Tables for
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 1.2, pp. 9-10   | 1 | 2 |

Section 1.2.6. Technological developments (1958 to the 1980s)

M. G. Rossmanna*

aDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907–1392, USA
Correspondence e-mail:

1.2.6. Technological developments (1958 to the 1980s)

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In the early 1960s, there were very few who had experience in solving a protein structure. Thus, almost a decade passed after the success with the globins before there was a noticeable surge of new structure reports. In the meantime, there were persistent attempts to find alternative methods to determine protein structure.

Blow & Rossmann (1961[link]) demonstrated the power of the single isomorphous replacement method. While previously it had been thought that it was necessary to have at least two heavy-atom compounds, if not many more, they showed that a good representation of the structure of haemoglobin could have been made by using only one good derivative. There were also early attempts at exploiting anomalous dispersion for phase determination. Rossmann (1961[link]) showed that anomalous differences could be used to calculate a `Bijvoet Patterson' from which the sites of the anomalous scatterers (and, hence, heavy-atom sites) could be determined. Blow & Rossmann (1961[link]), North (1965[link]) and Matthews (1966[link]) used anomalous-dispersion data to help in phase determination. Hendrickson stimulated further interest by using Cu Kα radiation and employing the anomalous effect of sulfur atoms in cysteines to solve the entire structure of the crambin molecule (Hendrickson & Teeter, 1981[link]). With today's availability of synchrotrons, and hence the ability to tune to absorption edges, these early attempts to utilize anomalous data have been vastly extended to the powerful multiple-wavelength anomalous dispersion (MAD) method (Hendrickson, 1991[link]). More recently, the generality of the MAD technique has been greatly expanded by using proteins in which methionine residues have been replaced by selenomethionine, thus introducing selenium atoms as anomalous scatterers.

Another advance was the introduction of the `molecular replacement' technique (Rossmann, 1972[link]). The inspiration for this method arose out of the observation that many larger proteins (e.g. haemoglobin) are oligomers of identical subunits and that many proteins can crystallize in numerous different forms. Rossmann & Blow (1962[link]) recognized that an obvious application of the technique would be to viruses with their icosahedral symmetry. They pointed out that the symmetry of the biological oligomer can often be, and sometimes must be, `noncrystallographic' or `local', as opposed to being true for the whole infinite crystal lattice. Although the conservation of folds had become apparent in the study of the globins and a little later in the study of dehydrogenases (Rossmann et al., 1974[link]), in the 1960s the early development of the molecular replacement technique was aimed primarily at ab initio phase determination (Rossmann & Blow, 1963[link]; Main & Rossmann, 1966[link]; Crowther, 1969[link]). It was only in the 1970s, when more structures became available, that it was possible to use the technique to solve homologous structures with suitable search models. Initially, there was a good deal of resistance to the use of the molecular replacement technique. Results from the rotation function were often treated with scepticism, the translation problem was thought to have no definitive answer, and there were excellent reasons to consider that phasing was impossible except for centric reflections (Rossmann, 1972[link]). It took 25 years before the full power of all aspects of the molecular replacement technique was fully utilized and accepted (Rossmann et al., 1985[link]).

The first real success of the rotation function was in finding the rotational relationship between the two independent insulin monomers in the P3 unit cell (Dodson et al., 1966[link]). Crowther produced the fast rotation function, which reduced the computational times to manageable proportions (Crowther, 1972[link]). Crowther (1969[link]) and Main & Rossmann (1966[link]) were able to formulate the problem of phasing in the presence of noncrystallographic symmetry in terms of a simple set of simultaneous complex equations. However, real advances came with applying the conditions of noncrystallographic symmetry in real space, which was the key to the solution of glyceraldehyde-3-phosphate dehydrogenase (Buehner et al., 1974[link]), tobacco mosaic virus disk protein (Bloomer et al., 1978[link]) and other structures, aided by Gerard Bricogne's program for electron-density averaging (Bricogne, 1976[link]), which became a standard of excellence.

No account of the early history of protein crystallography is complete without a mention of ways of representing electron density. The 2 Å map of myoglobin was interpreted by building a model (on a scale of 5 cm to 1 Å with parts designed by Corey and Pauling at the California Institute of Technology) into a forest of vertical rods decorated with coloured clips at each grid point, representing the height of the electron density (Fig.[link]). Later structures, such as those of lysozyme and carb­oxy­peptidase, were built with `Kendrew' models (2 cm to 1 Å) based on electron-density maps displayed as stacks of large Plexiglas sheets. A major advance came with Fred Richards' invention of the optical comparator (a `Richards box' or `Fred's folly') in which the model was optically superimposed onto the electron density by reflection of the model in a half-silvered mirror (Richards, 1968[link]). This allowed for convenient fitting of model parts and accurate placement of atoms within the electron density. The Richards box was the forerunner of today's computer graphics, originally referred to as an `electronic Richards box'. The development of computer graphics for model building was initially met with reservation, but fortunately those involved in these developments persevered. Various programs became available for model building in a computer, but the undoubted champion of this technology was FRODO, written by Alwyn Jones (Jones, 1978[link]).


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The 2 Å-resolution map of sperm-whale myoglobin was represented by coloured Meccano-set clips on a forest of vertical rods. Each clip was at a grid point. The colour of the clip indicated the height of the electron density. The density was interpreted in terms of `Corey–Pauling' models on a scale of 5 cm = 1 Å. Pictured is John Kendrew. (This figure was provided by M. F. Perutz.)


Bloomer, A. C., Champness, J. N., Bricogne, G., Staden, R. & Klug, A. (1978). Protein disk of tobacco mosaic virus at 2.8 Å resolution showing the interactions within and between subunits. Nature (London), 276, 362–368.
Blow, D. M. & Rossmann, M. G. (1961). The single isomorphous replacement method. Acta Cryst. 14, 1195–1202.
Bricogne, G. (1976). Methods and programs for direct-space exploitation of geometric redundancies. Acta Cryst. A32, 832–847.
Buehner, M., Ford, G. C., Moras, D., Olsen, K. W. & Rossmann, M. G. (1974). Structure determination of crystalline lobster D-glyceraldehyde-3-phosphate dehydrogenase. J. Mol. Biol. 82, 563–585.
Crowther, R. A. (1969). The use of non-crystallographic symmetry for phase determination. Acta Cryst. B25, 2571–2580.
Crowther, R. A. (1972). The fast rotation function. In The Molecular Replacement Method, edited by M. G. Rossmann, pp. 173–178. New York: Gordon & Breach.
Dodson, E., Harding, M. M., Hodgkin, D. C. & Rossmann, M. G. (1966). The crystal structure of insulin. III. Evidence for a 2-fold axis in rhombohedral zinc insulin. J. Mol. Biol. 16, 227–241.
Hendrickson, W. A. (1991). Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. Science, 254, 51–58.
Hendrickson, W. A. & Teeter, M. M. (1981). Structure of the hydrophobic protein crambin determined directly from the anomalous scattering of sulphur. Nature (London), 290, 107–113.
Jones, T. A. (1978). A graphics model building and refinement system for macromolecules. J. Appl. Cryst. 11, 268–272.
Main, P. & Rossmann, M. G. (1966). Relationships among structure factors due to identical molecules in different crystallographic environments. Acta Cryst. 21, 67–72.
Matthews, B. W. (1966). The determination of the position of anomalously scattering heavy atom groups in protein crystals. Acta Cryst. 20, 230–239.
North, A. C. T. (1965). The combination of isomorphous replacement and anomalous scattering data in phase determination of non-centrosymmetric reflexions. Acta Cryst. 18, 212–216.
Richards, F. M. (1968). The matching of physical models to three-dimensional electron-density maps: a simple optical device. J. Mol. Biol. 37, 225–230.
Rossmann, M. G. (1961). The position of anomalous scatterers in protein crystals. Acta Cryst. 14, 383–388.
Rossmann, M. G. (1972). Editor. The Molecular Replacement Method. New York: Gordon & Breach.
Rossmann, M. G., Arnold, E., Erickson, J. W., Frankenberger, E. A., Griffith, J. P., Hecht, H. J., Johnson, J. E., Kamer, G., Luo, M., Mosser, A. G., Rueckert, R. R., Sherry, B. & Vriend, G. (1985). Structure of a human common cold virus and functional relationship to other picornaviruses. Nature (London), 317, 145–153.
Rossmann, M. G. & Blow, D. M. (1962). The detection of sub-units within the crystallographic asymmetric unit. Acta Cryst. 15, 24–31.
Rossmann, M. G. & Blow, D. M. (1963). Determination of phases by the conditions of non-crystallographic symmetry. Acta Cryst. 16, 39–45.
Rossmann, M. G., Moras, D. & Olsen, K. W. (1974). Chemical and biological evolution of a nucleotide-binding protein. Nature (London), 250, 194–199.

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