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- EMDB-35610: Cryo-EM structure of phosphoketolase from Bifidobacterium longum ... -

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Basic information

Entry
Database: EMDB / ID: EMD-35610
TitleCryo-EM structure of phosphoketolase from Bifidobacterium longum in dimeric assembly
Map data
Sample
  • Complex: Bifidobacterium longum XFPK in complex with TPP/Mg2+
    • Protein or peptide: Xylulose5phosphatefructose6phosphate phosphoketolase
  • Ligand: THIAMINE DIPHOSPHATEThiamine pyrophosphate
  • Ligand: MAGNESIUM ION
KeywordsCarbon metabolism / TPP-dependent enzyme / CYTOSOLIC PROTEIN
Function / homology
Function and homology information


aldehyde-lyase activity / carbohydrate metabolic process
Similarity search - Function
Xylulose 5-phosphate/Fructose 6-phosphate phosphoketolase / Xylulose 5-phosphate/Fructose 6-phosphate phosphoketolase, C-terminal / Xylulose 5-phosphate/Fructose 6-phosphate phosphoketolase, N-terminal / Xylulose 5-phosphate/Fructose 6-phosphate phosphoketolase, thiamine diphosphate binding site / Xylulose 5-phosphate/Fructose 6-phosphate phosphoketolase, conserved site / D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase / XFP C-terminal domain / XFP N-terminal domain / Phosphoketolase signature 1. / Phosphoketolase signature 2. ...Xylulose 5-phosphate/Fructose 6-phosphate phosphoketolase / Xylulose 5-phosphate/Fructose 6-phosphate phosphoketolase, C-terminal / Xylulose 5-phosphate/Fructose 6-phosphate phosphoketolase, N-terminal / Xylulose 5-phosphate/Fructose 6-phosphate phosphoketolase, thiamine diphosphate binding site / Xylulose 5-phosphate/Fructose 6-phosphate phosphoketolase, conserved site / D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase / XFP C-terminal domain / XFP N-terminal domain / Phosphoketolase signature 1. / Phosphoketolase signature 2. / Transketolase C-terminal/Pyruvate-ferredoxin oxidoreductase domain II / Thiamin diphosphate-binding fold
Similarity search - Domain/homology
Xylulose5phosphatefructose6phosphate phosphoketolase
Similarity search - Component
Biological speciesBifidobacteriaceae bacterium (bacteria) / Bifidobacterium longum subsp. longum F8 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.62 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_35610.png

Downloads & links

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Map

FileDownload / File: emd_35610.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.202
Minimum - Maximum-0.5134884 - 1.4549931
Average (Standard dev.)0.0037012738 (±0.047067095)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 318.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_35610_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_35610_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 : Bifidobacterium longum XFPK in complex with TPP/Mg2+

EntireName: Bifidobacterium longum XFPK in complex with TPP/Mg2+
Components
  • Complex: Bifidobacterium longum XFPK in complex with TPP/Mg2+
    • Protein or peptide: Xylulose5phosphatefructose6phosphate phosphoketolase
  • Ligand: THIAMINE DIPHOSPHATEThiamine pyrophosphate
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Bifidobacterium longum XFPK in complex with TPP/Mg2+

SupramoleculeName: Bifidobacterium longum XFPK in complex with TPP/Mg2+ / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bifidobacteriaceae bacterium (bacteria)
Molecular weightTheoretical: 750 KDa

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Macromolecule #1: Xylulose5phosphatefructose6phosphate phosphoketolase

MacromoleculeName: Xylulose5phosphatefructose6phosphate phosphoketolase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bifidobacterium longum subsp. longum F8 (bacteria)
Molecular weightTheoretical: 92.621125 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTSPVIGTPW KKLNAPVSEE ALEGVDKYWR VANYLSIGQI YLRSNPLMKE PFTREDVKHR LVGHWGTTPG LNFLIGHINR FIADHGQNT VIIMGPGHGG PAGTSQSYLD GTYTETFPKI TKDEAGLQKF FRQFSYPGGI PSHFAPETPG SIHEGGELGY A LSHAYGAI ...String:
MTSPVIGTPW KKLNAPVSEE ALEGVDKYWR VANYLSIGQI YLRSNPLMKE PFTREDVKHR LVGHWGTTPG LNFLIGHINR FIADHGQNT VIIMGPGHGG PAGTSQSYLD GTYTETFPKI TKDEAGLQKF FRQFSYPGGI PSHFAPETPG SIHEGGELGY A LSHAYGAI MDNPSLFVPA IVGDGEAETG PLATGWQSNK LVNPRTDGIV LPILHLNGYK IANPTILSRI SDEELHEFFH GM GYEPYEF VAGFDDEDHM SIHRRFAELW ETIWDEICDI KATAQTDNVH RPFYPMLIFR TPKGWTCPKY IDGKKTEGSW RSH QVPLAS ARDTEAHFEV LKNWLESYKP EELFDANGAV KDDVLAFMPK GELRIGANPN ANGGVIRNDL KLPNLEDYEV KEVA EYGHG WGQLEATRTL GAYTRDIIKN NPRDFRIFGP DETASNRLQA SYEVTNKQWD AGYISDEVDE HMHVSGQVVE QLSEH QMEG FLEAYLLTGR HGIWSSYESF VHVIDSMLNQ HAKWLEATVR EIPWRKPIAS MNLLVSSHVW RQDHNGFSHQ DPGVTS VLL NKCFHNDHVI GIYFATDANM LLAIAEKCYK STNKINAIIA GKQPAATWLT LDEARAELEK GAAAWDWAST AKNNDEA EV VLAAAGDVPT QEIMAASDKL KELGVKFKVV NVADLLSLQS AKENDEALTD EEFADIFTAD KPVLFAYHSY AHDVRGLI Y DRPNHDNFNV HGYEEEGSTT TPYDMVRVNR IDRYELTAEA LRMIDADKYA DKIDELEKFR DEAFQFAVDN GYDHPDYTD WVYSGVNTDK KGAVTATAAT AGDNE

UniProtKB: Xylulose5phosphatefructose6phosphate phosphoketolase

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Macromolecule #2: THIAMINE DIPHOSPHATE

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

ChemComp-TPP:
THIAMINE DIPHOSPHATE / Thiamine pyrophosphate

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 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

Concentration0.4 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: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 6289 / Average exposure time: 2.5 sec. / Average electron dose: 42.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3ai7

Initial angle assignmentType: OTHER
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionNumber classes used: 3 / Resolution.type: BY AUTHOR / Resolution: 2.62 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.0) / Number images used: 590627
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3ai7


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8io7:
Cryo-EM structure of phosphoketolase from Bifidobacterium longum in dimeric assembly

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