A bacteriophytochrome Pr/Pfr heterodimer as obtained by single-ms TR cryo-EM

A bacteriophytochrome Pr/Pfr heterodimer as obtained by single-ms TR cryo-EM | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article A bacteriophytochrome Pr/Pfr heterodimer as obtained by single-ms TR cryo-EM Marius Schmidt, Prabin Karki, William Budell, Rohit Kannachel, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8079638/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Phytochromes are red/far-red light photoreceptors found across many kingdoms of life1. Upon light absorption, they reversibly interconvert between a red-absorbing Pr state and a far-red–absorbing Pfr state. Bacterial phytochromes (BphPs) regulate diverse physiological responses to red light, typically through a two-component signaling pathway6 involving a histidine kinase (HK) and a response regulator. Because the HK domain is an integral part of the BphP, its enzymatic activity is directly light-controlled. A recent cryo-EM study reported a BphP heterodimer2 consisting of one protomer in the Pr state and the other in the Pfr state, formed after exposure to ambient white laboratory light. Here, we use the Spotiton technique3,4 to deliberately generate high concentrations of the Pr/Pfr heterodimer by shortly illuminating BphP particles on a cryo-EM grid which was vitrified about 10 ms later. The resulting full-length Pr/Pfr heterodimer structure closely matches the one reported previously2. Reanalysis of earlier cryo-EM data 5 yielded a complete Pr/Pr homodimer structure with both protomers in the Pr state. Comparison of the two structures reveals that the coiled-coil linker helices connecting the HK domain to the photosensory core unwind during the transition from the homodimer to the heterodimer. The unwinding is accompanied by an almost 180° rotation of the entire HK domain. This large-scale rotation likely modulates HK activity and provides insight into the mechanism of signal perception and transduction in BphPs. Because coiled-coil helices are common in motor proteins7,8, a similar unwinding mechanism may underlie the rotational movements of their “motor heads”, impacting their dynamics and energetics as well. Biological sciences/Biophysics/Motility/Motor protein function Physical sciences/Chemistry/Chemical biology/Mechanism of action Biological sciences/Biochemistry/Kinases Physical sciences/Chemistry/Photochemistry/Photocatalysis Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryInformation.docx Supplmentary Information Movie1LocalRefinementWithoutLobesMedia1.mp4 Supplementary Movie 1 Movie2rightmorph.mp4 Supplementary Movie 2 Movie3wrongmorphmp4v2.mp4 Supplementary Movie 3 Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8079638","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":542907384,"identity":"5615fa0b-90f5-4329-9b75-f91e53064707","order_by":0,"name":"Marius 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Upon light absorption, they reversibly interconvert between a red-absorbing Pr state and a far-red–absorbing Pfr state. Bacterial phytochromes (BphPs) regulate diverse physiological responses to red light, typically through a two-component signaling pathway6 involving a histidine kinase (HK) and a response regulator. Because the HK domain is an integral part of the BphP, its enzymatic activity is directly light-controlled. A recent cryo-EM study reported a BphP heterodimer2 consisting of one protomer in the Pr state and the other in the Pfr state, formed after exposure to ambient white laboratory light. Here, we use the Spotiton technique3,4 to deliberately generate high concentrations of the Pr/Pfr heterodimer by shortly illuminating BphP particles on a cryo-EM grid which was vitrified about 10 ms later. The resulting full-length Pr/Pfr heterodimer structure closely matches the one reported previously2. Reanalysis of earlier cryo-EM data 5 yielded a complete Pr/Pr homodimer structure with both protomers in the Pr state. Comparison of the two structures reveals that the coiled-coil linker helices connecting the HK domain to the photosensory core unwind during the transition from the homodimer to the heterodimer. The unwinding is accompanied by an almost 180° rotation of the entire HK domain. This large-scale rotation likely modulates HK activity and provides insight into the mechanism of signal perception and transduction in BphPs. 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