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X-Ray Crystallography

X-RAY Crystallography Reveals Three-Dimensional Structure in Atomic Detail

Protein structure and function has been enriched by x-ray crystallography, a technique that can reveal the precise three-dimensional positions of most of the atoms in a protein molecule.
Crystals of the protein of interest are needed because the technique requires that all molecules be precisely oriented.  Crystals can often be obtained by adding ammonium sulfate or another salt to a concentrated solution of protein to reduce its solubility.
Example, myoglobin crystallizes in a 3M ammonium sulfate.  Slow salting out favors the formation of highly ordered crystals instead of amorphous precipitates.  Some proteins crystallize readily, whereas others do so only after much effort has been expended in finding the right conditions.  Crystalliztion is an art; the best practitioners have great perseverance and patience, as well as a golden touch.  Increasingly large and complex proteins are being crystallized.
The three components in an x-ray crystallographic analysis are a source of x-rays, a protein crystal, and a detector.  A beam of x-rays of wavelength 1.54 A is produced by accelerating electrons against a copper target.  A narrow beam of x-rays strikes the protein crystal.  Part of it goes straight through the crystal; the rest is scattered in various directions.  The scattered (or diffracted) beams can be detected by x-ray film, the blackening of the emulsion being proportional to the intensity of the scattered x-ray beam, or by a solid-state electronic detector. The basic physical principles underlying the technique are

  1. Electrons scatter x-rays.  The amplitude of the wave scattered by an atom is proportional to its number of electrons.  Thus a carbon atom scatters six times as strongly as a hydrogen atom.
  2. The scattered waves recombine.  Each atom contributes to each scattered beam.  The scattered waves reinforce one another at the film or detector if they are in phase (in step) there, and they cancel one another if they are out of phase.
  3. The way in which the scattered waves recombine depends only on the atomic arrangement.

The protein crystal is mounted in a capillary and positioned in a precise orientation with respect to the x-ray beam and the film.  Processional motion of the crystal results in an x-ray photograph consisting of a regular array of spots called reflections.  The intensity of each spot is measured.  These intensities are the basic experimental data of an x-ray crystallographic analysis.  The next step is to reconstruct an image of the protein from the observed intensities.  In light microscopy or electron microscopy, the diffracted beams are focused by lenses to directly form an image.  However, lenses for focusing x-rays do not exist.  Instead, the image is formed by applying a mathematical relation called a Fourier transform.  For each spot, this operation yields a wave of electron density, whose amplitude is proportional to the square root of the observed intensity of the spot.  Each wave also has a phase- that is, the timing of its crests and troughs relative to those of other waves.  The phase of each wave determines whether it reinforces or cancels the waves contributed by other spots.  These phases can be deduced from the well-understood diffraction patterns produced by heavy-atom reference markers such as uranium or mercury at specific sites in the protein.

The stage is then set for the calculation of an electron-density map, which gives the density of electrons at a large number of regularly spaced points in the crystal.  This three-dimensional electron-density distribution is represented by a series of parallel sections stacked on top of each other.  Each section is a transparent plastic sheet (or a layer in a computer image) on which the electron-density distribution is represented by contour lines.
The next step is to interpret the electron-density map.  A critical factor is the resolution of the x-ray analysis, which is determined by the number of scattered intensities used in the Fourier synthesis.  A resolution of 6 A reveals the course of the polypeptide chain but few other structural details.  The reason is that polypeptide chains pack together so that their centers are between 5 and 10 A apart.  Maps at higher resolution are needed to delineate groups of atoms, which lie from 2.8 to 4.0 A apart, and individual atoms, which are between 1.0 and 1.5 A apart.  The ultimate resolution of an x-ray analysis is determined by the degree of perfection of the crystal.  For proteins, this limiting resolution is usually about 2 A.
The structures of more than 300 proteins have been elucidated at atomic resolution.  Knowledge of their detailed molecular architecture has provided insight into how proteins recognize and bind other molecules, how they function as enzymes, how they fold, and how they evolved. 


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