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- EMDB-35613: Cryo-EM structure of cyanobacteria phosphoketolase -

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Basic information

Entry
Database: EMDB / ID: EMD-35613
TitleCryo-EM structure of cyanobacteria phosphoketolase
Map data
Sample
  • Complex: Cyanobacteria XPK complexed with TPP/Mg2+
    • Protein or peptide: Probable phosphoketolase
  • Ligand: MAGNESIUM ION
  • Ligand: THIAMINE DIPHOSPHATEThiamine pyrophosphate
KeywordsCarbon metabolism / TPP-dependent enzyme / CYTOSOLIC PROTEIN
Function / homology:
Function and homology information
Biological speciesSynechococcus (bacteria) / Synechococcus elongatus (strain ATCC 33912 / PCC 7942 / FACHB-805) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.63 Å
AuthorsChang C-W / Tsai M-D
Funding support Taiwan, 2 items
OrganizationGrant numberCountry
Academia Sinica (Taiwan)Grant No. AS-CFII-108-110 Taiwan
Academia Sinica (Taiwan)Grant No. AS-KPQ-109-TPP2 Taiwan
CitationJournal: Nat Metab / Year: 2023
Title: An ATP-sensitive phosphoketolase regulates carbon fixation in cyanobacteria.
Authors: Kuan-Jen Lu / Chiung-Wen Chang / Chun-Hsiung Wang / Frederic Y-H Chen / Irene Y Huang / Pin-Hsuan Huang / Cheng-Han Yang / Hsiang-Yi Wu / Wen-Jin Wu / Kai-Cheng Hsu / Meng-Chiao Ho / Ming- ...Authors: Kuan-Jen Lu / Chiung-Wen Chang / Chun-Hsiung Wang / Frederic Y-H Chen / Irene Y Huang / Pin-Hsuan Huang / Cheng-Han Yang / Hsiang-Yi Wu / Wen-Jin Wu / Kai-Cheng Hsu / Meng-Chiao Ho / Ming-Daw Tsai / James C Liao /
Abstract: Regulation of CO fixation in cyanobacteria is important both for the organism and global carbon balance. Here we show that phosphoketolase in Synechococcus elongatus PCC7942 (SeXPK) possesses a ...Regulation of CO fixation in cyanobacteria is important both for the organism and global carbon balance. Here we show that phosphoketolase in Synechococcus elongatus PCC7942 (SeXPK) possesses a distinct ATP-sensing mechanism, where a drop in ATP level allows SeXPK to divert precursors of the RuBisCO substrate away from the Calvin-Benson-Bassham cycle. Deleting the SeXPK gene increased CO fixation particularly during light-dark transitions. In high-density cultures, the Δxpk strain showed a 60% increase in carbon fixation and unexpectedly resulted in sucrose secretion without any pathway engineering. Using cryo-EM analysis, we discovered that these functions were enabled by a unique allosteric regulatory site involving two subunits jointly binding two ATP, which constantly suppresses the activity of SeXPK until the ATP level drops. This magnesium-independent ATP allosteric site is present in many species across all three domains of life, where it may also play important regulatory functions.
History
DepositionMar 10, 2023-
Header (metadata) releaseJun 28, 2023-
Map releaseJun 28, 2023-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35613.png

Downloads & links

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Map

FileDownload / File: emd_35613.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.274
Minimum - Maximum-0.45252487 - 1.3356061
Average (Standard dev.)0.0041659577 (±0.047915656)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 314.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_35613_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35613_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 : Cyanobacteria XPK complexed with TPP/Mg2+

EntireName: Cyanobacteria XPK complexed with TPP/Mg2+
Components
  • Complex: Cyanobacteria XPK complexed with TPP/Mg2+
    • Protein or peptide: Probable phosphoketolase
  • Ligand: MAGNESIUM ION
  • Ligand: THIAMINE DIPHOSPHATEThiamine pyrophosphate

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Supramolecule #1: Cyanobacteria XPK complexed with TPP/Mg2+

SupramoleculeName: Cyanobacteria XPK complexed with TPP/Mg2+ / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Synechococcus (bacteria)
Molecular weightTheoretical: 890 KDa

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Macromolecule #1: Probable phosphoketolase

MacromoleculeName: Probable phosphoketolase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Synechococcus elongatus (strain ATCC 33912 / PCC 7942 / FACHB-805) (bacteria)
Strain: ATCC 33912 / PCC 7942 / FACHB-805
Molecular weightTheoretical: 89.133211 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTSTLQATDI ATLSPNEQAA IDAWWRAANY LSVGQIYLRD NPLLQEPLRP EHIKQRLLGH WGSDPGLSFV YVHLNRLIRR LDLNLIYVT GPGHGAPALL ANAWLEGTYS EVYPNCQQST AGLQQFFKQF SFPGGIGSHC TPETPGSIHE GGELGYSLSH A FGAALDNP ...String:
MTSTLQATDI ATLSPNEQAA IDAWWRAANY LSVGQIYLRD NPLLQEPLRP EHIKQRLLGH WGSDPGLSFV YVHLNRLIRR LDLNLIYVT GPGHGAPALL ANAWLEGTYS EVYPNCQQST AGLQQFFKQF SFPGGIGSHC TPETPGSIHE GGELGYSLSH A FGAALDNP DLIVACVIGD GEAETGPLAT SWHSNKFLNP AQDGAVLPIL HLNGYKIANP TLLSRISHEE LRSLFIGYGY EP FFVEGND PAILHGVMAS TLATCVQKIQ AIQAAARSGE SSDRPMWPMI VLRTPKGWTG PATIKGHVVE GSWRSHQVPM ADV LTNPEH LQLLEDWLRS YRPEELFDAS GAPVAELQAI APIGDRRMSA NPVTNGGLLR RALTLPDFRD QAVSVPAPGK SRAD STRPL GQFLREVIRH NPDNFRLFGP DETASNRLDA VYEVTSKVWL GDRIPEDEDG GHLSDRGRVM EILSEHTLEG WLEAY LLTG RHGFFATYEA FAHVIDSMVN QHAKWLDVSK REVDWRAPVS SLNILLSSTV WRQDHNGFSH QDPGFIDLVT NKSARV TRI YLPPDANCLL SVADHCLRST DYINVIVADK QSHLQYLDAE AAARHCAKGI GIWDWASNDQ GASPDVVIAS CGDVVTL EA LAATALLREH FPDLKIRFVN VVDLFRLQPD TEHPHGLSDR DFDSLFTVDK PIIFNFHGYP WLIHKLAYRR HNHNNLHV R GYKEVGNINT PLELAIRNQV DRFNLAIDVI DRVPHLRDRG AHVKEWLKDQ IHDHIQYAYQ EGIDRPEINQ WQWPF

UniProtKB: UNIPROTKB: A0A8T9U4A0

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: THIAMINE DIPHOSPHATE

MacromoleculeName: THIAMINE DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: TPP
Molecular weightTheoretical: 425.314 Da
Chemical component information

ChemComp-TPP:
THIAMINE DIPHOSPHATE / Thiamine pyrophosphate

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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

Concentration0.4 mg/mL
BufferpH: 7.2
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec.
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 3983 / Average exposure time: 4.5 sec. / Average electron dose: 59.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: OTHER
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.63 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.0) / Number images used: 30649
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8io8


Chain - Source name: PDB / Chain - Initial model type: experimental model
Details: cyanobacteria XPK complexed with AMPPNP from the same study
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8ioa:
Cryo-EM structure of cyanobacteria phosphoketolase

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