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- PDB-8ho8: The cryo-EM structure of cellobiose phosphorylase from Clostridiu... -

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Basic information

Entry
Database: PDB / ID: 8ho8
TitleThe cryo-EM structure of cellobiose phosphorylase from Clostridium thermocellum in complex with cellobiose
ComponentsCellobiose phosphorylase
KeywordsTRANSFERASE / Cellobiose phosphorylase
Function / homology
Function and homology information


cellobiose phosphorylase / cellobiose phosphorylase activity / carbohydrate binding / carbohydrate metabolic process
Similarity search - Function
Glycoside hydrolase family 65, C-terminal / Glycosyl hydrolase family 65, C-terminal domain / Cellobiose phosphorylase, N-terminal / Putative carbohydrate binding domain / : / Glycosyl hydrolase 94 / Glycosyl hydrolase 94, supersandwich domain / Glycosyl hydrolase 36, catalytic domain / Glycosyl hydrolase 94 superfamily, catalytic domain / Glycoside hydrolase family 65, N-terminal domain superfamily ...Glycoside hydrolase family 65, C-terminal / Glycosyl hydrolase family 65, C-terminal domain / Cellobiose phosphorylase, N-terminal / Putative carbohydrate binding domain / : / Glycosyl hydrolase 94 / Glycosyl hydrolase 94, supersandwich domain / Glycosyl hydrolase 36, catalytic domain / Glycosyl hydrolase 94 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-cellobiose / Cellobiose phosphorylase
Similarity search - Component
Biological speciesAcetivibrio thermocellus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.24 Å
AuthorsIriya, S. / Kuga, T. / Sunagawa, N. / Igarashi, K.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)22J12566 Japan
CitationJournal: To Be Published
Title: The cryo-EM structure of cellobiose phosphorylase from Clostridium thermocellum in complex with cellobiose
Authors: Iriya, S. / Kuga, T. / Sunagawa, N. / Igarashi, K.
History
DepositionDec 9, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)188,4304
Polymers187,7452
Non-polymers6852
Water5,567309
1


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

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Components

#1: Protein Cellobiose phosphorylase


Mass: 93872.453 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acetivibrio thermocellus (bacteria) / Strain: YM4 / Gene: cbp / Plasmid: pET28 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8VP44, cellobiose phosphorylase
#2: Polysaccharide beta-D-glucopyranose-(1-4)-beta-D-glucopyranose


Type: oligosaccharide, Oligosaccharide / Class: Metabolism / Mass: 342.297 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: oligosaccharide / References: beta-cellobiose
DescriptorTypeProgram
DGlcpb1-4DGlcpb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][b-D-Glcp]{[(4+1)][b-D-Glcp]{}}LINUCSPDB-CARE
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 309 / 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: Cellobiose phosphorylase / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.18 MDa / Experimental value: YES
Source (natural)Organism: Acetivibrio thermocellus (bacteria) / Strain: YM4
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pET28
Buffer solutionpH: 6.5 / Details: 20 mM BisTris-HCl, 80 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM2,2-Bis(hydroxymethyl)-2,2',2''-nitrilotriethanol; 2-Bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol; Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methaneC8H19NO51
280 mMsodium chlorideNaCl1
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: 279 K / Details: Vitrification carried out in nitrogen atmosphere.

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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 magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 5.121 sec. / Electron dose: 49 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 2907

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
2SerialEMimage acquisition
4cryoSPARC3.3.2CTF correction
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARC3.3.2final Euler assignment
13cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1453883
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 869598 / Symmetry type: POINT

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