Lecture 2
Crystals: Theory and Practice
Dr. Susan Yates
Wednesday, February 2, 2011
Steps in Solving an X-ray Structure
What is a Crystal?
Crystal acts as an X-ray diffraction amplifier
Crystals
• Crystals consist of a
structural motif, repeated at
regular spacings
• Unit cell
• The smallest repeating unit
that can generate the entire
crystal using only
translation operations
• Mathematical concept - one
molecule does not need to
fit neatly in this “box”
Crystal Lattice
• Lattice
• The set of points in the
crystal that are
equivalent to each
other
• Geometric
arrangement of the
points in space at
which the
atoms/molecules/ions
of a crystal occur
Crystal Lattice
Precipitating Proteins
• In a concentrated protein solution, proteins
interact with water and with other proteins
• Proteins stay in solution as long as the
interactions they make with water are
energetically more favourable than those
they make with other proteins
• If you alter this equilibrium (e.g. by
competing water away using high salt
concentrations - salting out), proteins start
to bind one another and precipitate
Protein Precipitation
• When a precipitation agent is added to a
concentrated protein solution, protein-protein
interactions become energetically more favourable
than protein-solvent interactions
• Proteins bind one another and come out of solution
• No preferred way that the proteins interact
• Result is a precipitate with no long range order
Protein Crystallization
• Crystallization differs from precipitation because
each molecule in the “precipitate” interacts with its
neighbours in the same way as every other molecule
• The result is a highly ordered arrangement - a
crystal
Crystallizing a Protein
• Growing a protein crystal requires controlled
precipitation
• Interactions between individual protein
molecules in a crystal are stabilized by
energetically favourable contacts
• The forces involved include hydrogen bonds,
salt bridges, hydrophobic effect etc.
• Tricky to find this condition and prevent
formation of non-specific aggregates
Energy Barrier to Crystallization
∆G=∆H-T∆S
favourable
unfavourable
crystal represents
the lowest freeenergy
Crystallization: Solubility
• Protein solubility varies with
the concentration of salts,
polyethylene glycols and
other substances in the
protein solution
• A protein will quickly form
an amorphous precipitate if
the solubility is lowered
drastically
• A protein might crystallize if
the concentration is slightly
above the solubility limit
Crystallization Phase Diagram
Crystallization
Clear
Crystallization Process
• Productive crystallization process fluctuates
between Nucleation and Clear zones, largely
due to decreased protein concentration since
protein sample is consumed in crystal
formation
Crystallizing Agents
• Salts
• e.g. Sulfates, phosphates
• Long chain organic polymers
• Polyethylene glycols (PEG)
• Organic solvents
• Generally hydrophilic alcohols, ethers or
ketones
• e.g. Methyl-pentanediol, isopropanol
Methods to Precipitate a Protein
• High salt
• The salt ions order water molecules around them,
leaving less unstructured water to solubilize the protein
• Organic solvents
• These effectively dilute water with a less polar, less Hbond capable solvent with lower dielectric etc.
• Long chain organic polymers
• PEG prefers to writhe over a large volume of space
• Taking the protein out of solution frees up more space
for PEG and is energetically favoured
Other Factors
Influencing Crystallization
• Protein concentration
• Need less precipitant to precipitate the more
concentrated the protein
• pH
• Changing the pH adds/removes protons from individual
residues, possibly creating new salt bridges/H-bonds
• Temperature
• As temperature changes, so do the enthalpic and
entropic contributions to ∆Gcrystallization
• Presence of ligands
• Ligands may lock the protein into one conformation,
which can help crystallization
Vapour Diffusion
• Small volumes of precipitant and
protein mixed together into a
drop which is equilibrated against
a larger reservoir of solution
containing precipitant or
dehydrating agent
• Reservoir or crystallization
solution can be a mixture of
many combinations
Hanging drop
• Buffer (type and conc), pH,
precipitant (type and conc),
temperature, protein conc, ionic
strength etc.
Sitting drop
Vapour Diffusion
Vapour Diffusion
• Slowly increases protein
and precipitant
concentrations
• 12 h to 4 days to
equilibrate
• Mix protein solution with
precipitant solution (1:1)
and equilibrate against
excess of the latter
• Need 1 µL of 10 mg/ml
protein solution per
experiment (well)
Typical Crystallization Procedures
• Screening
• Start with commercial
screening kits derived from
extensive practical experience;
there are hundreds mixtures
covering wide range of
conditions
Culture plate
• Optimization
• Once a lead condition is found
from the screening process,
expansion (pH and
concentration of precipitant
etc.) will be carried out
Micro-crystals
Small crystals
Good single
(~0.1-0.3 mm)
The Practicalities of Growing
Macromolecule Crystals
If you are lucky half of all your
protein constructs will crystallize
Crystal Screening
• Combinations of precipitating agents and
factors that might lead to a crystal is near
infinite
• A typical protein will only crystallize in a small
fraction of these conditions
• When screening you look for crystal leads
• Anything that appears crystalline
• Unlikely to get big, picture perfect crystals
• Not all proteins crystallize!
• Often you have to go back, purify your protein
further, make a new construct…
Finding Initial Conditions
• Check crystal set-ups every day in first week
• Possible results
• Clear, precipitate, crystal and many others
(turbid, bubbles, clothing fibers)
• If almost all or almost no drops are clear,
raise or lower protein concentration,
respectively
• Focus on set-ups that show some precipitate,
but not a heavy yellow or brown precipitate
indicative of protein denaturation
Crystal Refinement
• Refinement is the process by which known crystals
are improved once initial crystals have been found by
screening
• Fine-tuning the conditions
•
•
•
•
Changing the PEG concentration from 30% to 35%
Increasing the pH from 5.5 to 6.5
Adding 4% glycerol
Increasing salt concentration from 200 mM to 300 mM
• The process is generally iterative
• Stop when the crystals are single and big enough to
undergo diffraction testing (50-300 µm)
Improving Size and Diffraction
• Systematic variation of all concentrations and
pH
• Additive screens and detergent screens
• Temperature
• Seeding with crushed crystals (micro
seeding)
• Dialysis, batch, sitting drop
• … check old set-ups for different crystal form
Crystallization Examples
• Lysozyme
• 100 mg/ml protein in
50 mM sodium acetate
pH 4.5
• 30% (w/v) PEG 5000,
1.0 M NaCl, 50 mM
sodium acetate pH 4.5
• Glucose isomerase
• 20-30 mg/ml protein in
water or 50 mM buffer
pH 6.8
• 1.5-2.5 M ammonium
sulfate pH 6-9
Crystal Growth
• Lysozyme crystal growth in a few hours time
• Most will take days to weeks
Example of a Protein Crystal
• The contacts between
molecules are generally
tenuous, involving only a
handful of residues
• Protein crystals contain
large solvent channels,
typically making up 40%
- 70% of its volume
• The crystalline order is
destroyed by exposing a
crystal to air (solvent
evaporates) or to
mechanical stress
(behaves somewhat like
watermelon flesh)
Crystal Galleries
Dust/fibre assisted
nucleation
Crystal Galleries
Crystal Galleries
“Things” in Drops
(Other than Crystals)
Clear
Junk
Ppt (Hope)
Ppt (No Hope)
Skins
Phase Separation
It Will Not Crystallize…
•
•
•
•
•
Check purity and stability
Remove cysteins and other trouble makers
Remove flexible parts
Try single domains
Try physiologically relevant complexes
• Be creative!
Truth Behind Crystallization
Obtaining Well-Diffracting Crystals
Take-home message
Getting a crystal can be hard
Goal
Three-dimensional single crystal
•
•
•
•
A good protein sample
Principles of crystal growth
Crystallization techniques
Strategies to obtain well-diffracting crystals
(quickly?)
• Practical considerations
Small Molecule and Large Crystal
• The world's largest (701 lbs)
fast-growth crystal, grown at
Lawrence Livermore National
Laboratory
• The pyramid-shaped
potassium dihydrogen
phosphate crystal measures
~26x21x23”
• The enormous crystal was
sliced into ½” thick plates
and used in a giant laser that
will “help maintain the safety
and reliability of the nation's
nuclear weapons stockpile”
Next time…
• Instrumentation
• Waves and Diffraction