Part I - Introduction

Chapter 1

Myoglobin

> A recent report Philip Anfinrud et al. has demonstrated the "filming" of myoglobin getting rid of toxic carbon monoxide, using picosecond crystallography. By exposing a crystal to ultra-short flashes (100 picoseconds) of X-ray light, it was possible to resolve the positions of all 1,432 atoms that make up the protein and to watch as carbon monoxide oxygen moves through the molecule.

> Myoglobin is the protein that stores oxygen in muscle tissue, which plays a central role in the production of energy in muscles. Every time mammals contract a muscle, myoglobin releases oxygen, which is used in the production of energy. Muscle cells use myoglobin as a peak-load buffer when blood cannot supply oxygen fast enough, for example when the circulation is blocked during muscle contraction. The oxygen molecule is initially confined in a cavity called the heme-pocket, where it is chemically bound to an iron atom. In the experiment carbon monoxide was used here as a replacement for oxygen for technical reasons, and a mutant version of the protein was studied because the highly strained atomic structure increases the speed that the CO molecule is removed compared with that of ordinary myoglobin, but this was thought to be a relaible model.

> The carbon monoxide was shown to move out of the protein swiftly, spending most of its time captured in 5 tiny cavities inside the protein. In the first cavity near the iron atom, the CO molecule makes an extremely brief visit lasting only a tenth of a billionth of a second. The iron atom would naturally try to rebind the carbon monoxide, but nearby molecules block the CO from going back to the iron. The film has shown that the motion between the 5 cavities is very fast and the carbon monoxide molecule reaches the fifth cavity after 30 nanoseconds and then it disappears into the solvent surrounding the protein. Interestingly, another CO molecule, released from a myoglobin molecule nearby, will eventually diffuse back towards the iron, most likely through another route. The iron accepts the incoming CO due to the fact that the structure of the protein has changed to allow for the rebinding.

> This ground breaking work was carried out at the ESRF using the White Beam Station (ID9), which was designed for time-resolved experiments in macromolecular crystallography and liquids, and also for high pressure research. Its unique feature is the focused white beam, which can be used for time-resolved diffraction on macromolecules. The migration of the carbon monoxide molecule through the myoglobin molecule after photolysis is shown below. The sites that are currently occupied by the carbon monoxide are marked by solid yellow circles. Sites from which the carbon monoxide has departed are depicted by a dotted yellow circle.

> The original details are available from the ESRF website, and the work has also been published:

> F. Schotte, M. H. Lim, T. A. Jackson, A. V. Smirnov, J. Soman, J. S. Olson, G. N. Phillips, M. Wulff, and P. A. Anfinrud,

> Science, 300(5627), 1944-1947 (2003).

Chapter 2

The Oxford Cryosystems PheniX

> The Oxford Cryosystems PheniX is designed so that the chamber moves with the gonoimeter. This means that all scattered X-rays can be collected in the full 2 theta range. The X-ray source is on the left and the position sensitive detector (PSD) on the right. The PSD scans through 2q (anticlockwise) while the goniometer in the centre scans through q (also anticlockwise). As the goniometer moves, so does the sample chamber, but there is a hinge between the sample chamber and the lines which mean that the chamber can move freely but the lines don't get tangled.

> Not only is the design inovative, the system is also stable and can run for relatively long periods of time without needing attention.

Thanks to Drs. Simon Allen and Richard Gover for the film footage.