Crystallization

What you need• Pure Protein

– Native PAGE - IEF– SDS PAGE - Mass Spec– N-terminal sequencing

• Single aggregation state– Monomeric, dimeric, heteromeric– Dynamic Light Scattering (DLS)

• Stable for a reasonable period of time– Dependent on stabilization buffer, 4°C vs Room temperture

• Concentrated– Need a concentration that will precipitate in screens about 30-40% of

the time.

What you can do to improve your chances of crystallization

• Make a bunch of protein (2-50mg/L is nice)• Evaluate your construct prior to starting,

Avoid:– Floppy ends – Long tags

Effect of protein concentration• The more concentrated the protein the more it interacts

with itself in the crystallization solution and can form crystals.

• If it’s too concentrated it can aggregate and precipitates too readily in the crystallization conditions.

• A protein should precipitate about 30-40% of the time in a screen.

• Rule of thumb is that the protein should be concentrated enough to precipitate in condition #4 and #6 of the Hampton Screen #1.

Reality of Protein Concentration• People have been able to crystallize

proteins at concentrations from 0.5 – 50 mg/ml.

• Larger proteins require less concentrated solutions.

• Small highly soluble proteins require more concentrated solution.

Theory of Crystallization• The protein is mixed with a crystallization

solution containing various precipitants. (usually 2l and 2l) (called the drop)

• The diluted protein and crystallization mixture is incubated “over” the concentrated crystallization solution (called the well).

• Over time, the drop will equilibrate with the well concentrating both the protein and the precipitants.

• As the protein concentrates it can come out of solution as either a precipitate, a phase transition (oil), or as a crystal.

Theory of Crystallization II

Increasing protein concentration

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Types of crystallization setups

Hanging drop Sitting DropCover slip(attached with grease)

Clear tape

Drop(Protein/precipitant mix)

Well(precipitant)

What’s in the “well.”• “Precipitant”

– Buffers• Screens usually contain 100 mM Buffer pH 4.5-9

– Salts• Huge variety here, usually at 200 mM in screens

– Precipitants• Poly Ethylene Glycols (PEGS)

– 10-30%– MW 400 to 10,000 with very different results

• Salts (AmSO4, LiCl, NaFormate, NaCitrate @ 1-3M)• Ethanol, MPD, iso-propanol

Types of screens

• Hampton Research®: Crystal Screen I and II• Emerald Biosystems®: Wizard Screen I and II• Usually 24-50 solutions• Both Companies go on to produce a variety of

specialty screens– PEG/ION– CRYO (conditions ready for freezing)– AmSO4

– MPD

Evaluating Screens

• Light, Medium or Heavy precipitate– Note whether precipitate is always in a certain type of condition,

<pH 7, PEGS, Salts etc.• Oiling out

– Phase separation • Aggregates

– Protein is forming gloopy bunches, might lead to crystals if conditions are tweaked.

• Microcrystalline– Best start you can have other large crystals. Usually just need to

reduce the main precipitant concentration. – Depending on how small the crystals it may be difficult to see the

crystals and might appear as a precipitate. Experience will tell.

What to do if you have a hit!• Usually the screens have too much precipitate

– Example: Screen may contain 30% PEG 4K but crystals optimum is 10-15%

– Less precipitate usually means larger crystals from fewer nucleations.• Check other salts, buffers and precipitants near to the current

condition.– Check everything you can think of. Do not rely on the variety in the

screens and assume that all the other conditions have been checked.– Change pH over range 0.2. – Check concentration of best salt (~50-300mM)

• Once stable conditions have been found/repeated, then try small amounts of “addatives.”

Additives

• I usually use this term to mean an additional chemical added in small amounts to alter the crystallization condition.

• 20-100l of any of the following is a a good start. (There are also purchased screens)– MPD, Ethanol, iso-propanol, PEG 400, DTT/ME, 1,4-

Dioxane, Ethylene Glycol, Glycerol, NaCl, SB-12

What to do if you don’t• Evaluate the Screen carefully.

– What pH’s are soluble, what salts– Does your protein need more salt/glycerol to remain stable?

• Crystallize in the presence of ligand.• Change the stabilization buffer or protein

concentration.• Did you really check the purity thoroughly before?• Try chemical modification of your protein.

– Iodoacetate, Iodoamide• Try and different constructs

– +/- tags

Value of ligands, substrates or inhibitors

• Very important to try to crystallize in the presence and absence of ligands.

• Ligands can bind to protein and cause conformational changes.

• Might try different ratios of ligand to protein.• Add multiple substrates at a time. • Try an inactive protein with substrate rather than

an inhibitor with the wild-type protein.

Types of protein modifications

• Huge variety I won’t cover here.• If your protein is too soluble you can modify with

iodoacetimide.• If you protein isn’t soluble enough, you can try to

modify with iodoacetate.• 5-10mM overnight at room temp. No reductants.

Then wash out chemical. Modification is hard to evaluate, may not be uniform across protein population.

Qualities of a Good Crystal

• Single (not attached to others, grown in cluster etc. – this might be fine, or might be evidence of problems)

• Straight edges.– Curves are generally bad– Experienced crystallographers can tell the point group of

a crystal from the crystal itself.• Reasonable size, 100-400nm• Despite what I’ve said here some great looking

crystals don’t diffract well and some horrible looking crystals diffract great. You never know.

Great crystals!

Good example of oiling out

Crystals are probably too small

Clusters This cluster probably okay