive crystallization process fluctuates between Nucleation and Clear zones, largely due to decreased protein concentration since protein sample is consumed in crystal formation

Crystallizing Agents• Salts

• e.g. Sulfates, phosphates

• Long chain organic polymers

• Polyethylene glycols (PEG)

• Organic solvents

• Generally hydrophilic alcohols, ethers or ketones

• e.g. Methyl-pentanediol, isopropanol

Methods to Precipitate a Protein• High salt

• The salt ions order water molecules around them, leaving less unstructured water to solubilize the protein

• Organic solvents

• These effectively dilute water with a less polar, less H-bond capable solvent with lower dielectric etc.

• Long chain organic polymers

• PEG prefers to writhe over a large volume of space

• Taking the protein out of solution frees up more space for PEG and is energetically favoured

Other FactorsInfluencing Crystallization

• Protein concentration

• Need less precipitant to precipitate the more concentrated the protein

• pH

• Changing the pH adds/removes protons from individual residues, possibly creating new salt bridges/H-bonds

• Temperature

• As temperature changes, so do the enthalpic and entropic contributions to ∆Gcrystallization

• Presence of ligands

• Ligands may lock the protein into one conformation, which can help crystallization

Vapour Diffusion• Small volumes of precipitant and protein mixed together into a drop which is equilibrated against a larger reservoir of solution containing precipitant or dehydrating agent

• Reservoir or crystallization solution can be a mixture of many combinations

• Buffer (type and conc), pH, precipitant (type and conc), temperature, protein conc, ionic strength etc.

Hanging drop

Sitting drop

Vapour Diffusion

Vapour Diffusion• Slowly increases protein and precipitant concentrations

• 12 h to 4 days to equilibrate

• Mix protein solution with precipitant solution (1:1) and equilibrate against excess of the latter

• Need 1 µL of 10 mg/ml protein solution per experiment (well)

Typical Crystallization Procedures• Screening

• Start with commercial screening kits derived from extensive practical experience; there are hundreds mixtures covering wide range of conditions

• Optimization

• Once a lead condition is found from the screening process, expansion (pH and concentration of precipitant etc.) will be carried out

Culture plate

Micro-crystals

Small crystals

Good single

(~0.1-0.3 mm)

If you are lucky half of all your

protein constructs will crystallize

The Practicalities of GrowingMacromolecule Crystals

Crystal Screening• Combinations of precipitating agents and factors that might lead to a crystal is near infinite

• A typical protein will only crystallize in a small fraction of these conditions

• When screening you look for crystal leads

• Anything that appears crystalline

• Unlikely to get big, picture perfect crystals

• Not all proteins crystallize!

• Often you have to go back, purify your protein further, make a new construct…

Finding Initial Conditions• Check crystal set-ups every day in first week

• Possible results

• Clear, precipitate, crystal and many others (turbid, bubbles, clothing fibers)

• If almost all or almost no drops are clear, raise or lower protein concentration, respectively

• Focus on set-ups that show some precipitate, but not a heavy yellow or brown precipitate indicative of protein denaturation

Crystal Refinement• Refinement is the process by which known crystals are improved once initial crystals have been found by screening

• Fine-tuning the conditions

• Changing the PEG concentration from 30% to 35%

• Increasing the pH from 5.5 to 6.5

• Adding 4% glycerol

• Increasing salt concentration from 200 mM to 300 mM

• The process is generally iterative

• Stop when the crystals are single and big enough to undergo diffraction testing (50-300 µm)

Improving Size and Diffraction• Systematic variation of all concentrations and pH

• Additive screens and detergent screens

• Temperature

• Seeding with crushed crystals (micro seeding)

• Dialysis, batch, sitting drop

• … check old set-ups for different crystal form

Crystallization Examples

• Lysozyme

• 100 mg/ml protein in 50 mM sodium acetate pH 4.5

• 30% (w/v) PEG 5000, 1.0 M NaCl, 50 mM sodium acetate pH 4.5

• Glucose isomerase

• 20-30 mg/ml protein in water or 50 mM buffer pH 6.8

• 1.5-2.5 M ammonium sulfate pH 6-9

Crystal Growth• Lysozyme crystal growth in a few hours time

• Most will take days to weeks

Example of a Protein Crystal

• The contacts between molecules are generally tenuous, involving only a handful of residues

• Protein crystals contain large solvent channels, typically making up 40% - 70% of its volume

• The crystalline order is destroyed by exposing a crystal to air (solvent evaporates) or to mechanical stress (behaves somewhat like watermelon flesh)

Crystal Galleries

Dust/fibre assisted nucleation

Crystal Galleries

Crystal Galleries

Clear Junk

Ppt (Hope) Ppt (No Hope)

Skins

Phase Separation

“Things” in Drops (Other than Crystals)

It Will Not Crystallize…• Check purity and stability

• Remove cysteins and other trouble makers

• Remove flexible parts

• Try single domains

• Try physiologically relevant complexes

• Be creative!

Truth Behind Crystallization

Obtaining Well-Diffracting CrystalsTake-home message

Getting a crystal can be hard

Goal

Three-dimensional single crystal

• A good protein sample

• Principles of crystal growth

• Crystallization techniques

• Strategies to obtain well-diffracting crystals (quickly?)

• Practical considerations

Small Molecule and Large Crystal• The world's largest (701 lbs) fast-growth crystal, grown at Lawrence Livermore National Laboratory

• The pyramid-shaped potassium dihydrogen phosphate crystal measures ~26x21x23”

• The enormous crystal was sliced into ½” thick plates and used in a giant laser that will “help maintain the safety and reliability of the nation's nuclear weapons stockpile”

Next time…• Instrumentation

• Waves and Diffraction