Wisdom From Albert

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Wisdom From Albert

• "The important thing is not to stop questioning.
  Curiosity has its own reason for existing."
• "If A is a success in life, then A equals x plus
  y plus z. Work is x y is play and z is keeping
  your mouth shut."

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• Proteins have a Hierarchy of structure Please
  describe that hierarchy and how each hierarchy
  fits into the next.

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Bits of Chapter 5

• Hydrodynamics Covered in Biophysical
• CD spectra
• Circularly polarized light interacts with Chiral
  molecules
• Helices and Beta sheets are Chiral even if they
  are made out of glycine.
• Why
• helices more chiral than beta sheets
• why?

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Bits of Chapter 5

• Fiberous proteins
• historically important
• First structures elucidated by x-rays
• Gave us terms Apha helix Beta sheet
• should review
• Take home Quiz/homwork 6. Based on their
  molecular structures why can you wash silk in hot
  water and have to wash wool in cold?

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Protein structure

• How do we look at proteins

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How do we determine the structure of proteins

• X ray Crystallography
• NMR
• Neutron diffraction
• Electron diffraction.

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X-ray Crystallography

• About the Unit Cell
• Why are X-rays used ?
• Why X-ray diffraction ?
• Growing Crystals
• X-ray diffraction

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About the Unit Cell

• Crystals are three dimensional ordered structures
  than can be described as a repetition of
  identical unit cells.
• The unit cell is made up of the smallest possible
  volume that when repeated, is representative of
  the entire crystal.
• This unit contains an integer number of protein
  molecules
• The dimensions of a unit cell can be described
  with 3 edge lengths (a,b,c) and 3 angles (alpha,
  beta, gamma).
• The 3D location of atoms within a unit cell can
  be listed as their x, y, z Cartesian Coordinates.
  
• Space groups describe the symmetry of a unit
  cell, of which there are 230 variations

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Why are X-rays used ?

• Resolution is limited to ½ ? Atoms have
  dimensions in Å
• Visible light has wavelengths of 100s of
  nanometers
• X-Rays have wavelengths of Å

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Why X-ray diffraction ?

• No known way to focus x-rays with a lens.
• Diffraction patterns can be interpreted
  mathematically.
• We use computers as a virtual lens, so on a
  monitor we can look at the structure of a
  molecule.
• The x-ray diffraction from one unit cell would
  not be significant. Crystals are important
  because by definition they have a repeated unit
  cell within them.
• The repetition of unit cells within a crystal
  amplifies the diffraction enough to give results
  that computers can turn into a picture

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Growing Crystals

• Mostly a trial and error process
• Closely related proteins may require different
  conditions to crystallize
• Some consistent principles
• Protein must be relatively pure
• Requires some sort of small ion salt
• Precipitant usually involved.
• Some way of gradually increasing the ppt in the
  system.
• pH is usually close to the pI of the protein

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Growing Crystals

• More general principles
• High concentration of protein
• Low temperature tends to favor crystallization
• Partial factorial screens now common
• Dumb Luck
• Numerous cases of finding conditions by accident
  during purifications on proteins

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Methods of Crystal growth

• There are various methods of growing protein
  crystals
• Vapor Diffusion -(Hanging Drop Method) This is
  probably the most common ways of crystal growth.
  A drop of protein solution is suspended over a
  reservoir containing buffer and precipitant.
  Water diffuses from the drop to the solution
  leaving the drop with optimal crystal growth
  conditions.
• Batch crystallization A saturated protein
  solution left in a sealed container to let the
  crystals grow.
• Micro batch crystallization A drop of protein
  solution is put in inert oil and left to grow.
  Here there probably is some diffusion of proteins
  into the oil, lowering the saturation over time.

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Methods of Crystal growth

• There are various methods of growing protein
  crystals
• Vapor Diffusion -(Hanging Drop Method) A drop
  of protein solution is suspended over a reservoir
  containing buffer and precipitant. Water diffuses
  from the drop to the solution leaving the drop
  with optimal crystal growth conditions.
• Batch crystallization A saturated protein
  solution left in a sealed container to let the
  crystals grow. (Almost always the way dumb luck
  works)
• Micro-batch crystallization A drop of protein
  solution is put in inert oil and left to grow.
  Here there probably is some diffusion of proteins
  into the oil, lowering the saturation over time.

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Methods of Crystal growth

• Free interface diffusion A container has levels
  of varying saturation. Microcrystals/ppt form
  initially in the highly saturated part, but as
  the solution mixes, it eventually only supports
  crystal growth.
• Dialysis Similar to the previous, but with a
  semi-permeable membrane separating the layers.
• Macro-seeding A crystal is grown in a highly
  saturated solution and placed in a less saturated
  one where only growth of the crystal will occur.
• Micro-seeding A few crystals are grown, then
  crushed, and put into a final solution that
  combines them into a few nice crystals. This
  involves quite a bit of experimentation with
  solutions' concentrations to get the desired
  number of crystals.

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X-ray diffraction

• Single Wavelength diffraction
• 1.542 Å most commen Resolution 0.771 Å
• Multiple exposures required
• Heavy atom derivitives required.
• Data is relatively easy to interprete.
• Ability to tell if the Data is worth anything
• Equipment fits in a regular lab

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X-ray diffraction

• Laue Diffraction
• Multiple wavelengths used
• Requires a cyclotron to generate X-rays
• Data is much more complex
• Single good exposure often results in a structure
• Able to use poorer quality crystals
• Phase may be determined by over-tones
• Able to image transient structures.

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X-ray diffraction

• Reflections occur in reciprocal space
• Reflections follow Braggs Law
• 2d sinTn n?
• Symmetry elements critical to solving the
  structure.