Journal:IUCrJ:S2052252525006645
From Proteopedia
(Difference between revisions)
| Line 5: | Line 5: | ||
<b>Molecular Tour</b><br> | <b>Molecular Tour</b><br> | ||
Time-resolved X-ray crystallography enables the capture of molecules movies which show how proteins in action, and is currently undergoing a renaissance due to the development of serial crystallography at synchrotron and XFEL beamlines. Crucial to such experiments are efficient and effective methods to uniformly initiate time-dependent processes within microcrystals, such as ligand binding, enzymatic reactions, or signalling. A common approach is the use of light to activate a reaction across the whole crystal at the same timepoint, however only a small proportion of protein reactions are light-activated. Diverse reactions can be made amenable to light-activation using photocaged substrates, which are molecules soaked into the crystal in advance, and release a reaction substrate upon laser activation. To collect high-quality time-resolved data, the reaction should be initiated in as many unit cells as possible within the crystal to provide structures with high occupancy of the activated states for easier interpretation, but without any damage caused by excessive light intensity, and without multiphoton excitation causing off-pathway reactions which do not reflect biological reality. | Time-resolved X-ray crystallography enables the capture of molecules movies which show how proteins in action, and is currently undergoing a renaissance due to the development of serial crystallography at synchrotron and XFEL beamlines. Crucial to such experiments are efficient and effective methods to uniformly initiate time-dependent processes within microcrystals, such as ligand binding, enzymatic reactions, or signalling. A common approach is the use of light to activate a reaction across the whole crystal at the same timepoint, however only a small proportion of protein reactions are light-activated. Diverse reactions can be made amenable to light-activation using photocaged substrates, which are molecules soaked into the crystal in advance, and release a reaction substrate upon laser activation. To collect high-quality time-resolved data, the reaction should be initiated in as many unit cells as possible within the crystal to provide structures with high occupancy of the activated states for easier interpretation, but without any damage caused by excessive light intensity, and without multiphoton excitation causing off-pathway reactions which do not reflect biological reality. | ||
| + | |||
| + | <scene name='10/1087716/020_Fig_5c/1'>Fig_5c</scene> | ||
This work characterises photocage release of nitric oxide and binding of this ligand to two heme protein systems, cytochrome c′-beta and dye-decolourising peroxidase B, using a fixed-target sample delivery system. The differences between the structures of Methylococcus capsulatus cytochrome c′-beta as determined by traditional rotation crystallography at cryogenic temperatures, and via room-temperature serial crystallography, both in presence and absence of NO are described. The two bacterial proteins used here are ideal test cases, as the binding of NO results in the otherwise empty pockets on the distal side of the heme results in obvious changes in electron density. These structures have also allowed investigation of different methods for determining occupancy of the bound NO ligand, and how occupancy correlates to laser power. | This work characterises photocage release of nitric oxide and binding of this ligand to two heme protein systems, cytochrome c′-beta and dye-decolourising peroxidase B, using a fixed-target sample delivery system. The differences between the structures of Methylococcus capsulatus cytochrome c′-beta as determined by traditional rotation crystallography at cryogenic temperatures, and via room-temperature serial crystallography, both in presence and absence of NO are described. The two bacterial proteins used here are ideal test cases, as the binding of NO results in the otherwise empty pockets on the distal side of the heme results in obvious changes in electron density. These structures have also allowed investigation of different methods for determining occupancy of the bound NO ligand, and how occupancy correlates to laser power. | ||
Revision as of 09:48, 12 August 2025
| |||||||||||
This page complements a publication in scientific journals and is one of the Proteopedia's Interactive 3D Complement pages. For aditional details please see I3DC.
