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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-4
  • 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: Nature / Year: 2024
Title: Computational design of soluble and functional membrane protein analogues.
Authors: Casper A Goverde / Martin Pacesa / Nicolas Goldbach / Lars J Dornfeld / Petra E M Balbi / Sandrine Georgeon / Stéphane Rosset / Srajan Kapoor / Jagrity Choudhury / Justas Dauparas / ...Authors: Casper A Goverde / Martin Pacesa / Nicolas Goldbach / Lars J Dornfeld / Petra E M Balbi / Sandrine Georgeon / Stéphane Rosset / Srajan Kapoor / Jagrity Choudhury / Justas Dauparas / Christian Schellhaas / Simon Kozlov / David Baker / Sergey Ovchinnikov / Alex J Vecchio / Bruno E Correia /
Abstract: De novo design of complex protein folds using solely computational means remains a substantial challenge. Here we use a robust deep learning pipeline to design complex folds and soluble analogues of ...De novo design of complex protein folds using solely computational means remains a substantial challenge. Here we use a robust deep learning pipeline to design complex folds and soluble analogues of integral membrane proteins. Unique membrane topologies, such as those from G-protein-coupled receptors, are not found in the soluble proteome, and we demonstrate that their structural features can be recapitulated in solution. Biophysical analyses demonstrate the high thermal stability of the designs, and experimental structures show remarkable design accuracy. The soluble analogues were functionalized with native structural motifs, as a proof of concept for bringing membrane protein functions to the soluble proteome, potentially enabling new approaches in drug discovery. In summary, we have designed complex protein topologies and enriched them with functionalities from membrane proteins, with high experimental success rates, leading to a de facto expansion of the functional soluble fold space.
History
DepositionApr 15, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 26, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_abbrev / _citation.journal_id_ISSN ..._citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _citation_author.name / _em_admin.last_update
Revision 1.2Jul 3, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.3Jul 24, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.4Oct 16, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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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
Water00
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


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)
Has protein modificationY

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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 mMNaClNaCl1
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 FIELD / 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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