History:

1. First used in 1913 for ZnS (a small molecule) by the Braggs
2. Max F. Perutz began work on the structure of hemoglobin (mol. wt. of 67,000 daltons) in 1936
3. Perutz' student, J. C. Kendrew, began the structure of myoglobin shortly thereafter

Structure of myoglobin was completed in 1957; hemoglobin in 1959; Nobel prizes were awarded in 1962!

Principles:

Bragg's law = indicates the condition for waves in phase; these give constructive interference

o-----o-----o-----o-----o-----o-----o-----o-----o
l d
(0.1 nm) q
o-----o-----o-----o-----o-----o-----o-----o-----o

Where l is the wavelength of light
d is the distance between elements in the array
q is the angle of incidence of the X-ray

Diffraction results in areas (spots) of high intensity where constructive interference occurs and other areas of low intensity where destructive interference occurs.

Laboratory procedure:

1. must have a purified protein (or nucleic acid)

2. must be able to crystallize this molecule into a crystal from that is stable to irradiation by X-rays

3. mount the crystal in the X-ray machine

                       x-rays	I
 I______I  ---------------D    	I   film or array of detectors	
	I

5. use a computer to carry out Fourier transforms on this density data

6. from these spots (reflections) and the Fourier transform, a contour map of electron density is created

7. an actual physical model is then constructed so as to place the nuclei of each of the non-hydrogen atoms in the center of one of each of the areas of high electron density (originally done by hand is now done by computer).

In addition to the immense amount of work required, Perutz and Kendrew faced two major problems that had to be solved in order to obtain a structure:

A. The phase determination (i.e. what is up and down). This was achieved by the process of heavy atom (U or Pb) replacement.

B. Obtaining adequate computer power for the massive number of computations, the Fourier transform calculations. The first electronic computers were not developed until after World War II.

By 1957, Kendrew had measured 400 reflections, and from them, created a structure for myoglobin at the level of 0.6 nm resolution. (No individual amino acid residues were visible)

Later he analyzed 9600 reflections to produce a density map at 0.2 nm resolution.

Finally, in 1962 he analyzed 25,000 reflections to produce a density map at 0.14 nm. Resolution of this level allowed assignment of 1200 of the 1260 non-hydrogen atoms.

Today the technique of X-ray crystallography has been refined such that preparing robust crystals of the appropriate size is the most difficult task in obtaining a molecular structure. The advent of arrays of detectors connected directly to powerful computers, permits the generation of molecular structures on the computer display in a matter of weeks.

Validity of X-ray crystal structures:

How do we know that what we see in the crystal structure is valid for the molecule in solution?

Evidence:

1. crystalline Mb can bind oxygen
2. absorption spectra for crystalline and solution Mb are the same, and they are very sensitive to environment.
3. -helix content in solution (ORD-CD) is approximately that found in crystal structures.
4. crystals with different lattice forms (different molecular packing) yield same X-ray protein structure.