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- PDB-8u4x: Cryo-EM structure of PsBphP in Pr state -

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Basic information

Entry
Database: PDB / ID: 8u4x
TitleCryo-EM structure of PsBphP in Pr state
Componentshistidine kinase
KeywordsPLANT PROTEIN / Pseudomonas syringae bacteriophytochrome
Function / homology
Function and homology information


detection of visible light / histidine kinase / phosphorelay sensor kinase activity / photoreceptor activity / regulation of DNA-templated transcription
Similarity search - Function
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 ...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-related protein, C-terminal / 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 / histidine kinase
Similarity search - Component
Biological speciesPseudomonas syringae pv. tomato str. DC3000 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.81 Å
AuthorsBasore, K. / Burgie, E.S. / Vierstra, D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM127892 United States
CitationJournal: Nat Commun / Year: 2024
Title: Signaling by a bacterial phytochrome histidine kinase involves a conformational cascade reorganizing the dimeric photoreceptor.
Authors: E Sethe Burgie / Katherine Basore / Michael J Rau / Brock Summers / Alayna J Mickles / Vadim Grigura / James A J Fitzpatrick / Richard D Vierstra /
Abstract: Phytochromes (Phys) are a divergent cohort of bili-proteins that detect light through reversible interconversion between dark-adapted Pr and photoactivated Pfr states. While our understandings of ...Phytochromes (Phys) are a divergent cohort of bili-proteins that detect light through reversible interconversion between dark-adapted Pr and photoactivated Pfr states. While our understandings of downstream events are emerging, it remains unclear how Phys translate light into an interpretable conformational signal. Here, we present models of both states for a dimeric Phy with histidine kinase (HK) activity from the proteobacterium Pseudomonas syringae, which were built from high-resolution cryo-EM maps (2.8-3.4-Å) of the photosensory module (PSM) and its following signaling (S) helix together with lower resolution maps for the downstream output region augmented by RoseTTAFold and AlphaFold structural predictions. The head-to-head models reveal the PSM and its photointerconversion mechanism with strong clarity, while the HK region is interpretable but relatively mobile. Pr/Pfr comparisons show that bilin phototransformation alters PSM architecture culminating in a scissoring motion of the paired S-helices linking the PSMs to the HK bidomains that ends in reorientation of the paired catalytic ATPase modules relative to the phosphoacceptor histidines. This action apparently primes autophosphorylation enroute to phosphotransfer to the cognate DNA-binding response regulator AlgB which drives quorum-sensing behavior through transient association with the photoreceptor. Collectively, these models illustrate how light absorption conformationally translates into accelerated signaling by Phy-type kinases.
History
DepositionSep 11, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 21, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: histidine kinase
B: histidine kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)165,9364
Polymers164,7652
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 histidine kinase


Mass: 82382.555 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas syringae pv. tomato str. DC3000 (bacteria)
Gene: bphP / Plasmid: pBAD
Details (production host): CDS includes N-terminal TEV protease cleavable hexahistidine tag
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q885D3
#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
Has protein modificationY

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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: PsBphP / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Pseudomonas syringae pv. tomato str. DC3000 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pBAD
Buffer solutionpH: 7.5
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 2 rounds of glow-discharge: 1st: Copper side with air 2nd: carbon side with added amylamine
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Preliminary grid screening was performed manually.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 96000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Cs: 0.01 mm / C2 aperture diameter: 150 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 84 K / Temperature (min): 82 K
Image recordingAverage exposure time: 4.28 sec. / Electron dose: 51.78 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 9173
EM imaging opticsSpherical aberration corrector: Microscope is outfitted with a Cs image corrector with two hexapole elements.
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCparticle selection
2PHENIX1.21rc1_4903:model refinement
3EPUimage acquisition
5cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstructionlocal refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3233515
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1666098 / Algorithm: FOURIER SPACE
Details: Particles were symmetry expanded around the C2 axis before the final 3D refinement.
Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028037
ELECTRON MICROSCOPYf_angle_d0.55410930
ELECTRON MICROSCOPYf_dihedral_angle_d5.6621105
ELECTRON MICROSCOPYf_chiral_restr0.0421222
ELECTRON MICROSCOPYf_plane_restr0.0041429

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