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

© International Union of Crystallography 2006
International Tables for Crystallography (2006). Vol. F. ch. 18.4, pp. 401-402   | 1 | 2 |

Section 18.4.7. Relation to biological chemistry

Z. Dauter,a* G. N. Murshudovb and K. S. Wilsonc

a National Cancer Institute, Brookhaven National Laboratory, Building 725A-X9, Upton, NY 11973, USA,bStructural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, England, and CLRC, Daresbury Laboratory, Daresbury, Warrington, WA4 4AD, England, and cStructural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, England
Correspondence e-mail:  dauter@bnl.gov

18.4.7. Relation to biological chemistry

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A question arises as to what biological issues are addressed by analysis of macromolecular structures at atomic resolution. For any protein, the overall structure of its fold, and hence its homology with other proteins, can already be provided by analyses at low to medium resolution. However, proteins are the active entities of cells and carry out recognition of other macromolecules, ligand binding and catalytic roles that depend upon subtle details of chemistry, for which accurate positioning of the atoms is required. Even at atomic resolution, the accuracy of structural definition is less than what would ideally be required for the changes observed during a chemical reaction. At lower resolutions, structure–function relations require yet further extrapolation of the experimental data.

To understand the function of many macromolecules, such as enzymes, it is not sufficient to determine the structure of a single state. Alongside the native structure, those of various complexes will also be required. The differences between the states provide additional information on the functionality. For an understanding of the chemistry involved, atomic resolution has tremendous advantages in terms of accuracy, as reliable judgments can be based on the experimental data alone.

Advantages of atomic resolution include the following:

  • (1) The positions of all atoms that possess defined conformations are more accurately defined. This means that all bond lengths and angles in the structure have lower standard uncertainties (EU 3-D Validation Network, 1998[link]). For regions of the molecule where the conformation is representative this is of purely quantitative significance, but where the stereochemistry deviates from the expected value this accuracy takes on a special significance, which poses questions to the theoretical chemist. Such deviations from standard geometry often play an important role in biological function.

  • (2) The better the ADP definition, notably its anisotropy, the greater the insight into the static or thermal flexibility of individual regions of the molecule. Macromolecules are crucially dependent upon flexibility for properties, such as induced fit in substrate or ligand recognition, allosteric responses or responses to the biological environment. More detailed definition of the position and mobility of flexible regions may be assisted by atomic resolution analysis.

  • (3) A few amino-acid side chains play an active role in catalysis (those that do include histidine, aspartic and glutamic acids and serine) throughout protonation–deprotonation events, and hydrogen atoms are crucial to their function. Hydrogen atoms are usually treated as riding on their parent atoms and should be included in the model, even at medium resolution; unfortunately, those hydrogen atoms that are of interest can only rarely be treated as rigidly bonded at a predictable position. However, atomic resolution allows many hydrogen atoms to be clearly identified in the refined electron density. In addition, the presence or absence of hydrogen may be inferred by accurate estimation of the bond lengths between atoms, e.g. within the carboxylate groups.

  • (4) The relative orientation of reacting moieties is crucial to enzyme catalysis. If chemical hypotheses of mechanism are to be subjected to appropriate Popperian scrutiny (Popper, 1959[link]), then precise definition of atomic coordinates in native and complex structures is necessary.

  • (5) Enzyme catalysis provides a reduction of the activation energy of the reaction, which can be achieved by distortion of the conformation of the substrate bound to the enzyme, the so-called Michaelis complex, towards the transition state or by the stabilization of the latter by the enzyme. For both, the study of complexes of inhibitors or substrate analogues at a sufficient resolution to clarify the fine detail of the structures is required.

  • (6) Adaptation of the enzyme to the substrate is postulated by the induced-fit theory of catalysis. The level of adjustment can be very small, and energy calculations again require that this be precisely defined.

  • (7) In metalloproteins, the ligand field and hence geometry and bond lengths around the metal ion are essential indicators of any variation in valence electrons between different states. For example, bond lengths between oxidized and reduced states of metal ions vary by the order of 0.1 Å or less, and clear distinction between alternative oxidation states requires an accuracy only provided by atomic resolution.

Almost all atomic resolution analyses require data recorded from cryogenically frozen crystals. This does pose some problems of biological relevance, as proteins in vivo have adapted to operate at ambient cellular temperatures. The required structure is that of the protein and surrounding solvent at the corresponding temperature. The trade-off is that cryogenic structures may be better defined, but only because of the increased order of protein and solvent at low temperature. This has to be weighed against the lack of fine detail in a medium-resolution analysis at room temperature.

A question often raised with regard to the worth of atomic resolution data concerns the effort required in refining a protein at such resolution. To define all details, such as alternative conformations, hydrogen-atom positions and solvent, is certainly time consuming, especially if an anisotropic model is adopted. However, the advantages outweigh the disadvantages, as even if a full anisotropic model is not refined to exhaustion, nevertheless all density maps will be clearer if the resolution is better, resulting in an improved definition of the features of interest.

References

First citation EU 3-D Validation Network (1998). Who checks the checkers? Four validation tools applied to eight atomic resolution structures. J. Mol. Biol. 276, 417–436.Google Scholar
 First citation Dauter, Z., Wilson, K. S., Sieker, L. C., Meyer, J. & Moulis, J.-M. (1997). Atomic resolution (0.94 Å) structure of Clostridium acidurici ferredoxin. Detailed geometry of [4Fe-4S] clusters in a protein. Biochemistry, 36, 16065–16073.Google Scholar
 First citation Johnson, C. K. (1976). ORTEPII. A FORTRAN thermal-ellipsoid plot program for crystal structure illustration. Report ORNL-5138. Oak Ridge National Laboratory, Tennessee, USA.Google Scholar
 First citation Murshudov, G. N., Vagin, A. A., Lebedev, A., Wilson, K. S. & Dodson, E. J. (1999). Efficient anisotropic refinement of macromolecular structures using FFT. Acta Cryst. D55, 247–255.Google Scholar
 First citation Popper, K. R. (1959). The logic of scientific discovery. London: Hutchinson.Google Scholar








































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