A modern X-ray diffraction experiment

Dr Armin Wagner and Dr David Allan, Principal Beamline Scientists at Diamond Light Source, demonstrate the capabilities of one of the synchrotron X-ray diffraction beamlines at Diamond. Crystals of B12 have been grown from solution and must be selected one at a time, mounted on the equipment and carefully exposed to X-rays in a set of different orientations.

1 Selecting a crystal

Growing a good crystal can still be difficult! Crystals must be handled very carefully to avoid damaging them.

Liquid nitrogen is often used to preserve the crystals once they are removed from the solution from which they grew. Here a purple liquid nitrogen container is ready to receive the selected crystal.

Beamline I19 is one of the experimental stations at the Diamond synchrotron. An X-ray shutter is kept safely closed while preparations are made. The crystal is transported in the liquid nitrogen dewar to the experiment ‘hutch’ and carefully mounted at the centre of the diffractometer. Sometimes a robot is used to load samples from a pre-prepared set.

The fundamental geometry of crystal diffraction remains unchanged, but modern equipment is far more automated, has electronic detectors rather than film and utilises much more powerful and better focussed sources of X-rays.

David and Armin adjust the position of the crystal so it will stay in the narrow X-ray beam even when it is rotated. From the control room, they open the X-ray shutter and monitor the results remotely. They can immediately judge the quality of the crystal and carry out initial data analysis.

As the B12 crystal was slowly rotated, about 1000 separate images were collected. Here are just two of them. Each dark spot is a reflection from regularly- spaced atoms in the crystal.

From data to structure

The X-ray photographs taken by Dorothy Hodgkin and her group have been replaced by digital images from a detector, just as digital cameras are replacing film. The several stages of data analysis are now far easier than when Vitamin B12 was first solved. Dorothy’s group pioneered the use of computers for crystallography.

What a computer can now do in milliseconds used to take days, if not weeks. Calculations were done by hand or with the help of ‘Beevers Lipson strips’ and later with punched card machines.

Viewing molecules in 3D was also difficult. In 1945 Dorothy Hodgkin built a 3-D model of penicillin on Perspex sheets.

The positions of the reflections are analysed to find the set of repeat distances in the structure, called the ‘unit cell’. A correct analysis will match most of the reflections in the images (left and centre). They can also be plotted on a three-dimensional grid (right).

These calculations now take at most a few seconds of computer time, and the results are easy for the scientists to inspect.

The intensity of each reflection spot depends on the atomic positions in the molecule. These intensities are now measured from the images by computer, replacing extremely laborious work.

The process of working back to the structure is mathematically very complex. The crystallographer can now choose from a range of computer programs and tactics. The predicted intensities are compared with the actual observed values to test the structure that is obtained.

Finally a molecular structure of Vitamin B12 is obtained.