Research Article: Combining X-ray and neutron crystallography with spectroscopy

Date Published: February 01, 2017

Publisher: International Union of Crystallography

Author(s): Hanna Kwon, Oliver Smith, Emma Lloyd Raven, Peter C. E. Moody.


The use of neutron crystallography and in situ spectroscopy to study enzyme mechanism is discussed.

Partial Text

In biological processes, enzymes act as catalysts to increase the rate of reaction. These reactions always involve changes in energy and electron configurations. To understand these processes, we also need to be able to know the location of H atoms and how they are transferred between macromolecules, substrates and solvent molecules. The textbook energy profile of a reaction will show peaks of transition states (of the highest energy or least stability) and troughs of intermediates (more stable than the transition states but still less stable than reactants or products). Unfortunately, this means that the most interesting states are the least stable and the hardest to study. For crystal structures to report on mechanism we need to establish how we can isolate and monitor intermediates in the crystal; this monitoring has to be able to not only establish that a trapped intermediate is stable during the time taken for crystallographic structure determination but also that the process of data collection itself does not change it. A clear understanding of the chemistry of an enzymatic reaction also needs knowledge of the positions of H atoms. Furthermore, computational analysis and simulations of enzyme mechanisms require a full description of hydrogen positions.

X-ray crystallography relies on the scattering of X-rays by electrons; the scattering from an individual atom or ion is proportional to the number of electrons that it has. For crystallographic calculations the atomic electron density is described as a two-term or five-term Gaussian distribution [for further discussion, see the write-up for the CCP4 program SFALL ( and International Tables for Crystallography (2006 ▸)]. The neutral H atom has only one electron, but this will be shared when covalently bonded to a heavier atom; a positive hydrogen ion would have no electrons at all, although a discrete H+ ion will only have an evanescent existence. The problem of weak density is further compounded by the short length of a bond to an H atom (typically ∼1.1 Å), so resolving the very weak hydrogen electron density from a bonded ‘heavy’ (i.e. non-H) atom requires sub-ångstrom data. Difference maps calculated using sub-ångstrom data clearly do show density from H atoms (for a recent review of what can be seen at atomic resolution, see Neumann & Tittmann, 2014 ▸). Unfortunately, such data are not often available. When errors and the smearing effects of atomic displacement are considered, it is not safe to assume that an absence of density correlates to the absence of a hydrogen at a point (for an example, see Fig. 1 ▸). The high X-ray dose required to obtain atomic resolution data may also have an effect on the chemistry of the protein being studied (see §3.2).

As the most catalytically relevant and therefore informative states of the reaction pathway of an enzyme are expected to be the least stable, ways have to be found to follow reactions in the crystal and then to manipulate conditions such that these interesting states can be trapped and maintained for structural study.

Single-crystal spectroscopy can allow the monitoring and validation of intermediate catalytic states in the crystal. In favourable circumstances these can be cryo-trapped and the structures determined. Single-crystal spectroscopy is also useful to monitor crystals for photoreduction during X-ray data collection. X-ray crystallography does not directly give the positions of H atoms, but neutron crystallography does. Neutron crystallography also does not induce photoreduction. However, neutron crystallography is not as accessible as X-ray crystallography. A carefully considered combination of methods is likely to be the best approach to understanding biological chemistry through structure, and new techniques (such as X-ray free-electron lasers) will increase the scope of what can be discovered.




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