Macromolecular Crystallography

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Macromolecular Crystallography
Marc SCHILTZ EPFL Laboratoire de
Cristallographie Cours BLOC 2006

• Bio-macromolecular crystallography
• solving structures of

• Proteins
• Nucleic acids (DNA, RNA)
• Large molecular assemblies (Viruses, Ribosome )

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Nucleic acids DNA

• information storage genetic information

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The structure of DNA
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Nucleic acids RNA

• Can do everything information storage
  catalysis

Transfer RNA
Hammerhead RNA
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Proteins (I)

• Polymers of 20 amino acids

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Proteins (II)

• Adopt a well-defined 3D structure

Secondary structure elements
Alcohol Dehydrogenase
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Proteins (III)

• The folding mystery 3D structure is coded
  by 1D sequence

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Proteins (IV)

• Proteins are bio-catalysts (enzymes) they
  control almost every chemical process in living
  organisms

• The 3D structure defines the biological
  function

Alcohol Dehydrogenase Active (catalytic) site
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Structure-based drug design
HIV protease complexed with Xv638 Of Dupont
Pharmaceuticals
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Myoglobin Hemoglobin
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Steps to solve a macromolecular structure by
X-ray crystallography
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What makes protein crystallography different ?
The crystals

• Difficult to obtain
• Trial and error approach
• Need a high degree of purity and rather large
  quantities of proteins
• Contain large amounts (3080) of disordered
  water
• Peculiar state of mater (gel-like)
• Rapidly dehydrate in free air
• Are mechanically fragile and sensitive to X-rays
• Are rarely of the same diffraction quality as
  small-molecule crystals

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What makes protein crystallography different ?
X-ray data collection

• Problems
• Large unit cells (cell dimensions 50500 Å)
• Very large number of reflections
  (10.0001.000.000)
• Weak intensities
• Fast deterioration of the crystals in the X-ray
  beam
• Goals
• Fast X-ray data collection with good signal/noise
• High incident beam intensity
• Avoid spatial overlapping of diffraction spots
• Slow-down the radiation damage
• Solutions
• Rotation method with area detectors
• Synchrotron radiation
• Cryo-cooling of crystals

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What makes protein crystallography different ?
X-ray data collection
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What makes protein crystallography different ?
The phase problem

• Direct methods (ab initio methods)
• Are not successful except in special cases
• Usually not enough data at high angles (high
  resolution)
• The basic hypothesis of a uniform distribution of
  atoms in the crystal unit cell is unrealistic
• Search methods (molecular replacement)
• Need a good enough model (a closely related
  structure)
• Numerically challenging (3 rotations 3
  translations)
• Physical methods (de novo methods)
• Isomorphous replacement labeling with heavy
  atoms
• Anomalous (resonant) scattering

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Phasing physical methods
No wonder we lose the phase if there is nothing
to compare with it ! Let us see what happens if
we add a standard to it, a coherent background.
() The interference of the object wave and of
the coherent background or reference wave will
then produce interference fringes. There will be
maxima wherever the phases of the two waves were
identical. (Gabor, 1972)
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Isomorphous replacement (I)
If FH is known, and IP IPH have been measured,
the phase jP can be determined up to a twofold
ambiguity.
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Isomorphous replacement (II)

• Reality is less bright
• FPH FP FH Non-Isomorphism
• Measurement errors on IP and IPH
• FH may not be known very accurately
• Phase probability distributions

Im
Re
FPH FP FH
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Anomalous Scattering (I)
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Anomalous Scattering (II)
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Electron density map model building
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Outlook

• Tremendous technical and methodological
  progresses have been achieved
• Molecular biology genetic engineering
  technologies
• Area detectors Synchrotron radiation
• Cryo-cooling of crystals
• Anomalous diffraction Se-incorporation
• Theoretical developments in phasing methods
• Numerical methods Computing power
• Sample preparation and crystallization remain
  bottlenecks
• The future
• Structural genomics proteomics
• Membrane proteins
• Eucaryotic systems
• Methodolgical challenges
• Crystalization, crystallization,
  crystallization.. more physical chemistry !
• Time-resolved diffraction
• Diffraction on micro-crystals
• Protein powder diffraction

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Myoglobin Hemoglobin
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Heamoglobin
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• Deoxyhaemoglobin
• Heam group is doomed
• Iron is out-of-plane
• High-spin state

• Oxyhaemoglobin
• Heam group is planar
• Iron is in-plane
• Low-spin state

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Small changes translate to large movements
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Small changes translate to large movements
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Virus structures
Tobacco Mosaic Virus (TMV)
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The structure of AdenovirusElectron microscopy
X-ray crystallography
Combining EM image reconstruction and X-ray
crystal structures of coat proteins
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Photosynthetic reaction centre