The document provides information on protein crystallization, including what is needed to crystallize a protein, how to improve crystallization chances, the theory behind crystallization setups and conditions, evaluating crystallization screens, optimizing conditions if hits are found, adding ligands or modifying the protein if no crystals are obtained initially, and qualities of good crystals. The goal is to crystallize pure, concentrated, monodisperse protein in conditions that will cause it to come out of solution and form ordered crystal lattices for structure determination.

1. Crystallization

2. 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.

3. 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

4. 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.

5. 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.

6. Theory of Crystallization
• The protein is mixed with a crystallization
solution containing various precipitants. (usually
2µl and 2µl) (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.

7. Theory of Crystallization II
Increasing precipitant concentration
precipitant
Crystalline?
soluble
Increasing protein concentration

8. Types of crystallization setups
Hanging drop Sitting Drop
Cover slip Drop
(attached with grease) (Protein/precipitant mix)
Clear tape
Well
(precipitant)

9. 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

10. 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

11. 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.

12. 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.”

13. Additives
• I usually use this term to mean an additional
chemical added in small amounts to alter the
crystallization condition.
• 20-100µl of any of the following is a a good start.
(There are also purchased screens)
– MPD, Ethanol, iso-propanol, PEG 400, DTT/βME, 1,4-
Dioxane, Ethylene Glycol, Glycerol, NaCl, SB-12

14. 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

15. 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.

16. 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.

17. 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.

18. Clusters This cluster probably okay
Crystals are
probably too Good example
small of oiling out
Great crystals!