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- PDB-9euy: Cryo-EM structure of the full-length Pseudomonas aeruginosa bacte... -

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Basic information

Entry
Database: PDB / ID: 9euy
TitleCryo-EM structure of the full-length Pseudomonas aeruginosa bacteriophytochrome in its Pfr state
ComponentsBacteriophytochrome
KeywordsCYTOSOLIC PROTEIN / Photosensor / Photoreceptor / Phytochrome / Bacterial protein
Function / homology
Function and homology information


osmosensory signaling via phosphorelay pathway / detection of visible light / phosphorelay response regulator activity / protein kinase activator activity / histidine kinase / phosphorelay sensor kinase activity / photoreceptor activity / regulation of DNA-templated transcription / ATP binding / identical protein binding
Similarity search - Function
: / Phytochrome / Phytochrome, PHY domain superfamily / PAS fold-2 / PAS fold / Phytochrome, central region / Phytochrome region / Phytochrome chromophore attachment domain / Phytochrome chromophore attachment site domain profile. / His Kinase A (phospho-acceptor) domain ...: / Phytochrome / Phytochrome, PHY domain superfamily / PAS fold-2 / PAS fold / Phytochrome, central region / Phytochrome region / Phytochrome chromophore attachment domain / Phytochrome chromophore attachment site domain profile. / His Kinase A (phospho-acceptor) domain / His Kinase A (phosphoacceptor) domain / Signal transduction histidine kinase, dimerisation/phosphoacceptor domain / Signal transduction histidine kinase, dimerisation/phosphoacceptor domain superfamily / Histidine kinase domain / Histidine kinase domain profile. / GAF domain / Domain present in phytochromes and cGMP-specific phosphodiesterases. / GAF domain / GAF-like domain superfamily / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase-like ATPases / PAS domain superfamily / Histidine kinase/HSP90-like ATPase / Histidine kinase/HSP90-like ATPase superfamily
Similarity search - Domain/homology
Chem-LBV / Bacteriophytochrome
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsBodizs, S. / Westenhoff, S.
Funding support Sweden, 1items
OrganizationGrant numberCountry
Swedish Research Council Sweden
CitationJournal: Structure / Year: 2024
Title: Cryo-EM structures of a bathy phytochrome histidine kinase reveal a unique light-dependent activation mechanism.
Authors: Szabolcs Bódizs / Petra Mészáros / Lukas Grunewald / Heikki Takala / Sebastian Westenhoff /
Abstract: Phytochromes are photoreceptor proteins in plants, fungi, and bacteria. They can adopt two photochromic states with differential biochemical responses. The structural changes transducing the signal ...Phytochromes are photoreceptor proteins in plants, fungi, and bacteria. They can adopt two photochromic states with differential biochemical responses. The structural changes transducing the signal from the chromophore to the biochemical output modules are poorly understood due to challenges in capturing structures of the dynamic, full-length protein. Here, we present cryoelectron microscopy (cryo-EM) structures of the phytochrome from Pseudomonas aeruginosa (PaBphP) in its resting (Pfr) and photoactivated (Pr) state. The kinase-active Pr state has an asymmetric, dimeric structure, whereas the kinase-inactive Pfr state opens up. This behavior is different from other known phytochromes and we explain it with the unusually short connection between the photosensory and output modules. Multiple sequence alignment of this region suggests evolutionary optimization for different modes of signal transduction in sensor proteins. The results establish a new mechanism for light-sensing by phytochrome histidine kinases and provide input for the design of optogenetic phytochrome variants.
History
DepositionMar 28, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 11, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Bacteriophytochrome
B: Bacteriophytochrome
hetero molecules


Theoretical massNumber of molelcules
Total (without water)165,3614
Polymers164,1902
Non-polymers1,1712
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Bacteriophytochrome / Phytochrome-like protein


Mass: 82094.867 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: bphP, PA4117 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9HWR3, histidine kinase
#2: Chemical ChemComp-LBV / 3-[2-[(Z)-[3-(2-carboxyethyl)-5-[(Z)-(4-ethenyl-3-methyl-5-oxidanylidene-pyrrol-2-ylidene)methyl]-4-methyl-pyrrol-1-ium -2-ylidene]methyl]-5-[(Z)-[(3E)-3-ethylidene-4-methyl-5-oxidanylidene-pyrrolidin-2-ylidene]methyl]-4-methyl-1H-pyrrol-3- yl]propanoic acid / 2(R),3(E)- PHYTOCHROMOBILIN


Mass: 585.670 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C33H37N4O6 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Homodimeric complex of the protein bacteriophytochrome containing its cofactor biliverdin
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.16 MDa / Experimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria) / Cellular location: cytoplasm
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pET21b(+)
Buffer solutionpH: 7.8 / Details: 80 mM Tris, 10 mM MgCl2, 150 mM CH3CO2K, pH 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
180 mMTrisC4H11NO31
210 mMMagnesium chlorideMgCl21
3150 mMPotassium acetateCH3CO2K1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Current: 20 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blotting for 5 s from both sides

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 2 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.7 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 30336
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv4.2.1particle selection
2EPUv3.5.0image acquisition
4cryoSPARCv4.2.1CTF correction
7ISOLDE1.5model fitting
9PHENIXmodel refinement
10cryoSPARCv4.2.1initial Euler assignment
13cryoSPARCv4.2.13D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 239820 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model

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