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- PDB-9bei: Cryo-EM structure of synthetic claudin-4 complex with Clostridium... -

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Basic information

Entry
Database: PDB / ID: 9bei
TitleCryo-EM structure of synthetic claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain, sFab COP-2, and Nanobody
Components
  • Anti-fab nanobody
  • COP-2 Fab Heavy chain
  • COP-2 Fab Light chain
  • Claudin-4CLDN4
  • Heat-labile enterotoxin B chain
KeywordsMEMBRANE PROTEIN/IMMUNE SYSYTEM / Claudin / Fab / Toxin / MEMBRANE PROTEIN / MEMBRANE PROTEIN-IMMUNE SYSYTEM complex
Function / homologyClostridium enterotoxin / Clostridium enterotoxin / toxin activity / extracellular region / Heat-labile enterotoxin B chain
Function and homology information
Biological speciesClostridium perfringens (bacteria)
Homo sapiens (human)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.16 Å
AuthorsVecchio, A.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138368 United States
CitationJournal: To be published
Title: Computational design of soluble functional analogues of integral membrane proteins
Authors: Goverde, C.A. / Pacesa, M. / Goldbach, N. / Dornfeld, L.J. / Balbi, P.E. / Georgeon, S. / Rosset, S. / Kapoor, S. / Choudhury, J. / Deuparea, J. / Schellhaas, C. / Kozlov, S. / Baker, D. / ...Authors: Goverde, C.A. / Pacesa, M. / Goldbach, N. / Dornfeld, L.J. / Balbi, P.E. / Georgeon, S. / Rosset, S. / Kapoor, S. / Choudhury, J. / Deuparea, J. / Schellhaas, C. / Kozlov, S. / Baker, D. / Ovchinnikov, S. / Vecchio, A.J. / Correia, B.E.
History
DepositionApr 15, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Claudin-4
B: Heat-labile enterotoxin B chain
H: COP-2 Fab Heavy chain
K: Anti-fab nanobody
L: COP-2 Fab Light chain


Theoretical massNumber of molelcules
Total (without water)98,6265
Polymers98,6265
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Claudin-4 / CLDN4


Mass: 22078.986 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#2: Protein Heat-labile enterotoxin B chain


Mass: 14591.295 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium perfringens (bacteria) / Gene: cpe / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P01558
#3: Antibody COP-2 Fab Heavy chain


Mass: 25263.010 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#4: Antibody Anti-fab nanobody


Mass: 13175.438 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#5: Antibody COP-2 Fab Light chain


Mass: 23517.057 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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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: Synthetic human claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain, sFab COP-2, and nanobody against COP-2
Type: COMPLEX
Details: Assembled complex of 5 proteins (Fab is 2 proteins) expressed from insect cells and E coli
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.103 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.4 / Details: 20 mM Hepes pH 8.0, 150 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepes1
2150 mMNaClSodium chlorideNaClSodium chloride1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: UltraAuFoil 1.2/1.3 grids (Quantifoil) were glow discharged for 30 s at 15 mA in a Pelco easiGlow (Ted Pella Inc) instrument
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K
Details: 3.5 microL of complex was applied onto grids and blotted for 3 s at 4 degrees C under 100 percent humidity then plunge frozen into liquid ethane cooled by liquid nitrogen.

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 120000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 400 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 49.4 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1159

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4.1particle selection
2EPUimage acquisition
4cryoSPARC4.4.1CTF correction
7PHENIX1.20.1-4487model fitting
9PHENIX1.20.1-4487model refinement
10cryoSPARC4.4.1initial Euler assignment
11cryoSPARC4.4.1final Euler assignment
12cryoSPARC4.4.1classification
13cryoSPARC4.4.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1848208
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21296 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 322 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDDetailsInitial refinement model-IDSource nameType
17DTM

7dtm

B7DTMBcCPE1PDBexperimental model
27DTM

7dtm

H7DTMHCOP-2 Heavy1PDBexperimental model
37DTM

7dtm

L7DTMLCOP-2 Light1PDBexperimental model
4Syn claudin-4AlphaFoldin silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036791
ELECTRON MICROSCOPYf_angle_d0.7349220
ELECTRON MICROSCOPYf_dihedral_angle_d7.383947
ELECTRON MICROSCOPYf_chiral_restr0.0461024
ELECTRON MICROSCOPYf_plane_restr0.0061191

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