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- EMDB-50008: Local refinement of the full-length Pseudomonas aeruginosa bacter... -

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Basic information

Entry
Database: EMDB / ID: EMD-50008
TitleLocal refinement of the full-length Pseudomonas aeruginosa bacteriophytochrome in its Pr state
Map dataLocal refinement used for the composite reconstruction EMD-19981
Sample
  • Complex: Homodimeric complex of the protein bacteriophytochrome containing its cofactor biliverdin
KeywordsPhotosensor / Photoreceptor / Phytochrome / Bacterial protein / CYTOSOLIC PROTEIN
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsBodizs S / Westenhoff S
Funding support Sweden, 1 items
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
DepositionApr 1, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateSep 11, 2024-
Current statusSep 11, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50008.png

Downloads & links

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Map

FileDownload / File: emd_50008.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal refinement used for the composite reconstruction EMD-19981
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 400 pix.
= 331.2 Å		0.83 Å/pix.
x 400 pix.
= 331.2 Å		0.83 Å/pix.
x 400 pix.
= 331.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.2605159 - 1.7203455
Average (Standard dev.)0.000011463249 (±0.020637676)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 331.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_50008_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_50008_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homodimeric complex of the protein bacteriophytochrome containing...

EntireName: Homodimeric complex of the protein bacteriophytochrome containing its cofactor biliverdin
Components
  • Complex: Homodimeric complex of the protein bacteriophytochrome containing its cofactor biliverdin

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Supramolecule #1: Homodimeric complex of the protein bacteriophytochrome containing...

SupramoleculeName: Homodimeric complex of the protein bacteriophytochrome containing its cofactor biliverdin
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Pseudomonas aeruginosa (bacteria) / Location in cell: cytoplasm
Molecular weightTheoretical: 160 KDa

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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

Concentration1.5 mg/mL
BufferpH: 7.8
Component:
ConcentrationFormulaName
80.0 mMC4H11NO3Tris
10.0 mMMgCl2Magnesium chloride
150.0 mMCH3CO2KPotassium acetate

Details: 80 mM Tris, 10 mM MgCl2, 150 mM CH3CO2K, pH 7.8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting for 5 s from both sides.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 2 / Number real images: 30336 / Average exposure time: 1.7 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 2.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
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

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v4.2.1) / Number images used: 889461
Initial angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. v4.2.1)
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model

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