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- PDB-6lxv: Cryo-EM structure of phosphoketolase from Bifidobacterium longum -

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Entry
Database: PDB / ID: 6lxv
TitleCryo-EM structure of phosphoketolase from Bifidobacterium longum
ComponentsPhosphoketolase
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
THIAMINE DIPHOSPHATE / Phosphoketolase
Similarity search - Component
Biological speciesBifidobacterium longum subsp. longum F8 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsNakata, K. / Miyazaki, N. / Yamaguchi, H. / Hirose, M. / Miyano, H. / Mizukoshi, T. / Kashiwagi, T. / Iwasaki, K.
Funding support Japan, 1items
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, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 17, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 12, 2022Group: Database references / Category: citation / citation_author / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Phosphoketolase
B: Phosphoketolase
C: Phosphoketolase
D: Phosphoketolase
E: Phosphoketolase
F: Phosphoketolase
G: Phosphoketolase
H: Phosphoketolase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)751,32324
Polymers747,6008
Non-polymers3,72316
Water53,4512967
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area55210 Å2
ΔGint-287 kcal/mol
Surface area177480 Å2

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Components

#1: Protein
Phosphoketolase /


Mass: 93450.016 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bifidobacterium longum subsp. longum F8 (bacteria)
Gene: BIL_11880 / Plasmid: pET24 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: D6D942, fructose-6-phosphate phosphoketolase
#2: Chemical
ChemComp-TPP / THIAMINE DIPHOSPHATE / Thiamine pyrophosphate


Mass: 425.314 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C12H19N4O7P2S / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 2967 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Phosphoketolase with thiamine-diphophate / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bifidobacterium longum subsp. longum F8 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pET24
Buffer solutionpH: 9
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: MOLYBDENUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Nominal defocus max: 2750 nm / Nominal defocus min: 1000 nm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 45 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 2897
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 426149
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 194517 / Algorithm: FOURIER SPACE / Symmetry type: POINT

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