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1.
Figure 5

Figure 5. From: Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches.

Time-resolved wide-angle X-ray scattering data from solubilized samples of bacteriorhodopsin. (a) Integration in rings and subtraction of ‘laser off’ images from ‘laser on’ images yields the difference scattering curves as a function of the time delay (Δt) between photoactivation and the X-ray probe. (b) Basis spectra of an intermediate and late conformational state extracted from spectral decomposition of the data shown in (a). (c) Structural interpretation of these data using a rigid-body minimization routine illustrated in terms of the bacteriorhodopsin photocycle (Andersson et al., 2009 ▶). Reproduced from Structure (Andersson et al., 2009 ▶), copyright (2009), with permission from Elsevier.

Sebastian Westenhoff, et al. Acta Crystallogr A. 2010 March 1;66(Pt 2):207-219.
2.
Figure 3

Figure 3. From: Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches.

Structural results from intermediate trapping studies of bacteriorhodopsin. Four structures of resting (Belrhali et al., 1999 ▶) (purple, Protein Data Bank entry 1qhj), early (Edman et al., 1999 ▶) (blue, 1qkp), intermediate (Royant et al., 2000 ▶) (green, 1eop) and late (Luecke et al., 1999 ▶) (yellow, 1c8s) conformations are shown. These intermediate conformations were trapped by illuminating crystals at 110 K, 170 K and during thawing, respectively. A clear evolution of the retinal can be observed for these structures, which moves towards the cytoplasm as the temperature is raised. Moreover, significant displacements of Trp-182, Asp-85 and Arg-82 are also observed, as are rearrangements of water molecules recorded in the corresponding Protein Data Bank entries (not shown for reasons of clarity).

Sebastian Westenhoff, et al. Acta Crystallogr A. 2010 March 1;66(Pt 2):207-219.
3.
Figure 2

Figure 2. From: Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches.

Intermediate trapping studies of the non-haem iron protein superoxide reductase from D. barsii (Katona et al., 2007 ▶). (a) Structure of the iron peroxide intermediate bound in an end-on configuration in the active site of superoxide reductase. The F obsF calc omit (green) maps are contoured at 4.5σ. The peroxo moiety is hydrogen-bonded to Lys48 and two water molecules of the active site, which assist the protonation en route to the product formation. The structural model and electron density derive from entry 2ji3 of the Protein Data Bank. (b) Off-resonance Raman spectra collected from single crystals and solutions of hydrogen-peroxide-treated superoxide reductase. Single crystals of superoxide reductase (top spectrum) were treated in crystallization buffer with the addition of 10 mM H2O2 for 3 min and subsequently flash frozen in a cryoprotected buffer. SOR solutions (bottom spectrum) were treated similarly (for details see Katona et al., 2007 ▶).

Sebastian Westenhoff, et al. Acta Crystallogr A. 2010 March 1;66(Pt 2):207-219.
4.
Figure 4

Figure 4. From: Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches.

Time-resolved wide-angle X-ray scattering of the haemoglobin:carbon monoxide complex. (a) Sketch of the experimental set-up at the dedicated time-resolved beamline at the European Synchrotron Radiation Facility. Polychromatic X-ray pulses are generated in an undulator and a rotating chopper (triangle) is used to isolate a chosen pulse train. Protein samples are held within a glass capillary (red) and are excited by laser pulses (green) incident perpendicular to the X-ray beam. Concentric diffusive X-ray scattering rings are collected on a charge-couple device (CCD) detector. (b) Integration in rings and subtraction of ‘laser off’ images from ‘laser on’ images yields difference scattering curves, which are the fingerprint of the structural rearrangements in the protein. Difference scattering recorded from haemoglobin in complex with carbon monoxide 100 µs after photoexcitation (red) are compared with ‘static’ differences between haemoglobin with carbon monoxide bound and deoxyhaemoglobin (black). (c) Surface representation of the expected time-dependent structural changes in haemoglobin. Regions of the proteins that are involved in the changes are coloured red. Reproduced by permission from Macmillan Publishers: Nature Methods (Cammarata et al., 2008 ▶), copyright (2008).

Sebastian Westenhoff, et al. Acta Crystallogr A. 2010 March 1;66(Pt 2):207-219.
5.
Figure 6

Figure 6. From: Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches.

X-ray absorption spectra from iron-containing proteins. (a) Static X-ray absorption spectra from the iron K edge of PerR protein (Jacquamet et al., 2009 ▶). Extended X-ray absorption fine structure (EXAFS) records the modulation of an X-ray absorption spectrum in the energy region from 50 eV to approximately 1000 eV above the absorption edge, providing structural information on the neighbours of the absorbing atom and very accurate first-shell iron–ligand distances. X-ray absorption near-edge structure (XANES) focuses upon the smaller energy region up to approximately 50 eV above the edge and provides structural and electronic information for the absorbing atom. The pre-edge region of XANES is sensitive to the oxidation, spin state and geometric environment of the absorbing atom. (b) Time-resolved X-ray absorption spectra of myoglobin in complex with carbon monoxide and its interpretation in terms of structure. Iron K-edge XANES spectra (dashed line) were recorded 100 µs following photoexcitation, and spectra from the resting conformation are shown for comparison (black line). Spectral changes were interpreted as resulting from the movement of carbon monoxide away from the haem group (inset). Reprinted from Journal of Electron Spectroscopy and Related Phenomena (Wang et al., 2005 ▶), copyright (2005), with permission from Elsevier.

Sebastian Westenhoff, et al. Acta Crystallogr A. 2010 March 1;66(Pt 2):207-219.
6.
Figure 1

Figure 1. From: Time-resolved structural studies of protein reaction dynamics: a smorgasbord of X-ray approaches.

Time-resolved Laue diffraction and intermediate trapping studies of the photodissociation of carbon monoxide bound to the haem group of myoglobin. (a) F obs(light) − F obs(dark) difference Fourier electron density map of myoglobin (L29F) calculated for the time point 100 ps following photoactivation by a short laser pulse (Schotte et al., 2003 ▶; Aranda et al., 2006 ▶). Negative difference electron density (red) is observed 100 ps after photoactivation at the resting-state position of the carbon monoxide molecule (above the haem) and positive difference electron density (green) is observed nearby below Val68. (b) A similar map calculated for the time point 3.16 µs after photoactivation. At this time another binding pocket is visible as positive difference electron density (green) below the haem group near His93. In calculating these maps the crystallographic observations were taken from Protein Data Bank entries 2g0s (resting state), 2g0v (100 ps) and 2g14 (3.16 µs) (Aranda et al., 2006 ▶). (c) Difference Fourier maps calculated from low-temperature trapping studies on wild-type myoglobin:carbon monoxide complexes (Chu et al., 2000 ▶). The resting-state model is shown in cyan (Protein Data Bank entry 1dwr) and the photolysed state is shown in magenta (1dws). (d) Difference Fourier map calculated for the photorelaxed state, shown in blue (1dwt), from the same low-temperature study. All maps are contoured at 4.5σ. Agreement is apparent between the positions of the carbon monoxide molecule observed in the intermediate trapping and the Laue diffraction studies.

Sebastian Westenhoff, et al. Acta Crystallogr A. 2010 March 1;66(Pt 2):207-219.

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