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- PDB-8un2: Microbacterium testaceum C-glucosyl deglycosidase (CGD), wild typ... -

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Basic information

Entry
Database: PDB / ID: 8un2
TitleMicrobacterium testaceum C-glucosyl deglycosidase (CGD), wild type (cryo-EM)
ComponentsSugar phosphate isomerase
KeywordsLYASE / bacterial C-glucosyl deglycosidase / C-C bond cleavage / C-glucosyl flavonoids / Microbacterium tetsaceum NS220 / plant endosymbiont / N-terminal DUF6379 beta-sandwich domain / C-terminal TIM-barrel domain / homodimer
Function / homology
Function and homology information


isomerase activity / lyase activity
Similarity search - Function
Domain of unknown function DUF6379 / C-glycoside deglycosidase beta subunit / : / Xylose isomerase-like, TIM barrel domain / Xylose isomerase-like TIM barrel / Xylose isomerase-like superfamily
Similarity search - Domain/homology
: / C-deglycosylation enzyme beta subunit
Similarity search - Component
Biological speciesMicrobacterium testaceum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsFurlanetto, V. / Kalyani, D.C. / Hallberg, B.M. / Divne, C.
Funding support Sweden, 3items
OrganizationGrant numberCountry
Other governmentFormas 2017-00983 Sweden
Other governmentVR 2017-03877 Sweden
Other privateOscar and Lili Lamm Memorial Foundation DO2017-0020 Sweden
CitationJournal: To be published
Title: Microbacterium testaceum C-glucosyl deglycosidase (CGD), wild type (cryo-EM)
Authors: Furlanetto, V. / Kalyani, D.C. / Srivastava, V. / Hallberg, B.M. / Ezcurra, I. / Divne, C.
History
DepositionOct 18, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 23, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 23, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sugar phosphate isomerase
B: Sugar phosphate isomerase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,6464
Polymers97,5362
Non-polymers1102
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, confirmed by gel filtration, cryo-EM and X-ray crystallography
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Sugar phosphate isomerase


Mass: 48768.133 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Microbacterium testaceum (bacteria) / Strain: NS220 / Gene: NS220_14145 / Plasmid: pNIC-CTHO / Production host: Escherichia coli B (bacteria) / Strain (production host): (DE3)-T1 / References: UniProt: A0A147EV52
#2: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
Has ligand of interestN
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: C-glucosyl deglycosidase from Microbacterium testaceum
Type: COMPLEX / Details: wild type / Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Microbacterium testaceum (bacteria) / Strain: NS220
Source (recombinant)Organism: Escherichia coli B (bacteria) / Strain: (DE3)-T1 / Plasmid: pNIC-CTHO
Buffer solutionpH: 7.5 / Details: 25 mM HEPES pH 7.5, 150 mM NaCl, 0.15 mM MnCl2
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: monodisperse sample
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Details: Data collected on the Karolinska Institutet's Krios G3i equipped with a cold-FEG
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 200 nm / Cs: 2.7 mm / C2 aperture diameter: 20 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 56 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1Warp0.9particle selection
2EPU3.1image acquisition
4Warp0.9CTF correction
7Cootmodel fitting
9cryoSPARC4.3.1initial Euler assignment
11cryoSPARC4.3.1classification
12cryoSPARC4.3.13D reconstruction
13PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 734119 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingDetails: X-ray model of the same sample / Source name: Other / Type: experimental model

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