use to achieve this goal. The “diffract-and-destroy” approach has been developed at
XFELs to study macromolecular systems [42–45]. From these and related studies, it
is apparent that the direction-controlling, initial stages of a chemical reaction appear
at timescales faster than are accessible through the use of synchrotrons which
effectively have a 100 ps limit. The volume of data that is collected in one of
these studies is enormous. For example, in the study of the initial stages of the
photoexcitation of the photoactive yellow protein (PYP), which is triggered by the
trans-cis isomerisation of the coumarin chromophore, 2.5 Â 10
6 snapshots were
recorded to 1.6 Å [46]. With these advances, and with the large number of data sets
being collected accurate scaling between these data sets is required to obtain reliable
results. It has been found from scaling analyses that the anisotropy of absorption
following sample excitation can be pronounced and depends on the orientation of the
crystals in the laser beam [47] so that this factor has to be taken into consideration if
accurate analysis of molecular samples is to be achieved in the future.
3 The Beginnings of Time-Resolved Crystallography
3.1 Macromolecular Photocrystallography
The initial developments in time-resolved crystallography came in the area of
macromolecular crystallography because of the interest in important biological
processes. These studies required faster data collection and processing techniques
than had been standard. Biological crystals are also prone to decomposition particularly as a result of X-ray radiation damage, so the use of Laue methods could
achieve faster data collection with less crystal decay.
The first nanosecond time-resolved macromolecular crystallographic study using
Laue techniques, with a broad range of wavelengths (white beam), was reported in
1996 when Moffat and co-workers reported a study of the photodissociation mechanism of carbon monoxide in carbon monoxy-myoglobin (MbCO) [48]. The MbCO
system had previously been studied in depth by ultra-fast spectroscopic techniques,
and, as a result, the photoactivity of the complex in solution had been established
[49–51]. In the experiments carried out at the European Synchrotron Radiation
Facility (ESRF), Moffat et al. employed a pump-probe strategy consisting of an
initial 7.5 nm-wide laser pump pulse at λ ¼ 630 nm, followed by an X-ray probe
pulse timed to arrive after a specific delay (τ). This delay was varied so that six
different data sets were completed at intervals of between τ ¼ 4 ns and 1.9 ms. The
analysis of the data from the 4 ns and 1 μs data sets showed that regions of negative
electron density appeared where the coordinated CO molecule had been. These
observations confirmed the results of the earlier solution-based spectroscopic studies
that photolysis of the Fe–CO bond had occurred and indicated that a similar occurs in
the single crystal. The crystallographic data also showed a region of positive electron
density below the heme centre, suggesting that the iron atom moves out of the heme
plane as a result of the Fe–CO bond cleavage. This is consistent with the CO group
Time-Resolved Single-Crystal X-Ray Crystallography
251
XFELs to study macromolecular systems [42–45]. From these and related studies, it
is apparent that the direction-controlling, initial stages of a chemical reaction appear
at timescales faster than are accessible through the use of synchrotrons which
effectively have a 100 ps limit. The volume of data that is collected in one of
these studies is enormous. For example, in the study of the initial stages of the
photoexcitation of the photoactive yellow protein (PYP), which is triggered by the
trans-cis isomerisation of the coumarin chromophore, 2.5 Â 10
6 snapshots were
recorded to 1.6 Å [46]. With these advances, and with the large number of data sets
being collected accurate scaling between these data sets is required to obtain reliable
results. It has been found from scaling analyses that the anisotropy of absorption
following sample excitation can be pronounced and depends on the orientation of the
crystals in the laser beam [47] so that this factor has to be taken into consideration if
accurate analysis of molecular samples is to be achieved in the future.
3 The Beginnings of Time-Resolved Crystallography
3.1 Macromolecular Photocrystallography
The initial developments in time-resolved crystallography came in the area of
macromolecular crystallography because of the interest in important biological
processes. These studies required faster data collection and processing techniques
than had been standard. Biological crystals are also prone to decomposition particularly as a result of X-ray radiation damage, so the use of Laue methods could
achieve faster data collection with less crystal decay.
The first nanosecond time-resolved macromolecular crystallographic study using
Laue techniques, with a broad range of wavelengths (white beam), was reported in
1996 when Moffat and co-workers reported a study of the photodissociation mechanism of carbon monoxide in carbon monoxy-myoglobin (MbCO) [48]. The MbCO
system had previously been studied in depth by ultra-fast spectroscopic techniques,
and, as a result, the photoactivity of the complex in solution had been established
[49–51]. In the experiments carried out at the European Synchrotron Radiation
Facility (ESRF), Moffat et al. employed a pump-probe strategy consisting of an
initial 7.5 nm-wide laser pump pulse at λ ¼ 630 nm, followed by an X-ray probe
pulse timed to arrive after a specific delay (τ). This delay was varied so that six
different data sets were completed at intervals of between τ ¼ 4 ns and 1.9 ms. The
analysis of the data from the 4 ns and 1 μs data sets showed that regions of negative
electron density appeared where the coordinated CO molecule had been. These
observations confirmed the results of the earlier solution-based spectroscopic studies
that photolysis of the Fe–CO bond had occurred and indicated that a similar occurs in
the single crystal. The crystallographic data also showed a region of positive electron
density below the heme centre, suggesting that the iron atom moves out of the heme
plane as a result of the Fe–CO bond cleavage. This is consistent with the CO group
Time-Resolved Single-Crystal X-Ray Crystallography
251