Properties of Crystals

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Properties of Crystals Ellen
Moody March 15, 2001
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Why do we need to use crystals?
Two Main Reasons
1. Intensity is too weak from a single molecule
2. Need molecules in an ordered array
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Protein Crystals vs. Inorganic Crystals
Size

Inorganic up to several cm

Protein usually 0.2-0.4 mm in two
dimensions
Larger crystals are twined
Interactions
Inorganic electrostatic
attractions of fully charged ions
Protein H-bonds between hydrated protein
surfaces
Structural Integrity
Inorganic
tough

Protein Fragile. Growing, handling, and
mounting take a gentle hand
Crystallizing methods
Inorganic hot,
saturated solution is slowly cooled, or by adding
organic solvents

Protein Dissolved in
buffer containing a precipitant, water is removed
and protein crystallizes
Mosaic
Protein
isnt perfect, measurements must be taken over a
range of small angles
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Water Content
Dried crystals give no diffraction pattern
Many ordered water molecules are on the surface
of crystalline proteins
How many? About one per amino acid
X-ray diffraction done under humid conditions or
in the presence of mother liquor
NMR shows water moves around, but it is still
crucial to crystallography
Water explains small regions of disconnected
electron density
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Are proteins in a crystal the same as in solution?
Proteins retain their function in the crystal
Enzymes still function Conformational changes
occur
X-ray structures are compatible with other
structural evidence
Cross-linking experiments NMR structures Different
types of crystals
Remember, Proteins are still somewhat in aqueous
phase
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Growing crystals
Basic procedure Purified protein is dissolved in
an aqueous buffer containing a precipitant at a
concentration just below that necessary to
precipitate the protein. Then water is removed
by controlled evaporation to produce
precipitating condition, which are maintained
until crystal growth ceases.
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Three degrees of Solutions
Unsaturated no crystals will form or grow Low
supersaturated crystals will grow but no new
ones will form High supersaturated crystals
will both form and grow
In theory, start in the high supersaturated
solution and then move the small crystals to a
low supersaturated solution.
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Methods of Growing Protein Crystals
Many methods
Vapor Diffusion (Hanging Drop Method) Batch
crystallization Microbatch crystallization Macro
seeding Microseeding Free interface
diffusion Dialysis
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Vapor Diffusion Hanging Drop method
-Most common form of crystal growth -Drop of
protein solution and precipitant (50/50 mixture)
is placed on cover glass over buffer solution of
high concentration of precipitant -Water diffuses
from the less concentrated drop to the reservoir,
increasing the precipitant concentration in the
drop -equilibrium is reached at optimal growth
conditions without overshooting -crystals grow
(hopefully)
-crystals may need to be seeded
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Seeding
Macro a crystal is grown in a highly saturated
solution and placed in a less saturated one where
only growth of the crystal will occur
Micro a few crystals are grown, then crushed,
and put into a final solution that combines them
into a few nice crystal. This involves quite a
bit of experimentation with solutions
concentrations to get the desired number of
crystals
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Batch crystallization and Microbatch
Crystallization

• -A saturated protein solution left in a sealed
  container to let the crystals grow
• Micro a drop of protein solution is put in
  inert oil and left to grow.
• Some diffusion of proteins into the oil, lowering
  the saturation over time

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Methods for Growing Crystals, cont.
Free Interface Diffusion - A container has levels
of varying saturation. Crystals form initially
in the highly saturated part, but as the solution
mixes, it eventually only supports crystal
growth. Permits increase in Precipitant level
while holding protein concentrations steady
Dialysis - Similar to free interface diffusion,
but with a semi-permeable membrane separating the
layers.
Proteins are crystallized on such a small scale
that it is difficult to reproduce
concentrations. This makes crystallizing proteins
almost more of an art than a science, and
trial-and-error is the common approach.
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Derivative Crystals
A crystal of the protein with a ligand such as a
substrate analog, inhibitor, cofactor, heavy
metal atoms or ions
Two methods
1. Cocrystallize protein and ligand
2. Soak preformed protein crystals in
mother-liquor solutions containing ligand
Soaking is preferred method if comparisons will
be made to pure protein crystals
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Finding optimum conditions for Crystal Growth
What Factors? Protein purity concentration of
protein concentration of precipitant
pH Temperature Cleanliness Vibration Sound
Convection Source of protein Age of
Protein Presence of ligands
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Systematic procedure to find Optimal Conditions
Determine effect of pH with a given
precipitant Repeat at various temperature Repeat
these experiments with different precipitating
agents
Robotics are used to automate this
Test multiple conditions in a tray with multiple
wells.
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Response-surface procedure
Score the results of different crystallization
conditions.
Use mathematical function to plot a
multidimensional surface. Peaks are highest
scores
Sometimes leads to better crystallizing
conditions than were originally investigated.
Please see Rhodes, page 40, figure 3.4
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When all else fails, try these suggestions.
Limited digestion of protein may remove a
disordered structure.
Add a ligand to make protein more rigid.
Add detergents to membrane-associated proteins
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Density of Crystals
Used to determine protein molecular weight,
protein/water ratio, and number of protein
molecules in an asymmetric unit
Measured in a graduated cylinder containing a
density gradient
Protein/water ratio if protein molecular mass
and number of protein molecules per unit cell are
known, then remainder of the cell can be assumed
to be water.
Mp molecular weight of protein Dc density of
crystal Dw density of water N avogadros
number V volume of the unit cell np partial
specific volume n number of protein molecules
in cell
Mp NV(Dc-Dw) n(1-npDw)
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Mounting of Crystals
Classically transfer into a fine glass
capillary with a droplet of mother liquor
Low temperatures are good because it increase the
order of the protein in the crystal
Problem Ice forms at low temp
Solution Cyrocrystallography
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Cryrocrystallography
Place crystals in cryoprotected mother liquor for
5-15 seconds then flash freeze
Crystal is picked up with circular loop of nylon
fiber or glass wool
Loop is dipped into liquid nitrogen and freezes
and remains clear
Loop is then mounted on machine where is it held
in a stream of cold nitrogen gas (-100 oC)
Advantage reduction of radiation damage to
crystal and reduction of X-ray scattering from
water
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References
Gale Rhodes Homepage http//www.usm.maine.edu/rho
des/CMCC
http//www-structure.llnl.gov/Xray/101index.html B
ernhard Rupp, Lawrence Livermore National
Laboratory, Livermore, CA
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