Synchrotron radiation biomedical imaging and radiotherapy: from the UK to the Antipodes.

Although the general public might think of 'X-rays' as they are applied to imaging (radiography) and for the treatment of disease (radiotherapy), the use of synchrotron radiation (SR) X-ray beams in these areas of science was a minor activity 50 years ago. The largest gains in science from SR were seen to be in those areas where signals were weakest in laboratory instruments, such as X-ray diffraction and spectroscopy. As the qualities of SR X-rays were explored and more areas of science adopted SR-based methods, this situation changed. About 30 years ago, the clinical advantages of using SR X-ray beams for radiography, radiotherapy and clinical diagnostics started to be investigated. In the UK, a multi-disciplinary group, consisting of clinicians, medical physicists and other scientists working mainly with the Synchrotron Radiation Source (SRS) in Cheshire, started to investigate techniques for diagnosis and potentially a cure for certain cancers. This preliminary work influenced the design of new facilities being constructed around the world, in particular the Imaging and Medical Beam Line on the Australian Synchrotron in Melbourne. Two authors moved from the UK to Australia to participate in this exciting venture. The following is a personal view of some of the highlights of the early-year SRS work, following through to the current activities on the new facility in Australia. This article is part of the theme issue 'Fifty years of synchrotron science: achievements and opportunities'.

X-Ray Free-Electron Lasers for the Structure and Dynamics of Macromolecules.

X-ray free-electron lasers provide femtosecond-duration pulses of hard X-rays with a peak brightness approximately one billion times greater than is available at synchrotron radiation facilities. One motivation for the development of such X-ray sources was the proposal to obtain structures of macromolecules, macromolecular complexes, and virus particles, without the need for crystallization, through diffraction measurements of single noncrystalline objects. Initial explorations of this idea and of outrunning radiation damage with femtosecond pulses led to the development of serial crystallography and the ability to obtain high-resolution structures of small crystals without the need for cryogenic cooling. This technique allows the understanding of conformational dynamics and enzymatics and the resolution of intermediate states in reactions over timescales of 100 fs to minutes. The promise of more photons per atom recorded in a diffraction pattern than electrons per atom contributing to an electron micrograph may enable diffraction measurements of single molecules, although challenges remain.

Small Angle Scattering: Historical Perspective and Future Outlook.

Small angle scattering (SAS) is a powerful and versatile tool to elucidate the structure of matter at the nanometer scale. Recently, the technique has seen a tremendous growth of applications in the field of structural molecular biology. Its origins however date back to almost a century ago and even though the methods potential for studying biological macromolecules was realized already early on, it was only during the last two decades that SAS gradually became a major experimental technique for the structural biologist. This rise in popularity and application was driven by the concurrence of different key factors such as the increased accessibility to high quality SAS instruments enabled by the growing number of synchrotron facilities and neutron sources established around the world, the emerging need of the structural biology community to study large multi-domain complexes and flexible systems that are hard to crystalize, and in particular the development and availability of data analysis software together with the overall access to computational resources powerful enough to run them. Today, SAS is an established and widely used tool for structural studies on bio-macromolecules. Given the potential offered by the next generation X-ray and neutron sources as well as the development of new, innovative approaches to collect and analyze solution scattering data, the application of SAS in the field of structural molecular biology will certainly continue to thrive in the years to come.

Remembrance of Hugh E. Huxley, a founder of our field.

Hugh E. Huxley (1924-2013) carried out structural studies by X-ray fiber diffraction and electron microscopy that established how muscle contracts. Huxley's sliding filament mechanism with an ATPase motor protein taking steps along an actin filament, established the paradigm not only for muscle contraction but also for other motile systems using actin and unconventional myosins, microtubules and dynein and microtubules and kinesin.

A history of gap junction structure: hexagonal arrays to atomic resolution.

Gap junctions are specialized membrane structures that provide an intercellular pathway for the propagation and/or amplification of signaling cascades responsible for impulse propagation, cell growth, and development. Prior to the identification of the proteins that comprise gap junctions, elucidation of channel structure began with initial observations of a hexagonal nexus connecting apposed cellular membranes. Concomitant with technological advancements spanning over 50 years, atomic resolution structures are now available detailing channel architecture and the cytoplasmic domains that have helped to define mechanisms governing the regulation of gap junctions. Highlighted in this review are the seminal structural studies that have led to our current understanding of gap junction biology.

William Astbury and the biological significance of nucleic acids, 1938-1951.

Famously, James Watson credited the discovery of the double-helical structure of DNA in 1953 to an X-ray diffraction photograph taken by Rosalind Franklin. Historians of molecular biology have long puzzled over a remarkably similar photograph taken two years earlier by the physicist and pioneer of protein structure William T. Astbury. They have suggested that Astbury's failure to capitalize on the photograph to solve DNA's structure was due either to his being too much of a physicist, with too little interest in or knowledge of biology, or to his being misled by an erroneous theoretical model of the gene. Drawing on previously unpublished archival sources, this paper offers a new analysis of Astbury's relationship to the problem of DNA's structure, emphasizing a previously overlooked element in Astbury's thinking: his concept of biological specificity.

50 years of fiber diffraction.

In 1955 Ken Holmes started working on tobacco mosaic virus (TMV) as a research student with Rosalind Franklin at Birkbeck College, London. Afterward he spent 18months as a post doc with Don Caspar and Carolyn Cohen at the Children's Hospital, Boston where he continued the work on TMV and also showed that the core of the thick filament of byssus retractor muscle from mussels is made of two-stranded alpha-helical coiled-coils. Returning to England he joined Aaron Klug's group at the newly founded Laboratory of Molecular Biology in Cambridge. Besides continuing the TMV studies, which were aimed at calculating the three-dimensional density map of the virus, he collaborated with Pringle's group in Oxford to show that two conformation of the myosin cross-bridge could be identified in insect flight muscle. In 1968 he opened the biophysics department at the Max Planck Institute for Medical Research in Heidelberg, Germany. With Gerd Rosenbaum he initiated the use of synchrotron radiation as a source for X-ray diffraction. In his lab the TMV structure was pushed to 4A resolution and showed how the RNA binds to the protein. With his co-workers he solved the structure of g-actin as a crystalline complex and then solved the structure of the f-actin filament by orientating the g-actin structure so as to give the f-actin fiber diffraction pattern. He was also able to solve the structure of the complex of actin with tropomyosin from fiber diffraction.

Historical article: DNA polymorphism and the early history of the double helix.

Early X-ray diffraction patterns from oriented fibres indicated that DNA must have a simple, repetitious structure and encouraged some researchers, who were already convinced that DNA was the genetic material, to undertake more detailed diffraction analyses and speculative modelling. The pioneering experimental work by Wilkins in the Wheatstone Laboratory at King's College London in the late 1940s first inspired, and then was overtaken by, the conjectural modelling of Watson and Crick in the Cavendish Laboratory at Cambridge. Why this was allowed to happen is still something of a puzzle. Here, I explore the puzzle and expose a peculiar flaw in the details of the original Watson-Crick model that was left for Wilkins to resolve.

Notes of a protein crystallographer; our unsung heroes.

Dedicated to the people who designed, built, and currently work at synchrotron beamlines.

Fifty years of muscle and the sliding filament hypothesis.

This review describes the early beginnings of X-ray diffraction work on muscle structure and the contraction mechanism in the MRC Unit in the Cavendish Laboratory, Cambridge, and later work in the MRC Molecular Biology Laboratory in Hills Road, Cambridge, where the author worked for many years, and elsewhere. The work has depended heavily on instrumentation development, for which the MRC laboratory had made excellent provision. The search for ever higher X-ray intensity for time-resolved studies led to the development of synchrotron radiation as an exceptionally powerful X-ray source. This led to the first direct evidence for cross-bridge tilting during force generation in muscle. Further improvements in technology have made it possible to study the fine structure of some of the X-ray reflections from contracting muscle during mechanical transients, and these are currently providing remarkable insights into the detailed mechanism of force development by myosin cross-bridges.

Aaron Klug and the revolution in biomolecular structure determination.

Aaron Klug's group was one of the first to use a combination of X-ray diffraction and electron microscopy to study the structures of macromolecules. He helped to provide the intellectual framework for understanding the self-assembly of regular viruses and developed methods for analyzing their three-dimensional structures from electron microscope images, as well as the structures of helical polymers. He and his coworkers established the basic features of chromatin organization, including the structure of the repeating units (nucleosomes) and how they are stacked together. He studied a variety of molecules that interact with DNA or RNA, including disks of tobacco mosaic virus protein, a tRNA and a ribozyme, and also discovered the zinc-finger motif in nucleic acid-binding proteins. Thus, he has played a major part in developing the ideas and techniques that established structural molecular biology as an exciting new science during the second half of the twentieth century.

1964: The first model for the shape of a transfer RNA molecule. An account of an unpublished small-angle X-ray scattering study.

The shape of non-fractionated Escherichia coli transfer RNA molecules in solution was investigated using small-angle X-ray scattering during the years 1960-1962 at the Centre de Recherche sur les Macromolécules in Strasbourg. The innermost region of the scattering curve yielded the average molecular weight (Mr) and the radius of gyration (Rg) of the particles, whereas the experimental data at large angles could be approximated at best by the scattering curve of a kinked rod-shaped molecule. The simplest model that was compatible with Mr, Rg, and the mass per unit length of the rod was a boomerang-shaped particle made of two double helical stems connected by a sharp kink. This model that eventually proved similar to the high-resolution L-shaped structure, was presented in my Ph.D. dissertation (J. Witz, Etude de la structure de quelques polynucléotides en solution par diffusion centrale des rayons X, Ph.D. dissertation, University of Strasbourg, France, 1964) but has never been published in detail. It is the purpose of this note to recall this story.