Physical Biochemistry X-Ray Crystallography 1

Crystallisation of Macromolecules:
1st crystal structure – Myoglobin (1959)

Protein structure
 3D coordinates of atoms within a molecule
 Number of peptide chains within a functional unit
 Local secondary structure (α-helix, β-strand)
 Domain organisation and interactions
 Interactions with substrates, inhibitors, cofactors, ligands, drugs
 Interactions with solvent

Solving structures helps us to understand the function of a protein

 Active site chemistry
 Kinetic data
 Predicted function of key residues
 Modification of protein function
 Effect of mutation (on disease)

Electron density maps are generated through X-ray crystallography which are then interpreted

It is also useful to study complexes

 Protein-Small Molecule (Pharmacology)
 Protein – Protein (Cancer Proteins)
 Protein – DNA (DNA Repair)
 RNA – Protein (Ribosome)

Advantages of X-Ray Crystallography

 Any size of molecule can be used
 High resolution: up to 0.5Å (C–C bond is larger than this)
 Able to ‘see’ electrons

Disadvantages of X-Ray Crystallography

 Growing crystals can be difficult
 Cant make X-ray lenses – mathematical methods need to be used to solve phase problem
 Protein is in crystal, not solution, which may affect its conformation

Structural biology includes X-ray crystallography, neutron scattering, electron microscopy, NMR,
mass spec etc.

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Crystals are arrays of periodically repeating identical protein molecules ordered in 3D

 Molecules are liked by ionic interactions, H-bonds, hydrophobic interactions and through
solvent
 Small and usually colourless (can be given false colour if they bind certain metal ions)
 Solid, fragile
 High solvent content (~90%)

Growing Crystals:

Natural protein: protein is purified from cells using traditional biochemical techniques. More
difficult to work with, low yields but necessary if a large complex cannot be reconstituted.

Recombinant protein: Proteins are over-expressed in a cell system such as E. coli or yeast.
Easier to handle than large animals, easier to purify via tag technology (His, FLAG, GST, MBP,
TAP etc. are fused to recombinant proteins to aid affinity purification). Cloning and DNA
sequencing makes it easy to obtain the protein sequence, which is required for model building.

1. Clone gene of interest

2. Insert gene into expression vector
3. Transfect plasmid into host and grow host cells
4. Purify protein
 mg quantities
 High level of purification
 Remove unrelated small / macromolecules
5. Bring concentration to ~5-20mg/ml
6. Use an appropriate buffer that will not crystallise
 e.g. A phosphate buffer will crystallise with Mg2+ ions

It is good to remove a tag, especially if it is a large protein like GST, or it will massively influence
the crystal. Smaller tags such as His6 might not need to be removed.

In order to crystallise a protein, the solubility of the protein needs to be decreased until it either
precipitates or crystallises.
The protein sample must not be allowed to dry out
Many factors will influence whether or not a protein will crystallise, each parameter can be varied
systematically to see which conditions give the best crystals. Commercial screening kits known as
‘sparse matrix kits’ have preloaded conditions.

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Physical Biochemistry X-Ray Crystallography 1

Crystallisation parameters
 pH
 Precipitant (alcohols, salts, PEGs – polyethylene glycols etc.)
 Ionic strength
 Metal ions (Ca2+, Mg2+ etc.)
 Additives
 Detergents
 Temperature
 Hanging, sitting or under oil trays
 Drop size
To vary all of these parameters systematically would use a lot of protein and would take a long
time, so a systematic approach has to be used.

Crystallisation Phase Diagram:

 Undersaturation (soluble)
 We want to get protein into supersaturation/metastable zone (crystals can grow but not
nucleate)
 Nucleation zone (where crystal nuclei form) needs to be entered then come out of
 At low [protein], the solution will be below the saturation limit
 At high [protein], the protein will precipitate

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Sparse matrix screen kits use a selection of the crystallisation parameters. The conditions are
selected semi-randomly, but biased in favour of conditions that have worked well with other
proteins. Commercial kits allow screening of around 1000 semi-random conditions, which can be
repeated in trays at 4C and 20C. If this fails, then try another construct of the protein.

Crystallisation Methods:
If possible, use a robot

I) Batch Method
Precipitant is added directly to the protein crystallisation solution.
Traditionally carried out in a vial, not used much now
The [precipitant] (e.g. (NH4)2SO4) is varied
Increasing precipitant concentration will have large effects on the protein:
Clear solution  Large, single crystals  many microcrystals  amorphous precipitate

II) Free Interface Diffusion (FID)

Precipitant is layered on top of a protein solution in a capillary
Protein can be frozen first; as it thaws crystals will grow at the protein-precipitant interface
Good at screening as it can use small quantities of protein, but this means its hard to get crystals
from the plate. Often used on proteins that are rare and have been purified from natural sources.

Fluidgm system carries out FID on a nl scale. Very simple to use, but expensive.

III) Dialysis
Protein is dialysed against a precipitant using a semi-permeable membrane that allows only low
MW molecules to pass through. The concentration of precipitant will slowly increase over time,
allow the protein to precipitate out. Simple, but only really used if there has not been success in
other methods.
Bulk dialysis uses a tube of membrane (ml scale)
Micro dialysis uses ‘buttons’ (μl scale)

Advantages: dialysis solutions are easily exchangeable; if initial

conditions do not give crystals, membrane can be placed into a
different solution.
Disadvantages: can be difficult to recover crystals from drops

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IV) Micro Batch Methods

Protein and precipitant are mixed in wells in a microbatch tray
A layer of oil is placed over the drops to prevent evaporation
μl sample scale
Quick and easy to use (can be automated), u useful technique for screening initial conditions

V) Vapour Diffusion: Hanging Drop

Precipitant is placed in well, drop of protein hangs from cover slip that covers the well
Well is vacuum sealed
Over time, an equilibration process takes place
μl sample scale
Manual process
Initial precipitant concentration in the drops is ½ that of the well
Solvent evaporates from drop, making it shrink
Protein & precipitant concentrations gradually increase until the precipitant concentration
approaches that of the well.

VI) Vapour Diffusion: Sitting Drop

Similar to hanging drop
Drop is placed on a stand

Robots allow for high throughput analysis. Require small volumes (nl)
1st robot adds sparse matrix solutions into the wells
2nd robot will add a small amount of well solution to the stage, then add protein solution

Modern robotics can carry out

most processes including plate
storage and imaging.
Throughput is increased, but it is
expensive. Drops are imaged
over time to look for crystals.
Plates are stored in ‘hotels’.

i) Batch
ii) Vapour diffusion
iii) Dialysis
iv) FID

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Physical Biochemistry X-Ray Crystallography 1

It is unlikely to find perfect conditions in a screening process. Conditions may be found that
produce a crystalline substance or needles, but this is not optimal. These conditions are then
tweaked in order to obtain a good crystal that will diffract well.

Membrane Proteins
30% of proteins in the HG are membrane proteins, however the structural description of them lags
behind all other proteins.
Difficult to work with – in order to make these proteins soluble detergents have to be added. They
crystallise in a different way. Membrane proteins are insoluble when taken out of a membrane
environment.

A) Use 2D crystals – crystallise in lipid bilayers

Detergents are used to extract protein from host bilayer
Artificial lipids are then introduced, forming the 2D bilayer

B) 3D crystals can be formed using detergents to form protein-detergent complexes

Membrane proteins only have a small soluble region (extracellular part) which can be used to form
the crystal.

C) Lipid cubic phase method forms a 3D crystalline array

Cubic phase is a 3D array of monolein (lipid analogue) with two separate water filled areas
separated by a 3D bilayer arrangement.
The cubic phase feeds a lamellar phase with crystals in bilayers, protein has crystal contacts
within the membrane.
Spongey phase also exists

It is important to be able to tell the difference between small molecule (salt) crystals and protein
crystals. Proteins are chiral, so can change the polarisation of light. Two polarisers are used at
right angles to one another – all light is blocked except where a chiral molecule changes the
polarisation of light, known as bi-refringence.
 Crush test – prod crystal with a fine hair – protein crystals are soft, salt crystals are hard
 Stain using dye (e.g. coomassie blue)
 Wash, purify, then run on a gel
 X-rays – the only reliable test

If crystals are small / of poor quality / don’t refract X-rays

 Seeding – transfer microcrystals into a new drop

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Physical Biochemistry X-Ray Crystallography 1

 Screen again adding ligands

 Try same protein from a different source, such as thermophiles (protein will be
 stable)
 Limited proteolysis – look for stable, proteolysis resistant domain. Floppy regions are not
wanted as they will not produce good crystals
 Subcloning domains
 Deglycosylation – glycan chains are not good for forming crystal contacts
 Site directed mutagenesis Lys can be randomly mutated
 Membrane proteins can be co-crystallised with an antibody fragment / carrier protein /
protein complex

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