Structural Biology files From the SRF to the SRS
From the NINA SRF to the Synchrotron Radiation Source (SRS)
I joined the late Prof Ernst Eckard Koch at DESY in Hamburg as a DESY Fellow where I was able to participate in a variety of experiments ranging from spectroscopy to diffraction of molecular crystals. Koch was again one of the pioneers who together with Ruprecht Haensel and Christoph Kunz had established the synchrotron radiation facility on the DESY synchrotron. During my stay at DESY, the synchrotron radiation team established HASYLAB. On my return to the UK in October 1978, I took a conscious decision to use my physics and synchrotron radiation background at the interface of chemistry and biomedical science, thus joining an interdisciplinary team at Manchester derived from the Chemistry (Dave Garner, FRS) and Medical Biophysics (David Hukins) Departments who had just started the UK’s first biological XAFS project working on metalloenzymes and biological calcification. Again, I decided to locate myself at Daresbury where I had the good fortune of being given a temporary office (which became my office for the next 15 years) that was only two doors away from Sir John Pendry, FRS, who had put forward the most comprehensive modern theory of EXAFS (more of this later) (Lee & Pendry, Phys Rev B11, 2795-2811, 56-63 (1975). A year later, in 1979, I joined the national effort of establishing the world’s first dedicated synchrotron radiation source (SRS) as a full time scientific staff member of the Daresbury Laboratory where I remained until March 2008, when I became Max Perutz Professor of Molecular Biophysics at the University of Liverpool.
Establishment of XAFS as an important structural biology technique at the SRS. The two-year period 1978-80 was a steep learning curve getting to grips with X-ray instrumentation (no gratings but single perfect radiation-resistant crystals such as germanium or silicon; mirrors of different size, smoothness and quality and detectors ranging from solid-state devices to ionisation chambers), data analysis and interpretation of extracted EXFAS data. I was fortunate, as mentioned above, to have Sir John Pendry two doors away, whom l found most welcoming for a science discussion, prepared to translate difficult theoretical concepts into simple language for experimentalists such as me. Even though an approximation known as “the plane wave approximation” of the theory was readily usable requiring little computer time, I immediately grasped the importance of curvature of the electron wave for accurate structure determination and began to put effort into its full implementation in the form of EXCURVE (Binsted, Gurman and Strange played major roles, see Biochemistry, 31, 12117-12125 (1992) & J. Synchr. Rad. 3, 185-196 (1996)). From John, I also learnt an “open door policy” to encourage younger members of the team to come and talk, that I still maintain at the University. Likewise I was inducted into the new and emerging field of bioinorganic chemistry by David Garner, FRS, who was one of my PDRA supervisors for a year (1978-79) and then a great collaborator until the early 90s when my science interest and approach began to change. In these early years many leading biochemists placed their trust in us with their precious samples that they obtained with hard labour in a highly purified form in the hope that we would be able to provide some important structural information that would provide support to a particular mechanism. An initial success came from the late Bob Bray of the University of Sussex who had provided more than a gram of purified lyophilised molybdenum containing xanthine oxidase in two forms. We were successful in collecting data and extracting reliable structural information that resulted in the first significant biological XAFS publication from the UK (Biochemical J. 191, 499 (1980)).
Towards the end of 1980, Max Perutz approached me to see if I was prepared to help him resolve a serious challenge to his stereochemical mechanism of haem-haem interaction that had come about from some EXAFS work that was conducted in the United States by some leading influential scientists (Eisenberger et al Nature 274, 30-34 (1978)). I accepted the request despite the obvious difficulties (see below).
Note from Samar Hasnain: Max’s view on the James Lauterbrunner (in real life Peter Eisenberger) result was that his theory of a stereochemical mechanism was dead. Typical of him, not knowing the technique, he set about making arrangements for doing the XAFS measurements on a smaple prepared by him. He recruited his friends worldwide to get the measurements done at the Stanford Synchrotron in May 1980 on BL15 and BL23. But Max then faced the problem of data analysis. This brought him luckily to me in late 1980. I was aware of the controversy and had learnt of the difficulties of anyone looking at the data in the USA, for fear of their career. In fact an englishman who had done his PhD at Stanford was at the EMBL in Hamburg at the time; he could have analysed the data but decided not to, as he did not wish to rule out the possibility of working in the USA. In a way this was good for Max’s mechanism as the problem turned out not to be with the EXAFS results but with triangulation (see Nature 295, 535-538 (1982)). (Correction! In the second paragraph my affiliation is mentioned as Hamburg Synchrotron. It should have been Daresbury Synchrotron.)
Over the next nine months, I rigorously analyzed the data using the most accurate curved wave implementation of EXAFS theory where it took overnight computation on the best IBM computer available at Daresbury (Daresbury was one of the major national computer centres at the time) to complete a single iteration. In May 1981, I had the result, which confirmed the original EXAFS structural parameters. This compelled me to think where the problem regarding the lack of movement of iron from the porphyrin plane in Eisenberger’s study might originate. I set about looking at all of the chemical porphyrin compounds that had been used for comparison and as standards in the original study and our own. The answer was obvious – Eisenberger had used the triangulation method where the assumption was made that the distance of the centre of the porphyrin plane to nitrogens between the compounds and haemoglobin is transferable. Eisenberger had used a value of 2.045Å for centre to nitrogen distance rather than the more commonly used value of 2.02Å. I wrote a detailed letter to Max on 14th May 1981 describing the problem in detail. I received an instant response via a handwritten letter on 18th May expressing his excitement. With some additional data on related compounds collected and analysed, we quickly wrote the paper and submitted it to Nature on 22 September 1981. This was just a few months after the SRS had come into operation with its initial energy of 1.8GeV and two bending magnet beamlines, line 7 for for X-rays and line 6 for VUV and soft X-ray primarily for Surface Science experiments. The paper was accepted in December 1981 and published on 11th February 1982 (Nature 295, 535 – 538). It is remarkable that both of us were so focussed on getting the data analysed and resolving the problem scientifically that we never met prior to the publication. This was remedied by many visits including him staying at our home; he prepared a very large MbCO single crystal for the first angle resolved XANES study of a protein crystal using polarised X-rays from the SRS (Nature, 318,685-687 (1985)). This early work led to two distinct major contributions: first the realization that multiple scattering events in XAFS needed to be handled accurately and second that the combination of XAFS and crystallography would be very powerful for structure-function studies of metalloproteins, hence giving birth to a combined methods approach that I developed.
Through a BBSRC/MRC grant, we were eventually able to build a dedicated experimental beamline for combined crystallography and single crystal XAFS at the SRS that was opened by Cherie Blair on 28th January 2005 (Journal of Synchrotron Radiation 12, 455-466 (2005), Proceedings of National Academy of Sciences, 104, 6211-6216 (2007) and Journal of Molecular Biology, 378, 353-361 (2008)). The use of this combined approach has led to a global effort to pursue damage-free crystallographic data collection by using spectroscopic methods to validate redox states. Using the most advanced synchrotrons and X-ray lasers, the serial crystallography approach is being developed for obtaining damage-free structures of functional states of redox enzymes.