8QI9

CrPhotLOV1 dark state structure determined by serial synchrotron crystallography at room temperature

Summary for 8QI9

• Entry DOI: 10.2210/pdb8qi9/pdb
• Descriptor: Phototropin, FLAVIN MONONUCLEOTIDE (3 entities in total)
• Functional Keywords: phototropin, flavin, electron transport, flavoprotein
• Biological source: Chlamydomonas reinhardtii
• Total number of polymer chains: 1
• Total formula weight: 15794.70

Authors

Gotthard, G.,Mous, S.,Weinert, T.,Maia, R.N.A.,James, D.,Dworkowski, F.,Gashi, D.,Antonia, F.,Wang, M.,Panepucci, E.,Ozerov, D.,Schertler, G.F.X.,Heberle, J.,Standfuss, J.,Nogly, P. (deposition date: 2023-09-11, release date: 2024-07-24, Last modification date: 2024-09-11)

Primary citation

Gotthard, G.,Mous, S.,Weinert, T.,Maia, R.N.A.,James, D.,Dworkowski, F.,Gashi, D.,Furrer, A.,Ozerov, D.,Panepucci, E.,Wang, M.,Schertler, G.F.X.,Heberle, J.,Standfuss, J.,Nogly, P.
Capturing the blue-light activated state of the Phot-LOV1 domain from Chlamydomonas reinhardtii using time-resolved serial synchrotron crystallography.
Iucrj, 11:792-808, 2024

PubMed Abstract: Light-oxygen-voltage (LOV) domains are small photosensory flavoprotein modules that allow the conversion of external stimuli (sunlight) into intracellular signals responsible for various cell behaviors (e.g. phototropism and chloroplast relocation). This ability relies on the light-induced formation of a covalent thioether adduct between a flavin chromophore and a reactive cysteine from the protein environment, which triggers a cascade of structural changes that result in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the activation cascade of the LOV domain to be observed in real time, which has been elusive. In this study, we report a robust protocol for the production and stable delivery of microcrystals of the LOV domain of phototropin Phot-1 from Chlamydomonas reinhardtii (CrPhotLOV1) with a high-viscosity injector for time-resolved serial synchrotron crystallography (TR-SSX). The detailed process covers all aspects, from sample optimization to data collection, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the crystals obtained preserve the photoreactivity using infrared spectroscopy. Furthermore, the results of the TR-SSX experiment provide high-resolution insights into structural alterations of CrPhotLOV1 from Δt = 2.5 ms up to Δt = 95 ms post-photoactivation, including resolving the geometry of the thioether adduct and the C-terminal region implicated in the signal transduction process.

PubMed: 39037420
DOI: 10.1107/S2052252524005608

Experimental method

X-RAY DIFFRACTION (1.87 Å)