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- PDB-8yaq: Cryo-EM structure of cellodextrin phosphorylase from Clostridium ... -

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Basic information

Entry
Database: PDB / ID: 8yaq
TitleCryo-EM structure of cellodextrin phosphorylase from Clostridium thermocellum with cellodextrin ligands
ComponentsCellodextrin phosphorylase
KeywordsCARBOHYDRATE / cellulose / cellodextrin / phosphorolysis / synthesis
Function / homology
Function and homology information


glycosyltransferase activity / carbohydrate binding / carbohydrate metabolic process
Similarity search - Function
: / Glycosyl hydrolase 94 / Glycosyltransferase family 36 / Glycosyl hydrolase 36, catalytic domain / Glycosyl hydrolase 36 superfamily, catalytic domain / Glycoside hydrolase family 65, N-terminal domain superfamily / Galactose mutarotase-like domain superfamily / Six-hairpin glycosidase-like superfamily / Six-hairpin glycosidase superfamily
Similarity search - Domain/homology
beta-cellopentaose / beta-cellotriose / Cellodextrin phosphorylase
Similarity search - Component
Biological speciesAcetivibrio thermocellus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsKuga, T. / Sunagawa, N. / Igarashi, K.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)22J12566 Japan
Ministry of Education, Culture, Sports, Science and Technology (Japan)18H05494 Japan
CitationJournal: To Be Published
Title: Structure and dynamics of cellodextrin phosphorylase from Clostridium thermocellum determine chain length and crystalline packing of highly ordered cellulose II synthesized in vitro
Authors: Kuga, T. / Sunagawa, N. / Igarashi, K.
History
DepositionFeb 9, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cellodextrin phosphorylase
B: Cellodextrin phosphorylase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)226,7099
Polymers225,1992
Non-polymers1,5107
Water181
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Cellodextrin phosphorylase


Mass: 112599.523 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acetivibrio thermocellus (bacteria) / Strain: YM4 / Gene: cdp-ym4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q93HT8
#2: Polysaccharide beta-D-glucopyranose-(1-4)-beta-D-glucopyranose-(1-4)-beta-D-glucopyranose-(1-4)-beta-D- ...beta-D-glucopyranose-(1-4)-beta-D-glucopyranose-(1-4)-beta-D-glucopyranose-(1-4)-beta-D-glucopyranose-(1-4)-beta-D-glucopyranose


Type: oligosaccharide, Oligosaccharide / Class: Metabolism / Mass: 828.719 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: oligosaccharide / References: beta-cellopentaose
DescriptorTypeProgram
DGlcpb1-4DGlcpb1-4DGlcpb1-4DGlcpb1-4DGlcpb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,5,4/[a2122h-1b_1-5]/1-1-1-1-1/a4-b1_b4-c1_c4-d1_d4-e1WURCSPDB2Glycan 1.1.0
[][b-D-Glcp]{[(4+1)][b-D-Glcp]{[(4+1)][b-D-Glcp]{[(4+1)][b-D-Glcp]{[(4+1)][b-D-Glcp]{}}}}}LINUCSPDB-CARE
#3: Polysaccharide beta-D-glucopyranose-(1-4)-beta-D-glucopyranose-(1-4)-beta-D-glucopyranose


Type: oligosaccharide, Oligosaccharide / Class: Metabolism / Mass: 504.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: oligosaccharide / References: beta-cellotriose
DescriptorTypeProgram
DGlcpb1-4DGlcpb1-4DGlcpb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,3,2/[a2122h-1b_1-5]/1-1-1/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][b-D-Glcp]{[(4+1)][b-D-Glcp]{[(4+1)][b-D-Glcp]{}}}LINUCSPDB-CARE
#4: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: Homodimeric structure of cellodextrin phosphorylase from Clostridium thermocellum
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.24 MDa / Experimental value: YES
Source (natural)Organism: Acetivibrio thermocellus (bacteria) / Strain: YM4
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3) / Plasmid: pET28-b
Buffer solutionpH: 7.5
Details: 20 mM Tris-HCl, 120 mM NaCl, 0.1 mM DTT, 1mM cellohexaose
Buffer component
IDConc.NameFormulaBuffer-ID
1120 mMsodium chlorideNaCl1
220 mMtris(hydroxymethyl)aminomethaneC4H11NO31
30.1 mMDTTC4H10O2S21
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 280 K

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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 FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 4023

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4.0particle selection
2SerialEMimage acquisition
4cryoSPARC4.4.0CTF correction
7PHENIX1.2model fitting
9cryoSPARC4.4.0initial Euler assignment
10cryoSPARC4.4.0final Euler assignment
11cryoSPARC4.4.0classification
12cryoSPARC4.4.03D reconstruction
13PHENIX1.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3503460
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 140756 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 8H6H

8h6h


Accession code: 8H6H / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00316207
ELECTRON MICROSCOPYf_angle_d0.45321935
ELECTRON MICROSCOPYf_dihedral_angle_d3.7412139
ELECTRON MICROSCOPYf_chiral_restr0.042425
ELECTRON MICROSCOPYf_plane_restr0.0032826

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