Transcript Document

Lecture 1: Crystallization
Methods and Protein Crystal
Four major steps in crystallization
• Obtain large amounts of pure protein
• Choose a protein buffer in which the protein
is both soluble and stable
• Bring protein solution to supersaturation
where spontaneous nucleation can take
• Crystal growth now begins
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).
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
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
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”.
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)
H. Li, M.A. Perozzo, J.H. Konnert,
A, Nadarajah & M.L. Pusey, Acta
Crystallographica, D55,
1023-1035 (1999).
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)
Cessation of growth
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.
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,
1) 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
Misfolded population
Structural flexibility
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
3) Starting conditions (make-up of
the protein solution)
The main point is to KNOW what your starting
conditions are for purposes of reproducibility.
4) Precipitating agent (precipitant)
Ammonium sulfate
Sodium chloride
Potassium phosphate
Organic reagents
Polyethylene glycol
PEG 4000
PEG 6000
PEG 8000
5) Temperature
Temperature affects protein stability and also the dynamics of
how protein solution reaching supersaturated states.
• 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
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.
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:
Reducing agents
Still no crystals after thorough
screening. Now what?
• New constructs
Deletion mutants
Complexes with substrates
Protein complex with Fab fragments
Homologous proteins
Crystallization of membrane proteins
• The lipidic cubic phase method (Landau
and Rosenbusch)
• Cocrystallization with Fab fragments
Common Methods for Crystallization:
Vapor Diffusion
Slow Evaporation
Hanging Drop Vapor Diffusion
Most popular method among
protein crystallographers.
1. Crystal screen buffer is the
well solution (0.5 - 1 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.
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
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.
Dialysis buttons can be
purchased for a wide range
ofsample sizes (~ 5 - 350 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
Interpreting the Results of the Crystallization Experiment
View the Hampton Crystal Gallery to see in which labs
each of these crystals originated and which biological
molecule(s) they represent.
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:
Crystal Mounting
Capillary tubes
(Glass or Quartz)
(thin nylon)
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