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- PDB-8h6h: cryo-EM structure of cellodextrin phosphorylase from Clostridium ... -

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Basic information

Entry
Database: PDB / ID: 8h6h
Titlecryo-EM structure of cellodextrin phosphorylase from Clostridium thermocellum
ComponentsCellodextrin phosphorylase
KeywordsCARBOHYDRATE / cellulose / cellodextrin / phosphorolysis / synthesis
Function / homology
Function and homology information


transferase 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
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
DepositionOct 17, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 25, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)225,3416
Polymers225,1992
Non-polymers1424
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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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) / Gene: cdp-ym4 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q93HT8
#2: Chemical
ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Cl
Has ligand of interestN

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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 (CtCDP)
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
Buffer component
IDConc.NameFormulaBuffer-ID
1120 mMsodium chlorideNaClSodium chloride1
220 mMtris(hydroxymethyl)aminomethaneC4H11NO31
30.1 mMDTTC4H10O2S21
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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 FIELDBright-field microscopy / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 49 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
2SerialEMimage acquisition
4cryoSPARC3.3.2CTF correction
7PHENIX1.2model fitting
9cryoSPARC3.3.2initial Euler assignment
10cryoSPARC3.3.2final Euler assignment
12cryoSPARC3.3.23D reconstruction
13PHENIX1.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3503460
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 604474 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 86.98 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002916196
ELECTRON MICROSCOPYf_angle_d0.429821926
ELECTRON MICROSCOPYf_chiral_restr0.04032392
ELECTRON MICROSCOPYf_plane_restr0.00232844
ELECTRON MICROSCOPYf_dihedral_angle_d3.97342151

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