1. Lecture 1: Crystallization Methods and Protein Crystal Properties

2. Four major steps in crystallization
Obtain large amounts of pure protein samples
Choose a protein buffer in which the protein is both soluble and stable
Bring protein solution to supersaturation where spontaneous nucleation can take place
Crystal growth now begins

3. Solubility
As a rule, protein solubility will usually increase as you add salt to your aqueous solution, then begin to decrease when the salt concentration gets high enough to compete with the protein for hydration (interaction with water molecules).
HbCO (carboxyhemoglobin) solubility as a function of ionic strength in the presence of several different types of salts
Diagram from the website of Alan Clark, Victoria University of Wellington, New Zealand

4. Supersaturation
Supersaturation can be achieved by adding more of a substance (to a solution) than can normally be dissolved. This is a thermodynamically unstable state, achieved most often in protein crystallography by vapor diffusion or other slow evaporation techniques.
Zone 1 - Metastable zone.
The solution may not nucleate for a long time
but this zone will sustain growth.
It is frequently necessary to add a seed crystal.
Zone 2 - Nucleation zone.
Protein crystals nucleate and grow.
Zone 3 - Precipitation zone.
Proteins do not nucleate but precipitate out
of solution.
Diagram from the website for The University of Reading, Course FS460
Investigating Protein Structure and Function

5. Nucleation Common difficulties:
A phenomenon whereby a “nucleus”, such as a dust particle, a tiny seed crystal, or commonly in protein crystallography, a small protein aggregate, starts a crystallization process.
Nucleation poses a large energy barrier, which is easier to overcome at a higher level of supersaturation.
Common difficulties:
1. If supersaturation is too high, too many nuclei form, hence
an overabundance of tiny crystals.
2. In supersaturated solutions that don’t experience spontaneous nucleation, crystal growth often only occurs in the presence of added nuclei or “seeds”.

6. Crystal Growth Adding single molecules to the
surfaces of the nucleating lattice.
Illustrated here through the work of
Li and Nadarajah of
The Macromolecular
Crystallization Laboratory
at the University of Toledo.
AFM image of individual
lysozyme molecules on
the (110) face of a
tetragonal crystal.
(Li and Nadarajah)
The growth steps and growth units of Lysozyme.  The growth steps are at least bimolecular in height.  The minimum growth unit for this step must be a tetramer corresponding to a single turn of the 43 helix as shown here.(Nadarajah)
H. Li, M.A. Perozzo, J.H. Konnert,
A, Nadarajah & M.L. Pusey, Acta
Crystallographica, D55,
(1999).

7. Cessation of growth Mother liquor
Caused by the development of growth defects or the approach of the solution to equilibrium.
Mother liquor
The solution in which the crystal exists - this is often not the same as the original crystallization screening solution, but is instead the solution that exists after some degree of vapor diffusion, equilibration through dialysis, or evaporation.

8. Major factors that affect crystallization
1) Purity of proteins
2) Protein concentration
3) Starting conditions (make-up of the protein solution)
4) Precipitating agent (precipitant)
5) Temperature
6) pH
7) Additives: Detergents, reducing agents, substrates, co-factors, etc.

9. Purity of proteins
Sources of heterogeneity (other than unrelated proteins and nucleic acids as contaminants):
Partial proteolysis products
Oxidation of cysteines
Deamidation of Asn and Gln to Asp and Glu
Post-translational modifications
Oligomerization
Isoforms
Misfolded population
Structural flexibility

10. 2) Protein concentration
Consistency and reproducibility are the major issues
with protein concentration - you should have a reliable
assay for determining the concentration.
Extinction coefficient for tryptophan
Bradford Assay (BSA is used as a standard)
E. Coli expression systems are crystallographers’ most commonly used method of obtaining protein. Problems can arise from low expression yields:
Cytotoxic - your protein is killing your E. coli
Unstable plasmid or mRNA
Protein is misfolded (coexpress with GroEL?)
Some common eukaryotic codons are rare in E. coli

11. 3) Starting conditions (make-up of the protein solution)
The main point is to KNOW what your starting conditions are for purposes of reproducibility.

12. 4) Precipitating agent (precipitant)
Salts
Ammonium sulfate
Sodium chloride
Potassium phosphate
Organic reagents
MPD
Isopropanol
Polyethylene glycol
PEG 4000
PEG 6000
PEG 8000

13. 5) Temperature
Temperature affects protein stability and also the dynamics of how protein solution reaching supersaturated states.
Ideally:
An individual crystal screen should be kept at constant temperature
Each set of conditions should be screened at several temperatures
The easiest are 4 C and room temperature, also try 12 or 15 C

14. 6) pH Surface charges affect “crystal packing”.
(Crystal packing refers to the spatial arrangement of
molecules within the crystal, particularly in reference to their relationships to one another.)
Hydrophobic interactions are less important than electrostatic interactions in crystal packing.

15. 7) Additives:
Sometimes you can increase the stability of your protein,
and/or the homogeneity of its conformation by having relevant additives present in the crystal screen:
Detergents
Reducing agents
Substrates
Co-factors
etc.

16. Still no crystals after thorough screening. Now what?
New constructs
Deletion mutants
Complexes with substrates
Protein complex with Fab fragments
Homologous proteins
Fab

17. Crystallization of membrane proteins
The lipidic cubic phase method (Landau and Rosenbusch)
Cocrystallization with Fab fragments

18. Common Methods for Crystallization:
Vapor Diffusion
Slow Evaporation
Dialysis

19. Hanging Drop Vapor Diffusion
Most popular method among protein crystallographers.
1. Crystal screen buffer is the well solution ( mL)
2. Drop (on siliconized glass cover slip) is 1/2 protein solution, 1/2 crystal screen buffer (6-10 L). So, the concentration of precipitant in the drop is 1/2 the concentration in the well.
3. Cover slip is inverted over the top of the well and sealed
with vacuum grease (airtight).
4. The precipitant concentration in the drop will equilibrate with the precipitant concentration in the well via vapor diffusion.

20. Sitting Drop Vapor Diffusion
Same basic principles
as in hanging drop
method, except the drop
containing your sample
sits on a bridge within
the well. This allows for
a larger sample size (20 -
40 L), however protein
is frequently precious to
the crystallographer, so
there isn’t that much
demand for a larger sample
size.

21. Oil Immersion Micro Batch
This method is rising rapidly in popularity- typical sample size 1-6 L
Figure 1- Paraffin oil allows for little to no diffusion of water through the oil. This is a true batch experiment because all the reagents are present at a specific and relatively unchanging concentration.
Figure 2- Al’s oil is a 1:1 mixture of silicon oil and paraffin oil which allows for evaporation through slow diffusion through the oil. This is an evaporation
Method, and the concentration of the protein and reagents in the drop does increase over time.

22. Microdialysis
Dialysis buttons can be purchased for a wide range ofsample sizes (~ L).
In the dialysis experiment, the sample is often introduced to high salt concentrations within the button that are allowed to equilibrate with lower salt concentrations in the buffer over time. This is known as a “salting-in” method. It exploits the fact that not only does protein solubility tend to decrease with very high ionic strengths, it also has a minimum at very low ionic strength.

23. Interpreting the Results of the Crystallization Experiment

24. View the Hampton Crystal Gallery to see in which labs
each of these crystals originated and which biological
molecule(s) they represent.

25. The oscillation equipment
Rotates the crystal about an axis () perpendicular to the
x-ray beam (and normal to the goniometer). The diffraction
pattern from a crystal is a 3-D pattern, and the crystal must
be rotated in order to observe all the diffraction spots.
This nice diagram also comes from Bernhard Rupp’s Crystallography
101 website:

26. Crystal Mounting Capillary tubes (Glass or Quartz)
Cryo-loops (thin nylon)

27. Properties of protein crystals
Soft, easy to crush
Contain large solvent channels
Relatively large organic and inorganic molecules can diffuse inside
Anisotropic physical properties
Birefrigence due to anisotropic refraction indices
Ability to diffract X-ray due to regular spaced lattices