Xray crystallography

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X-ray crystallography Richard Neutze Richard.Neutz
e_at_chembio.chalmers.se Ph 773 3974 Laboratory
located on bottom floor of the Lundberg building,
Medical Hill.
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• Crystal Concept
• In 1611 Kepler suggested that snowflakes derived
  from a regular arrangement of minute brick-like
  units.
• -The essential idea of a crystal.

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• 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.

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• Theory of Diffraction
• 1910 von Laue derived theory of diffraction from
  a lattice.

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• Braggs 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
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• 1953 Double helix structure of DNA
• Crick Watson used X-ray diffraction to work
  out the way genes are encoded.

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• Diffraction pattern.
• Crystals diffraction pattern recorded by
  Rosalind Franklin.
• Revealed the symmetry of the helix pitch of
  helix.

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• First Protein Structure
• Myoglobin.
• Protein purified from whale blood.
• Max Perutz 1958.
• Showed a 75 a-helical fold.
• 155 amino acids, 17 kDa.

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• First Protein Complex
• Hemoglobin.
• Two copies each of a b chains of myoglobin in
  a complex.
• Solved by John Kendrew.

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• Structure of Nucleic Acid Protein complex.
• Nobel prize to Aaron Klug in 1982.
• Also contribution to electron microscopy.

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• 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.

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• Structure of F1-ATPase
• Revealed the details of the rotational mechanism
  of ATP-synthase.
• Nobel prize in 1997.

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• 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.

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• Structure of TBSV
• First Virus structure, tomato bushy stunt virus,
  1978.
• By Steve Harrison.
• Revealed icosohedral symmetry of a virus
  particle.

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• Ribosome
• 50 S and 70 S ribosome structures in 2000.
• Massive RNAProtein complexes.
• Revealed details of how proteins synthesyzed by
  RNA.

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Crystal definition A crystal is an object with
translational symmetry r(r) r(r) ax by
cz
Has crystal symmetry
Doesnt have crystal symmetry
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Proteins pack symmeterically within crystals
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• 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.

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• 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.
• Purification
• - 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.

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• 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.

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• Factors affecting protein solubility.
• pH
• - 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).
• Polar solvents.
• - eg. polyetheleneglycol (PEG) soaks up water.
• Temperature
• - Thermodynamic factors influence solubility.

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• 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.

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• 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
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• 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.

Protein precipitant solution
Vapour diffusion
Precipitant solution
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• 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.

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• Temperature
• Normally experiments performed in temperature
  controlled rooms.
• We have 20oC and 4oC rooms.

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Types of preciptiation
non-amorphous
amorphous
microcrystals
A skilled person can read the drops knows
what to try next.
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Crystals
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More Crystals
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• 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.

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• 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.

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• 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).

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• 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.

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• 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.

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• X-ray source Diffractometer
• Freeze a crystal on a loop mount in an X-ray
  beam.

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X-ray diffraction from a protein

• Large number of spots because unit cell large
• - typically 30 to 300 Å.

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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.

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• 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.

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Progress in structural determination
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X-ray crystallography summary

• Grow protein crystals.

• Use synchrotron X-rays.

• Collect diffraction data.

• Interpret electron density.

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• 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.