MILESTONES MILESTONE 12 The first of its kind In 1937, an ambitious project was able to publish the was initiated with the aim of structure of haemoglobin, determining the molecular structure at a resolution of 5.5 Å. of haemoglobin, the protein in red To obtain the structure, blood cells that transports oxygen. it was necessary to analyse Haemoglobin forms crystals that thousands of reflections diffract X-rays, but even a beautiful and, as Perutz pointed diffraction pattern still requires out, “we have in fact been phases to solve the three-dimensional very fortunate, because the structure (see Milestone 7) and it was development of computers has always this problem that Max Perutz and his just kept in step with the expanding colleagues were confronted with. needs of our X-ray analyses”. After position of the haem group within In 1954, David Green, completing this complex raw-data the structure. Camazine/Alamy © Scott Vernon Ingram and Perutz published analysis, the four polypeptide chains The advances represented by the seminal paper describing how, of haemoglobin could be traced this structural work were rapidly in principle, X-ray diffraction could into the calculated electron density, recognized within the scientific be used for the direct determination which were described as resembling community, and resulted in the award of a protein structure. To obtain the vapour trails of an airplane. of the Nobel Prize in Chemistry in the crucial phases, Perutz and his Intriguingly, each of these four chains 1962 to Perutz and Kendrew “for colleagues used the isomorphous resembled the structure of the much being the first to successfully identify replacement method, whereby they smaller iron- and oxygen-binding the structures of complex proteins.” introduced ‘heavy’ atoms (in this muscle protein myoglobin, for In his acceptance speech, Kendrew case mercury) into the haemoglobin which a preliminary 6-Å resolution looked forward to a day in the crystal, taking advantage of the structure had been reported in 1958 future when structural predictions complexes formed between by John Kendrew. would allow X-ray crystallographers mercury-containing compounds Myoglobin had presented the to “go out of business, perhaps and the free sulphur groups present researchers with different challenges with a certain sense of relief”; in haemoglobin. Comparing the from haemoglobin. Importantly, this day is yet to come. differences in intensities between the myoglobin did not have the Rebecca Kirk, Senior Editor, diffraction spots from a heavy-atom- sulphur atoms required to bind to Nature Communications containing crystal and the normal mercury atoms in the same way ORIGINAL RESEARCH PAPERS Green, D. W., crystal allowed them to determine as haemoglobin. Several hundred Ingram, V. M. & Perutz, M. F. The structure the location of the mercury atoms possible heavy-atom-containing of haemoglobin. IV. Sign determination and from that information the ligands were empirically tested, by the isomorphous replacement method. Proc. Royal Soc. Lond. A 225, 287–307 (1954) | phases of the X-rays — solving the and finally mercury- and gold- Perutz, M. F. et al. Structure of haemoglobin: a so-called ‘phase problem’. As William containing ligands were found to three-dimensional Fourier synthesis at 5.5-Å Lawrence Bragg pointed out, this bind isomorphously to myoglobin, resolution, obtained by X-ray analysis. Nature 185, 416–422 (1960) | Kendrew, J. C. et al. heavy-atom technique works because allowing its structure to be Structure of myoglobin: a three-dimensional “the molecule takes no more notice of determined. In 1960, John Kendrew Fourier synthesis at 2 Å resolution. Nature such an insignificant attachment than and his colleagues reported the 185, 422–427 (1960) FURTHER READING Kendrew, J. C. et al. A three- a maharaja’s elephant would of the structure of sperm whale myoglobin dimensional model of the myoglobin molecule gold star painted on its forehead”. to a resolution of 2 Å, which required obtained by X-ray analysis. Nature 181, 662–666 (1958) | Bragg, W. L. in Fifty Years of X-ray Diffraction It would have seemed from this the assessment of 10,000 reflections. Ch. 8 (ed. Ewald, P. P.) 120–136 (IUCR, Oosthoek, breakthrough that the structure Crucially, in this high-resolution 1962) | Perutz, M. F. Nobel Lecture: X-ray Analysis determination of haemoglobin was structure the details of the atomic of Haemoglobin (Nobel Foundation, 1962) | Kendrew, J. C. Nobel Lecture: Myoglobin and the just around the corner. In fact, it took interactions could be seen, including Structure of Proteins (Nobel Foundation, 1962) six years of hard work before Perutz the right-handed α-helices and the NATURE MILESTONES | CRYSTALLOGRAPHY AUGUST 2014 © 2014 Macmillan Publishers Limited. All rights reserved.