Crystallization:
Crystallizing inorganic compounds is achieved by cooling down a hot saturated solution. Polar organic
compounds can also be crystallized by adding organic solvents. But these methods cannot be applied
to proteins. They would denaturate. We instead dissolve the protein in a buffer containing a
precipitant at a concentration just below that needed to precipitate the protein. Then the
concentrations of protein and of precipitant are increase, for example by means of removing water.
Requirements:
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At least 10mg protein (concentration of about 10mg/ml)
Optimal buffer (pH, ionic strength, etc.; test ingredients for their compatibility)
Sometimes stabilization with the help of cofactors, ligands etc.
Activ protein (inactive proteins are useless)
The right buffer: i.e. phosphate buffers make salts with Ca2+, Mg2+, ...
Chemical and conformational purity/homogeneity
Increasing solubility of partially hydrophobic proteins:
 n-alkyl-β-glucosides
 n-alkyl-β-maltosides
 glycerol
Best methods of disruption (opening of the cell):
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Manual grinding (Mahlen) with aluminum oxid
Enzymatic disintegration (Zerfall/Auflösung) methods
Purity:
Even small contaminations often poison the emerging nuclei important for crystal formation.
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Reaching at least 95% purity:
o SDS-PAGE followed by Coomassie blue or silver staining:
detecting 1% impurity with silver
Obtaining only one isoform:
o IEF (isoelectric focusing determining pI)
Detecting microheterogenities:
o ES-MS (electro spray mass spectrometry):
precision = 0.01% of protein mass (which is 4-40,000Da)
detection of
- proteolysis
- partial processing
- posttranslational modifications
o N-terminal amino acid analysis and ES-MS
analysis of the whole amino acid sequence
o MALDI-MS (matrix assisted laser desorption MS)
less precised
detection of
- bound buffers
- bound detergents
- bound ligands etc

Conformational purity:
a. Checking monodispersity
Monodispersity means that all proteins are in the same oligomeric state
o Size exclusion chromatography (gel filtration)
last purification step
selects one oligomeric state
o DLS (dynamic light scattering)
detects the size distribution profile of small particles in a solution
(monodispersity) by means of light scattering
b. Checking if the protein is folded:
o NMR (nuclear magnetic resonance)
determines the protein structure. A magnetic field causes different nuclear spin
levels (different orientations of the magnetic moment with energy differences).
Electromagnetic radiation of appropriate frequency allows transition to a higher
level. The frequency is characteristic for the strength of magnetic field and of the
chemical environment of the nucleus.
o CD (circular dichroism) spectroscopy
revealing important characteristics of secondary structure by means of different
absorption of left or right handed circular polarized light
o Calorimetry: Differential thermal calorimetry
detecting exo- or endothermic changes by means of differences between the
temperatures of a sample and a reference
o Thermal shift assays (thermo-fluor measurements)
A fluorescence dye binds to the unfolded protein and emits light.
Ad microheterogeniy
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Posttranslational modifications like phosphorylation or glycolysis
Chemical modifications like methylation, C-C-bond formation etc.
Partial processing
Partial degradation
Partial denaturation
Solubility:
= the maximum concentration of a solute in a solvent under equilibrium conditions. Exceeding this
value yields a metastable oversaturated solution. Only the needed activation energy allows
precipitation of the solute.
Solubility is precisely defined under given conditions because it depends on among others:
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pH
T
Ionic strength
Precipitant concentration
Additives (small amounts of organic compounds, salts, detergents,ions, …)
Logarithm of solubility
Equilibrium maintained by thermodynamic forces:
The amount of solute precipitated to form crystals is the same as the amount of solute dissolved
from the crystal into the solution => no gain on concentration in one of the two phases.
Root of ionic strength
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Left side of the phase diagram: Salting in
Solubility increases as precipitant concentration increases.
Right side of the phase diagram: Salting out
Solubility decreases as precipitant concentration increases.
Energy barrier:
An oversaturated solution can exist as long as the activation energy for precipitation is not invested.
It represents a chemical system not in equilibrium. Thermodynamic forces regenerate the
equilibrium resulting in crystal growth after a defined time:
First a critical nucleus has to be formed (like the high energy intermediate in chemical reactions):
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The higher the protein concentration the larger the oversaturation the smaller the activation
energy.
The smaller the activation energy the faster the rate of nucleation.
o Because the probability of formation of the critical nucleus is higher.
o Because the needed nucleus for crystal growth is smaller.
Probability of nucleation can be looked up in phase diagrams:
If supersaturation is reached to fast an amorphous precipitate instead of a crystal forms.
Precipitating Agents:
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Inorganic salts:
Ammonium sulphate, sodium citrate, tartrates, …
They compete with the protein for the hydration shell and protein without this shell
precipitates.
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Organic solutes
Ethanol, 2-methyl-2,4-pentadiol (MPD), isopropanol, butanols
They change the dielectric constant and compete with the protein for the hydration shell.
Linear polymers with big solvatisation volumes:
PEG (polyethylene glycol: Mw 200-20,000Da)
Molecules with different solvatisation volumes separate into distinct phases.
Screening approaches:
Crystallization is mainly a trial and error procedure. We have to screen a high number of parameters
(T, pH, ionic strength, precipitant concentration, additives). For example we have to choose a
temperature value and screen different pH values at this temperature. By this way we hope to find
the optimal conditions for crystal growth.
Systematic search – full factorial:
All elements of the n-dimensional matrix are sampled. That means we have investigated each
possible combination of parameters.
N = nk
k = number of parameters
n = number of levels (= values) used for each parameter (for example: 4°C, 20°C, 28°C)
N = 23 = 8

Incomplete or fractional factorial:
To avoid the high number of experiments when the number of parameters increases we use only a
fraction of full design. But now we confuse the main effects of parameters. We can not proof if a
special effect on the result of the experiment comes from parameter a or b. But what we can do is to
quantify the results (the crystal size) and to express it as a function of n variables. Searching the
extreme values of this function yields the values of the parameters causing the best crystal.
The fraction of full factorial contains two-parameter-combinations uniformly distributed over the
matrix to yield a good function of the n variables. But first the parameter levels are chosen randomly.
When we have obtained the first results we can judge how to balance the levels to achieve uniform
sampling.
Random:
We only randomly choose the parameter and do not balance them out. We can only hope that this
method approximates incomplete factorial.
Sparse matrix:
We use parameter combinations that have functioned in the past.
Crystallization techniques:
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Batch Crystallization:
1μl precipitating agent is instantaneously mixed with 1μl protein solution.
The mixture is covered with oil to avoid
evaporation
.
Different concentrations of precipitating agent:
Point A: undersaturated solution – no crystal growth
Point B: Probability of nucleation is high enough: Critical nucleus is formed using protein of
the solution -> loss of protein concentration. In the metastable zone crystals can still grow
from the critical nucleus.
Point C: Protein only precipitates. But once reaching the nucleation zone crystals may still
grow.
Hanging and sitting drop – Vapour diffusion:
2μl precipitant and 2μl protein are mixed and placed upside down on a cover slip and (if
surface tension is to small) placed on a ridge (Erhöhung) respectively. The cover slip is fixed
with grease above a reservoir of precipitant higher concentrated than the drop.
Because of evapouration from the drop into the reservoir the protein as well as the
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precipitant concentration douples. The nucleation zone is reached and the critical nucleus is
formed consuming protein of the solution -> protein concentation decreases. But in the
metastable zone crystals can still grow from the formed critical nucleus.
Dialysis:
5-350μl protein solution are filled in a tube which is closed by a membrane by means of a
rubber ring. The tube is put in a reservoir with precipitant which can diffuse through the
membrane to the protein -> precipitant concentration increases (like in the salting out
process).
The concentration of the precipitant approaches that of the surrounding solution. Therefore
the total concentration of protein remain constant.
Dialysis allows you to change the precipitant concentration during the course of experiment
or increase the concentration of one precipitant while decreasing that of another one.
Aim of crystallization:
+ Regular growth
+ Regular shape
+ Ability to diffract X-rays
+ Optical clarity
Microscope (magnification about 100x)
+ Smooth faces
+ Sharp edges
+ Birefringence (= double refraction): Some crystals rotate plane polarized light.
 Rotate polarizer: The crystal will darken and brighten.
+ Containing protein (no salt)  Proof by means of SDS-PAGE: Dye stains only the protein.
+ Big enough
− Bundling or twinning (We have to work with one single crystal.)
− Polynucleation (many small crystals)
Optimization of crystallization conditions:
-
pH
Concentration and type of precipitating agent
Protein concentration
o Increasing ratio of protein to precipitant
o Decrease ratio of V(reservoir) to V(protein)
o Increase starting concentration
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Temperature
Ionic strength (add salts)
Additives
 Monodispersed protein
 stopping nucleation -> fewer but bigger crystals
 1-25% glycerol (acts as a cryoprotectant)
 Organic solvents like ethanol, dioxane
 Divalent cations like magnesium or calcium
 Detergents like β-octyl-glucoside under CMC
Slowing down equilibration rate by limiting evaporation
o Oil covers the reservoir
o Bigger drops (decreased surface/volume-ratio)
Limiting polynucleation by removing dust, amorphous precipitate in the sample by means of
centrifugation and a vibration free environment
Increasing crystal dimensions
o by means of bigger drops
 decreased surface/volume-ratio = slower equilibration rate
 more protein in the drops
o sitting instead of hanging drops
Seeding for bigger, more reproducible crystals
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Streak seeding:
1.
Prepare a preequilibrated saturated drop (below
spontaneous nucleation phase).
2.
Touch the crystal (which is too small) with a cat or rabbit
whisker (Schnurrbarthaar).
3.
Streak the whisker across the center of the preequilibrated
drop.
Microseeding:
1.
Prepare a preequilibrated saturated drop (below
spontaneous nucleation phase).
2.
Prepare a seed stock by crushing the crystals (which are to
small) in a glass homogenizer.
3.
Use this seed stock to prepare several dilutions: 1 in 1000 till
1 in 10,000,000.
4.
Add a drop from each of the dilutions to the preequilibrated
drop.
Macroseeding:
1.
Prepare a preequilibrated saturated drop (below
spontaneous nucleation phase).
2.
Choose a crystal that is to small but free of defects.
3.
Prepare wash solutions with decreasing concentrations of
precipitating agent.
4.
Serially transfer the crystal from one solution to the next one
of lower concentration.
5.
Transfer the seed (the washed crystal) to the preequilibrated
drop.
If everything else fails:
-
Back to wet-lab and gene-library
Limited proteolysis (N- or C-terminal domain phasing,
deletion of mobile loops)
o
o
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Adding:
Ligands or cofactors
Binding partners like other proteins, DNA, RNA
for the purpose of stabilization
Chemical modification like methylation of Lysines
Mutation of glu/lys-clusters into less flexible thr/ser
For the purpose of reduction of surface entropy
Membrane proteins:
They are imbedded within a lipid layer with their hydrophobic suface while their hydrophilic surface
is exposed to the solvent.
I.
II.
Use a detergent for solubilization from the lipid layer and
formation of a protein-membrane micelle in which the detergents cover the hydrophobic
surfaces of the protein
Create three different protein crystals:
a.
Add lipid bilayer to generate a 2D crystal:
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Electron microscopy
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Electron diffraction
b.
Add a lipidic cubic phase to generate a type 1 3D crystal

The lipidic cubic phase is a continuous lipidic 3D phase
containing circles of lipids whose hydrophobic parts are at the outside of the
circle interacting with that of the other circles.

Type 1 3D crystals are just stacked 2D crystals (hydrophobic
interaction with the membrane and polar interaction between the proteins).
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Lack of specific interactions: poor Order
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Obtaining large crystals is difficult

BR, halorhodopsin, sensory rhodopsin II -> related proteins
c.
Leave the detergent (critical selection) to generate a type 2
3D crystal
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Type 2 3D crystals form a lattice. (polar interactions between
proteins but no hydrophobic interaction because detergents cover the
hydrophobic surfaces)
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Poorly orderd
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Very high solvent content (65-80%)
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The same techniques and precipitants as for soluble proteins
are used.