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- EMDB-41941: Cryo-EM structure of PsBphP in Pfr state, Dimer of Dimers FL -

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Basic information

Entry
Database: EMDB / ID: EMD-41941
TitleCryo-EM structure of PsBphP in Pfr state, Dimer of Dimers FL
Map dataDimer of Dimers of PsBphP in Pfr state, DeepEMhancer post-processed map
Sample
  • Complex: PsBphP
    • Protein or peptide: histidine kinase
  • Ligand: 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
KeywordsPseudomonas syringae bacteriophytochrome / PLANT PROTEIN
Function / homology
Function and homology information


detection of visible light / histidine kinase / phosphorelay sensor kinase activity / photoreceptor activity / regulation of DNA-templated transcription / ATP binding
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, dimerisation/phosphoacceptor domain superfamily / Signal transduction histidine kinase-related protein, C-terminal / 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
Biological speciesPseudomonas syringae pv. tomato str. DC3000 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsBasore K / Burgie ES / Vierstra D
Funding support United States, 1 items
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 13, 2023-
Header (metadata) releaseAug 21, 2024-
Map releaseAug 21, 2024-
UpdateAug 21, 2024-
Current statusAug 21, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41941.png

Downloads & links

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Map

FileDownload / File: emd_41941.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDimer of Dimers of PsBphP in Pfr state, DeepEMhancer post-processed map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.66 Å/pix.
x 500 pix.
= 328.5 Å		0.66 Å/pix.
x 500 pix.
= 328.5 Å		0.66 Å/pix.
x 500 pix.
= 328.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.657 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.0018470942 - 2.132882
Average (Standard dev.)0.0007332726 (±0.019122867)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 328.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Dimer of Dimers of PsBphP in Pfr state, unsharpened map

Fileemd_41941_additional_1.map
AnnotationDimer of Dimers of PsBphP in Pfr state, unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Dimer of Dimers of PsBphP in Pfr state, half-map #1

Fileemd_41941_half_map_1.map
AnnotationDimer of Dimers of PsBphP in Pfr state, half-map #1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Dimer of Dimers of PsBphP in Pfr state, half-map #2

Fileemd_41941_half_map_2.map
AnnotationDimer of Dimers of PsBphP in Pfr state, half-map #2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : PsBphP

EntireName: PsBphP
Components
  • Complex: PsBphP
    • Protein or peptide: histidine kinase
  • Ligand: 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

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Supramolecule #1: PsBphP

SupramoleculeName: PsBphP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Pseudomonas syringae pv. tomato str. DC3000 (bacteria)

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Macromolecule #1: histidine kinase

MacromoleculeName: histidine kinase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas syringae pv. tomato str. DC3000 (bacteria)
Molecular weightTheoretical: 82.382555 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GMSQLDKDAF EVLLANCADE PIQFPGAIQP HGLLFTLKEP ELTILQVSAN VQSVLGKVPD QLAGQTLDCV LGAGWAEVIR STSANDSLV DVPRLLMSVE GVEFEALLHR SQEALVLELE IQDKAAQAIS YSERTGNMGR MLRQLHAAAD LQTLYEVSVR E IQRMTGYD ...String:
GMSQLDKDAF EVLLANCADE PIQFPGAIQP HGLLFTLKEP ELTILQVSAN VQSVLGKVPD QLAGQTLDCV LGAGWAEVIR STSANDSLV DVPRLLMSVE GVEFEALLHR SQEALVLELE IQDKAAQAIS YSERTGNMGR MLRQLHAAAD LQTLYEVSVR E IQRMTGYD RVLIYRFEEE GHGQVIAEAS APAMELFNGL FFPASDIPEQ ARELYRRNWL RIIPDANYTP VPLVPQLRPD TQ QQLDLSF STLRSVSPIH CQYMKNMGVL SSMSVSLIQG GKLWGLISCG HRTPLYVSHE LRSACQAIGQ VLSLQISAME ALE VSRQRE TKIQTLQQLH QMMATSDTDV FDGLAQQPQL LMDLVGATGV AIIEDRQTHC YGNCPEPSDI RALHTWMMAG GEPV YASHH LSSVYPPGEA YQTLASGVLA MSLPKPVDNG VIWFRPEVKQ SVQWSGDPNK PLNLDASDNT LRLQPRTSFE IWKVE MTGI ATKWSHGDVF AANDLRRSAL ENDLARQVSK EQQAVRARDE LVAVVSHDLR NPMTVISMLC GMMQKSFSSD GPHTSR RIS TAIDTMQQAA SRMNVLLEDL LDTSKIEAGR YTITPQPLEV SQIFEEAYTL LAPLAMDKSI EISFNAEPDI KVNADPE RL FQVLSNLIGN AIKFTPKLGR IGVAAMSNGD EVVFTVRDSG EGIPPEQLPH IFERYWTVKE GNPTGTGLGL YISQGIIK A HGGELAAQSQ VGHGSEFRFT VPIAH

UniProtKB: histidine kinase

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Macromolecule #2: 3-[2-[(Z)-[3-(2-carboxyethyl)-5-[(Z)-(4-ethenyl-3-methyl-5-oxidan...

MacromoleculeName: 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- ...Name: 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
type: ligand / ID: 2 / Number of copies: 2 / Formula: LBV
Molecular weightTheoretical: 585.67 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 82.0 K / Max: 84.0 K
Specialist opticsSpherical aberration corrector: Microscope is outfitted with a Cs image corrector with two hexapole elements.
DetailsPreliminary grid screening was performed manually.
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 3 / Number real images: 12593 / Average exposure time: 4.28 sec. / Average electron dose: 51.78 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 150.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4796622
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Software - details: local refinement / Number images used: 402598
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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