Slide1: X-ray crystallography
Ph: 773 3974
Laboratory located on bottom floor of the Lundberg building, Medical Hill. Slide2: Crystal Concept
In 1611 Kepler suggested that snowflakes derived from a regular arrangement of minute brick-like units.
-The essential idea of a crystal. Slide3: X-rays
Discovered by Rontgen 1895
Cause of tremdous scientific excitement.
1,500 scientific communications within first twelve months.
US scientists repeated experiments within four weeks. Slide4: Theory of Diffraction
1910 von Laue derived theory of diffraction from a lattice.
Slide5: Bragg’s Law of diffraction 1912
Diffraction observed if X-rays scattering from a plane add in phase.
Path difference DP = 2d sin q.
- d is the spaceing between planes & q is the angle of incidence.
Scatter in phase if path difference is nl
n is an integer & l is the X-ray wavelength.
2d sin q = n l
First structure (NaCl) in 1912. W. H. Bragg & W. L. Bragg Slide6: 1953: Double helix structure of DNA
Crick & Watson used X-ray diffraction to work out the way genes are encoded. Slide7: Diffraction pattern.
Crystals & diffraction pattern recorded by Rosalind Franklin.
Revealed the symmetry of the helix & pitch of helix.
Slide8: First Protein Structure
Protein purified from whale blood.
Max Perutz 1958.
Showed a 75% a-helical fold.
155 amino acids, ~ 17 kDa. Slide9: First Protein Complex
Two copies each of a & b chains of myoglobin in a complex.
Solved by John Kendrew. Slide10: Structure of Nucleic Acid & Protein complex.
Nobel prize to Aaron Klug in 1982.
Also contribution to electron microscopy. Slide11: Photosynthetic Reaction Centre Structure.
First membrane protein structure in 1985.
Nobel prize to Michel, Deisenhofer & Huber 1988.
Showed the technique of detergent solubilised membrane protein crystallisation. Slide12: Structure of F1-ATPase
Revealed the details of the rotational mechanism of ATP-synthase.
Nobel prize in 1997. Slide13: Structure of the K+ channels
Revealed the structural basis for ion transport across a membrane.
Deep physiological relevance.
Nobel prize in Chemistry 2003 to Roderic MacKinnon. Slide14: Structure of TBSV
First Virus structure, tomato bushy stunt virus, 1978.
By Steve Harrison.
Revealed icosohedral symmetry of a virus particle. Slide15: Ribosome
50 S and 70 S ribosome structures in 2000.
Massive RNA:Protein complexes.
Revealed details of how proteins synthesyzed by RNA.
Slide16: Crystal definition
A crystal is an object with translational symmetry:
r(r) = r(r) + a·x + b·y + c·z Has crystal symmetry Doesn’t have crystal symmetry Slide17: Proteins pack symmeterically within crystals Slide18: Prerequisites for protein crystallisation.
Need about 10 mg purified protein.
- Various forms of chromatography.
- Better than 95 % purity if possible.
Must be homogeneous.
- Protein isoforms & microhetrogenity very damaging to crystal growth.
Typically concentrate to about 20 mg/ml.
Must be stable throughout the experiment.
- Can take days, weeks or months to grow crystals. Slide19: Typical purification protocols.
Grow cells (E. coli, yeast etc).
Break cells (French press or sonication or lysozyme).
Separate eg. membranes from other things by centrifugation.
- Extract supernatent, resolubilise membrane proteins or inclusion bodies.
- Ion exchange chromotography; affinity chromotography (His tag); Gel filtration most common. Isoelectric focussing a less common option.
Check purity on a SDS gel.
- Other biophysical characterisation such as activity assays, dynamic light scattering, etc.
Frequently change buffer.
Concentrate to typically around 10 to 20 mg/mL.
Crystallisation setups. Slide20: Crystallisation concept
Protein solubility affected by adding "precipation agents"
- eg. salt, polyetheleneglycol etc.
In a controlled way take protein to supersaturation.
- Adding percipitant.
- Drying out the drop.
- Exchanging the buffer (dialysis).
Wait & regulatly observe the experiment under a microscope. Slide21: Factors affecting protein solubility.
- As pH changes certain groups (eg. Asp, Glu, Lys, His, Arg, Try) go from neutral to charged, or from charged to netural.
- Alters surface charges & interactions with water.
Salts affect protein surface charges & interact with water.
- Salting in (adding salt increases protein solubility).
- Salting out (adding salt decreases protein solubility).
- eg. polyetheleneglycol (PEG) soaks up water.
- Thermodynamic factors influence solubility.
Slide22: Solubility curve
Protein solubility depends on concentration.
- Eventually it will percipitate.
Adding a "precipitant" (or precipitating agent) can lower the protein solubility.
- This way achieve super-saturation.
Nucleation can occur.
- If too many nuclei then hundreds of tiny-crystals.
BUT If close to the solubility curve may achieve slow crystal growth. Slide23: Batch (& micro-batch) experiments.
Mix solubilised protein with a precipitant.
- Achieve directly a super-saturated sample.
- Protein concentration decreases as the crystals grow.
Simple (just mix).
- Easily scaled down to 100 nl levels (micro or nano-batch) & robot based approaches. Protein + precipitant solution Slide24: Precipitant solution Protein + precipitant solution Vapour diffusion Vapour diffusion
Soluble protein placed in a drop (~5 ml) above a buffer with higher precipitant agent concentration.
Drop & reservoir equilibrate by exchanging water (vapour diffusion).
- The most popular method
- Hanging drop & sitting drop.
Achieve supersaturation, nucleation & crystal growth. Slide25: Dialysis experiments
Soluble protein placed in a "dialysis button" covered with a dialysis membrane.
- Equilibrates with buffer in which the button is placed.
- Can increase or decrease the precipitant contentration.
Leads to supersaturation, nucleation & growth. Slide26: Temperature:
Normally experiments performed in temperature controlled rooms.
We have 20oC and 4oC rooms. Slide27: amorphous non-amorphous Types of preciptiation microcrystals A skilled person can ”read” the drops & knows what to try next. Slide28: Crystals Slide29: More Crystals Slide30: Screening & Optimisation
Begin with a commercial screen of 48 or 96 conditions.
- Samples a range of pH, percipitant agent & additive conditions successful for crystallisation.
If hits are promising then optimise around the conditions.
- vary pH.
- Salts (monovalent & divalent cations can help tremendously).
- Additives (many small molecules, eg. MPD, ethanol, Heptanetriol etc.).
A multi-dimensional search.
- If have a lot of protein can make a grid-search.
- If limited must try to be selective & effecient. Slide31: Crystallisation Robots
Can perform experiments down to 100 nl drops.
- More accurate than a person.
- Enables many more experiments to be made rapidly for the same amount of purified protein. Slide32: Problems
Just get percipitate:
- Protein denatured?
- Micro-hetrogeniety? (seriously disrupts crystal packing).
- Need better preparation & purification.
- Other protein sources to find more likely candidates.
Protein too flexible?
- Additives eg. metals or inhibitors to increase stability.
- Mutagenesis/other sources to increase stability.
- Break up & target sub-domains.
Too many micro-crystals.
- Micro-seeding (adding crushed up & diluted crystal seeds).
- Streak seeding (touching a crystal with a cat-whisker & streaking it through a new drop). Slide33: Crystals diffract to only very low resolution.
- Check protein preparation & try seeding.
- Try to slow down growth
* lower protein/percipitant concentrations.
* lower temperature.
Crystals grow as eg. very thin rods & cannot be used
- Seek out new crystal forms.
Crystals not reproducible.
- Sequence the crystal & check for contaminant.
Number of experiments?
- May need hundreds of experiments to optimise overexpression.
- May need to try dozens of different protein sources.
- May need to do thousands of crystal screens.
- May need tens-of-thousands of optimisation experiments.
- May be lucky with first experiments. Slide34: Cryo-techniques & freezing
Exposure to X-rays causes damage.
- Electrons removed from atoms.
- Free radicals created & highly reactive.
- With time the crystal "dies".
At low temperature the crystal life-time extended.
- Most X-ray data now recorded near 100 K.
Freeze crystals by plunging into liquid nitrogen (or liquid propane if problematic).
- Crystals frequently damaged by freezing.
- Become more moasic (ie. Broken up into tiny tiny nano-crystals each with slightly different orientations).
- Can lower the resolution.
Add a cryo-protectant before freezing.
- Typically glycerol or PEG400.
- Must also screen & optimise cryo-protectants. Slide35: X-ray source Diffractometer
Freeze a crystal on a loop & mount in an X-ray beam. Slide36: X-ray diffraction from a protein Large number of spots because unit cell large
- typically 30 to 300 Å. Slide37: Synchrotron Radiation Large international facilities.
- Brightest X-ray sources available.
Cost about 1 billion Euros.
- Sweden has a cheap one in Lund.
User communities of scientists travel to them. Slide38: Collecting data
Must rotate the crystal over many degrees so as to sample all angles.
Typically ~ 100 X-ray diffraction images in a “data set”. Slide39: Progress in structural determination Slide40: Grow protein crystals. Use synchrotron X-rays. Collect diffraction data. Interpret electron density. X-ray crystallography summary Slide41: Summary of Lecture
X-ray diffraction a very powerful tool for structural determination.
Crystallisation is as much an art as a science.
Sample must be pure & homogeneous.
Myoglobin was the first X-ray structure solved almost 50 years ago.
27,000 entries now in the protein data bank.