Original document:developed by Tristan Fiedler for

2003 ACA Summer School in Macromolecular Crystallography

Augmented 6 July 2004 by Andy HowardRebuilt for Biology 555, spring 2008

Protein CrystallizationTheory & Practice

krystallos?krustallos?

Journalist’s Criteria

WhoWhatWhenWhereWhyHow

Whither

Whence

(Wherefore)

Who makes macromolecular crystals?

Macromolecular crystals are almost always produced artificially, i.e., by human action

So “who” is “scientists”

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

What are macromolecular crystals?

Crystals are translationally ordered arrays of moleculesMacromolecular crystals are held together by relatively weak ionic intermolecular forcesSolvent content generally above 40%

When have we made them?

Story goes back into the mid-19th centurySystematic search for crystallization conditions dates from the 1950’sScreening kits: concept 1980’s, commercialization in 1990’sRobotics late 1990’sNanoscale techniques early 21st century

Very old (reproduction, genetic duplication)

Empirical (trial & error -- ‘Screens’)

‘Art’ vs ‘Science’

Crystallization

1853 Hemoglobin Lehman, CG. Lehrbuch der physiologische

Chemie. Leipzig

1926 Urease Sumner, JB. J Biol Chem. 69: 435

1930 Pepsin & other proteolytic enzymes Northrop, JH, Kunitz, M, Herriot RM. Crystalline Enzymes. Columbia

University Press, NY (review)

1934 Pepsin Diffraction Bernal JD & Crowfoot, D. Nature,

133:794

1935 Tobacco Mosaic Virus Stanley, WM. Science. 81:

644

Short History1946 Nobel (Chemistry)

Short history (concluded)

1946: Sumner Nobel prize

1958: Myoglobin structure

1959: Hemoglobin structure

1962: Perutz/Kendrew Nobel

1979: Carter&Carter paper

1985: first microgravity experiments

1990’s: commercial screening kits

Late 1990’s: viable commercial crystallization robots

Where do we grow them?

Under mild laboratory conditions

Contrast to inorganic small molecules, which are often grown from a melt

Even small organic crystals often exploit temperature dependence

Proteins usually avoid these techniques…

Inorganic - cooling a hot saturated substance

Polar organic - same or ppt from aqueous using organic solvents

Proteins - Yeoww!! (denature)

1.Dissolved in buffer + ppt [low]

2.controlled evaporation [higher]

Growing [Protein] Crystals

Why do we grow them?

Because we want to know the macromolecule’s structure!

Fundamental postulate:The structure of a protein in a crystal differs only slightly, and then only on the surface, from that its soluble or membrane-associated (biologically active) form

Do we believe this?

Short answer: yes

Skepticism was rampant through the 1970’s and has only gradually diminished

Various experimental demonstrations

Evidence that(xtal) = (solution)

Enzymatic activity of crystals (1970’s)

Similarity of multiple crystal forms

Comparisons to NMR structures

Consistency with other biophysical techniques

When is the postulate wrong?

Some external loops held in wrong positions (Interleukin-8)Much more common: crystal structure shows us one conformer; other conformers, and the transitions among them, are relevant

How to make 3-D crystals

In general it involves creation of three-dimensional order

In practice with macromolecules that means creating conditions in which intermolecular forces can be exerted in the same way on each molecule

These intermolecular forces arePolar

Often water-mediated

Weak

How do we grow macromolecular crystals?

Short answer: we gradually decrease the solubility of the protein in a way that produces ordered (crystalline) precipitation rather than disordered (amorphous) precipitation

Recognize the stages in crystallization

Stages of crystallization

Nucleation

Governed by short-range intermolecular interactions

We want a few stable nuclei, not a lot!

Growth

Adding one molecule at a time to the nucleus

Incorrect additions lead to instability and…

Cessation of growth

Know your protein

Cysteines

Substrates / Ligands

Proteolytic sensitivity

Metal binding

pH & Temp for stability / activity

Post Translational Modification

Protein Preparation

Purify from natural sources

Create an expression construct

Add Tags to aid purification

6-His, Biotin-Strept., Calmodulin Bind. Peptide, GST, Maltose Bind. Protein

Expression systems

E.coli - no post translational modifs

Yeast - euk, may be better for secreted pro’s

Baculovirus-Insect & Mammalian cells

Protein Preparation

Purification StrategyOptimize Protein ExpressionPreparation of soluble cell-free extractAMS/PEG fractionationAffinity Chromatography

Ion Exchange ChromatographySize Exclusion ChromatographyHomogeneity Analysis (SDS-PAGE, MS, DLS)

Protein Preparation

Protein vs SaltProperty Salt Protein

Size cm’s < 1mmlarger often twinned

Integrity ElectrostaticCharged Ions

Hydrogen bondsHydrated Molecules

Solvent content LowerHigher

allows ligand access & activity

Fragile ? (needle test)

Less More

Keep Protein Crystals Hydratedin “Mother Liquor”

Protein StorageOxidation, Deamination, Denaturation, Proteolysis, AggregationGeneral Rule : Store [x] & purified> 1 mg/mlReducing agents, in vivo pHKeep on ice / quick freeze in aliquotsFilter sterilize, Antimicrobial agents

Protein Preparation

Solubility CurveBelow S - no ppt

Zone 1 - Metastablerare nucleationsustains growth (seed)

Zone 2- Nucleationcrystals grow

Zone 3 - Precipitation

Does it always work this way?

No. Some proteins are more soluble in high salt than low.

Same general principles apply as long as we understand the dynamics

What does aggregation do?

In a sense, a crystal is an aggregate…

The formation of oligomeric, randomly oriented aggregates is not conducive to crystallization

We’ll see a useful tool next week for detecting aggregation

Second virial coefficient

Characterizes two-body interactions between protein molecules in dilute solution

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

What do we do with that?

It can be measured through static light-scattering and SANS measurements

Good correlation with nucleation conditions, at least with favorable proteins

Batch

Dialysis

Vapor Diffusion

Crystallization Methods

Dialysis

Double Dialysis

Vapor Diffusion

Vapor Diffusion Variants

Physical:Temp, pressure, surface, viscosity, vibrationChemical:pH, precipitant, ionic strength, metalsBiochemical:purity, ligands, post-TL, proteolysisEngineering:solubility, fusion proteins, heavy atom sites

Factors affecting Crystallization

Don’t be deceived!http://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial1.html

Beautiful - No diffraction

Ugly - 1.6 Angstroms !

Moral : Its the diffraction that counts

Good - nonamorphous, birefringent, redissolves

Bad - skin, does not redissolve, characteristic brownish tinge

Precipitateshttp://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial2.html

http://xray.bmc.uu.se/~terese/crystallization/tutorials/tutorial2.html

Whence came we?

It used to be really hard:Inadequate quantityInadequate purityUnsystematic approachesMacro quantities required

Motivation to improve crystallization approaches came as the field matured

Whither?

High-throughputBetter protein purity

Higher quantities when required

Approaches that don’t require large quantities have appeared

More systematic approachesAutomation at every stage, including visualization

Automated crystallization

Sample loading, distribution

visualization, decision-making

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Can we get away with not knowing our protein?

Often, yes: cf. Structural genomics projects

Ugly cases (e.g. transmembrane): we still argue that the more you know, the more likely you are to get good crystals

www.hamptonresearch.com

www.emeraldbiostructures.com

Protein Crystallization (ed. T. Bergfors) http://xray.bmc.uu.se/~terese/

Crystallization of Nucleic Acids and Proteins (ed. A. Ducruix & R. Giege)

http://www.hwi.buffalo.edu/High_Through/High_Through.html

International Tables for Crystallography. Vol. F

Part 3 : Techniques of Molecular Biology (S. Hughes & A. Stock)

Part 4 : Crystallization (R. Giege et al.)

References