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- EMDB-30007: Cryo-EM structure of phosphoketolase from Bifidobacterium longum -

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Basic information

Entry
Database: EMDB / ID: EMD-30007
TitleCryo-EM structure of phosphoketolase from Bifidobacterium longum
Map data
Sample
  • Complex: Phosphoketolase with thiamine-diphophate
    • Protein or peptide: Phosphoketolase
  • Ligand: THIAMINE DIPHOSPHATE
  • Ligand: CALCIUM ION
  • Ligand: water
Keywordslyase / ketolase / thiamine diphosphate / octamer / Bifidobacterium longum / lyase activity
Function / homology
Function and homology information


fructose-6-phosphate phosphoketolase / fructose-6-phosphate phosphoketolase 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
Biological speciesBifidobacterium longum subsp. longum F8 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsNakata K / Miyazaki N
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED) Japan
CitationJournal: J Struct Biol / Year: 2022
Title: High-resolution structure of phosphoketolase from Bifidobacterium longum determined by cryo-EM single-particle analysis.
Authors: Kunio Nakata / Naoyuki Miyazaki / Hiroki Yamaguchi / Mika Hirose / Tatsuki Kashiwagi / Nidamarthi H V Kutumbarao / Osamu Miyashita / Florence Tama / Hiroshi Miyano / Toshimi Mizukoshi / Kenji Iwasaki /
Abstract: In bifidobacteria, phosphoketolase (PKT) plays a key role in the central hexose fermentation pathway called "bifid shunt." The three-dimensional structure of PKT from Bifidobacterium longum with co- ...In bifidobacteria, phosphoketolase (PKT) plays a key role in the central hexose fermentation pathway called "bifid shunt." The three-dimensional structure of PKT from Bifidobacterium longum with co-enzyme thiamine diphosphate (ThDpp) was determined at 2.1 Å resolution by cryo-EM single-particle analysis using 196,147 particles to build up the structural model of a PKT octamer related by D symmetry. Although the cryo-EM structure of PKT was almost identical to the X-ray crystal structure previously determined at 2.2 Å resolution, several interesting structural features were observed in the cryo-EM structure. Because this structure was solved at relatively high resolution, it was observed that several amino acid residues adopt multiple conformations. Among them, Q546-D547-H548-N549 (the QN-loop) demonstrate the largest structural change, which seems to be related to the enzymatic function of PKT. The QN-loop is at the entrance to the substrate binding pocket. The minor conformer of the QN-loop is similar to the conformation of the QN-loop in the crystal structure. The major conformer is located further from ThDpp than the minor conformer. Interestingly, the major conformer in the cryo-EM structure of PKT resembles the corresponding loop structure of substrate-bound Escherichia coli transketolase. That is, the minor and major conformers may correspond to "closed" and "open" states for substrate access, respectively. Moreover, because of the high-resolution analysis, many water molecules were observed in the cryo-EM structure of PKT. Structural features of the water molecules in the cryo-EM structure are discussed and compared with water molecules observed in the crystal structure.
History
DepositionFeb 12, 2020-
Header (metadata) releaseFeb 17, 2021-
Map releaseFeb 17, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
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  • Surface view with fitted model
  • Atomic models: PDB-6lxv
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6lxv
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30007.png

Downloads & links

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Map

FileDownload / File: emd_30007.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 360 pix.
= 313.2 Å		0.87 Å/pix.
x 360 pix.
= 313.2 Å		0.87 Å/pix.
x 360 pix.
= 313.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.87 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.20026584 - 0.54527557
Average (Standard dev.)0.00035448186 (±0.012134807)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 313.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.870.870.87
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z313.200313.200313.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.2000.5450.000

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Supplemental data

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Sample components

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Entire : Phosphoketolase with thiamine-diphophate

EntireName: Phosphoketolase with thiamine-diphophate
Components
  • Complex: Phosphoketolase with thiamine-diphophate
    • Protein or peptide: Phosphoketolase
  • Ligand: THIAMINE DIPHOSPHATE
  • Ligand: CALCIUM ION
  • Ligand: water

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Supramolecule #1: Phosphoketolase with thiamine-diphophate

SupramoleculeName: Phosphoketolase with thiamine-diphophate / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bifidobacterium longum subsp. longum F8 (bacteria)

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Macromolecule #1: Phosphoketolase

MacromoleculeName: Phosphoketolase / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: fructose-6-phosphate phosphoketolase
Source (natural)Organism: Bifidobacterium longum subsp. longum F8 (bacteria)
Molecular weightTheoretical: 93.450016 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 AGDNEHHHHH H

UniProtKB: Phosphoketolase

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Macromolecule #2: THIAMINE DIPHOSPHATE

MacromoleculeName: THIAMINE DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 8 / Formula: TPP
Molecular weightTheoretical: 425.314 Da
Chemical component information

ChemComp-TPP:
THIAMINE DIPHOSPHATE

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 8 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 2967 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration10 mg/mL
BufferpH: 9
GridModel: Quantifoil R1.2/1.3 / Material: MOLYBDENUM / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 2897 / Average exposure time: 45.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.75 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
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: 426149
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: D4 (2x4 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 194517
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 4 / Avg.num./class: 50000 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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