having dissociated from the iron and moved away from its binding position. The data
sets collected with longer delays of above 1 μm did not show these electron density
features, which is consistent with the CO recombining with the heme unit and the
whole system re-relaxing back to the ground state, the whole process being completed within a few milliseconds. In a subsequent study, by the same research team,
in 2001, further data sets were obtained with time delays of between 1 ns and 1 μm,
and analysis of these data showed the position of transient docking sites within the
heme pocket in which the photodisassociated CO sat [52]. The relative positions of
these docking sites provided information on the photodissociation process and
kinetic data describing the ligand recombination process.
The success of this time-resolved approach in establishing the pathways of biological processes led to further ground-breaking studies of proteins in photoactivated
states with lifetimes down to hundreds of picoseconds [53, 54]. Using these pioneering
techniques, it has proved possible to construct “molecular movies” that describe the
full biological process in three dimensions [55].
While macromolecular time-resolved crystallography is not the focus of this
chapter, the research area has continued to lead the discipline in terms of innovation
taking full advantage of developments of, initially, synchrotron facilities [56–58]
and, more recently, of the power of the XFELs [59–64]. The molecular crystallographers have much to learn from their macromolecular colleagues not least in the
area of the treatment of crystal damage in high-intensity X-ray beams [65–67] and in
the adaption of multi-crystal data collection techniques [68].
3.2 Molecular Photocrystallography
Research into photoactivated changes in single crystals of molecular compounds
commenced in the 1960s when Schmidt and Cohen reported that a series of transcinnamic acid derivatives underwent irreversible [2 + 2] photodimerization cycloaddition reactions in the solid state [69–71]. In these pioneering studies, the authors
highlighted the importance of the surrounding crystalline environment on the pathway of the photodimerization reaction. They presented a series of key criteria that
needed to be satisfied if a single-crystal-to-single-crystal transformation was to
take place without significant crystal deterioration. These criteria were generalised
in the Topochemical Postulate, stating that the photoreaction will follow a minimum energy pathway that imparts the lowest level of steric strain to the surrounding
crystal environment. This meant that only transformations that proceeded
topotactically, that is, with the minimum amount of movement at the atomic level
could occur without crystal decay. Some subsequent improvements have been made
[72] and several exceptions found [73], but the Topochemical Postulate remains an
effective guide in the design of systems that undergo high levels of photoactivated
[2 + 2] cycloaddition. More recently, crystal engineering techniques have been
applied to the design of systems [73] that readily undergo photoactivated [2 + 2]
cycloaddition reactions in the solid state, but the basis of the Postulate remains
252
P. R. Raithby
sets collected with longer delays of above 1 μm did not show these electron density
features, which is consistent with the CO recombining with the heme unit and the
whole system re-relaxing back to the ground state, the whole process being completed within a few milliseconds. In a subsequent study, by the same research team,
in 2001, further data sets were obtained with time delays of between 1 ns and 1 μm,
and analysis of these data showed the position of transient docking sites within the
heme pocket in which the photodisassociated CO sat [52]. The relative positions of
these docking sites provided information on the photodissociation process and
kinetic data describing the ligand recombination process.
The success of this time-resolved approach in establishing the pathways of biological processes led to further ground-breaking studies of proteins in photoactivated
states with lifetimes down to hundreds of picoseconds [53, 54]. Using these pioneering
techniques, it has proved possible to construct “molecular movies” that describe the
full biological process in three dimensions [55].
While macromolecular time-resolved crystallography is not the focus of this
chapter, the research area has continued to lead the discipline in terms of innovation
taking full advantage of developments of, initially, synchrotron facilities [56–58]
and, more recently, of the power of the XFELs [59–64]. The molecular crystallographers have much to learn from their macromolecular colleagues not least in the
area of the treatment of crystal damage in high-intensity X-ray beams [65–67] and in
the adaption of multi-crystal data collection techniques [68].
3.2 Molecular Photocrystallography
Research into photoactivated changes in single crystals of molecular compounds
commenced in the 1960s when Schmidt and Cohen reported that a series of transcinnamic acid derivatives underwent irreversible [2 + 2] photodimerization cycloaddition reactions in the solid state [69–71]. In these pioneering studies, the authors
highlighted the importance of the surrounding crystalline environment on the pathway of the photodimerization reaction. They presented a series of key criteria that
needed to be satisfied if a single-crystal-to-single-crystal transformation was to
take place without significant crystal deterioration. These criteria were generalised
in the Topochemical Postulate, stating that the photoreaction will follow a minimum energy pathway that imparts the lowest level of steric strain to the surrounding
crystal environment. This meant that only transformations that proceeded
topotactically, that is, with the minimum amount of movement at the atomic level
could occur without crystal decay. Some subsequent improvements have been made
[72] and several exceptions found [73], but the Topochemical Postulate remains an
effective guide in the design of systems that undergo high levels of photoactivated
[2 + 2] cycloaddition. More recently, crystal engineering techniques have been
applied to the design of systems [73] that readily undergo photoactivated [2 + 2]
cycloaddition reactions in the solid state, but the basis of the Postulate remains
252
P. R. Raithby