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- EMDB-44479: Cryo-EM structure of synthetic claudin-4 complex with Clostridium... -

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Basic information

Entry
Database: EMDB / ID: EMD-44479
TitleCryo-EM structure of synthetic claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain, sFab COP-2, and Nanobody
Map data
Sample
  • Complex: Synthetic human claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain, sFab COP-2, and nanobody against COP-2
    • Protein or peptide: Claudin-4CLDN4
    • Protein or peptide: Heat-labile enterotoxin B chain
    • Protein or peptide: COP-2 Fab Heavy chain
    • Protein or peptide: Anti-fab nanobody
    • Protein or peptide: COP-2 Fab Light chain
KeywordsClaudin / 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 speciesHomo sapiens (human) / Clostridium perfringens (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.16 Å
AuthorsVecchio AJ
Funding support United States, 1 items
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 CA / Pacesa M / Goldbach N / Dornfeld LJ / Balbi PE / Georgeon S / Rosset S / Kapoor S / Choudhury J / Deuparea J / Schellhaas C / Kozlov S / Baker D / Ovchinnikov S / Vecchio AJ / Correia BE
History
DepositionApr 15, 2024-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateApr 24, 2024-
Current statusApr 24, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44479.png

Downloads & links

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Map

FileDownload / File: emd_44479.map.gz / Format: CCP4 / Size: 229.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.884 Å
Density
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.13631451 - 0.32407513
Average (Standard dev.)-0.000044602784 (±0.006148181)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions392392392
Spacing392392392
CellA=B=C: 346.528 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_44479_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44479_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Synthetic human claudin-4 complex with Clostridium perfringens en...

EntireName: Synthetic human claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain, sFab COP-2, and nanobody against COP-2
Components
  • Complex: Synthetic human claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain, sFab COP-2, and nanobody against COP-2
    • Protein or peptide: Claudin-4CLDN4
    • Protein or peptide: Heat-labile enterotoxin B chain
    • Protein or peptide: COP-2 Fab Heavy chain
    • Protein or peptide: Anti-fab nanobody
    • Protein or peptide: COP-2 Fab Light chain

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Supramolecule #1: Synthetic human claudin-4 complex with Clostridium perfringens en...

SupramoleculeName: Synthetic human claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain, sFab COP-2, and nanobody against COP-2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Assembled complex of 5 proteins (Fab is 2 proteins) expressed from insect cells and E coli
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 103 KDa

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Macromolecule #1: Claudin-4

MacromoleculeName: Claudin-4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.078986 KDa
SequenceString: MSSLETFREA RRLAREGLEL VREAARLPMW RVTAFIGSNI VTSQTIWEGL WMNCVVQSTG QMQCKVYDSL LALPEDLRRA RESFERAIE VAEKALELLE IGDPDSDAIE DEEERLQTIH EAGELLLKAA ELAREPTEAI ADRIIQDFYN PLVASGQKRE M GASLALAR ...String:
MSSLETFREA RRLAREGLEL VREAARLPMW RVTAFIGSNI VTSQTIWEGL WMNCVVQSTG QMQCKVYDSL LALPEDLRRA RESFERAIE VAEKALELLE IGDPDSDAIE DEEERLQTIH EAGELLLKAA ELAREPTEAI ADRIIQDFYN PLVASGQKRE M GASLALAR RGAELLEEAG RKLLGLEGGS LEHHHHHH

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Macromolecule #2: Heat-labile enterotoxin B chain

MacromoleculeName: Heat-labile enterotoxin B chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Clostridium perfringens (bacteria)
Molecular weightTheoretical: 14.591295 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
MSTDIEKEIL DLAAATERLN LTDALNSNPA GNLYDWRSSN SYPWTQKLNL HLTITATGQK YRILASKIVD FNIYSNNFNN LVKLEQSLG DGVKDHYVDI SLDAGQYVLV MKANSSYSGN YPYSILFQKF

UniProtKB: Heat-labile enterotoxin B chain

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Macromolecule #3: COP-2 Fab Heavy chain

MacromoleculeName: COP-2 Fab Heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 25.26301 KDa
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NFSSSSIHWV RQAPGKGLEW VASISSYSGY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARYWSWYNSS HYIYSALDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK D YFPEPVTV ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NFSSSSIHWV RQAPGKGLEW VASISSYSGY TSYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARYWSWYNSS HYIYSALDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK D YFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCDKTHT

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Macromolecule #4: Anti-fab nanobody

MacromoleculeName: Anti-fab nanobody / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 13.175438 KDa
SequenceString:
GSVQLQESGG GLVQPGGSLR LSCAASGRTI SRYAMSWFRQ APGKEREFVA VARRSGDGAF YADSVQGRFT VSRDDAKNTV YLQMNSLKP EDTAVYYCAI DSDTFYSGSY DYWGQGTQVT VS

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Macromolecule #5: COP-2 Fab Light chain

MacromoleculeName: COP-2 Fab Light chain / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 23.517057 KDa
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSYEWAPVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSYEWAPVT FGQGTKVEIK RTVAAPSVFI FPPSDSQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG N SQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMHepes
150.0 mMNaClSodium chlorideNaClSodium chloride

Details: 20 mM Hepes pH 8.0, 150 mM NaCl
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
Details: UltraAuFoil 1.2/1.3 grids (Quantifoil) were glow discharged for 30 s at 15 mA in a Pelco easiGlow (Ted Pella Inc) instrument
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: LEICA EM GP
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

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 120000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 1159 / Average electron dose: 49.4 e/Å2

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Image processing

Particle selectionNumber selected: 1848208
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7dtm

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
Final 3D classificationNumber classes: 1 / Software - Name: cryoSPARC (ver. 4.4.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.16 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.1) / Number images used: 21296
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

7dtm

chain_id: B, source_name: PDB, initial_model_type: experimental modelcCPE

7dtm

chain_id: H, source_name: PDB, initial_model_type: experimental modelCOP-2 Heavy

7dtm

chain_id: L, source_name: PDB, initial_model_type: experimental modelCOP-2 Light
source_name: AlphaFold, initial_model_type: in silico modelSyn claudin-4
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 322
Output model

PDB-9bei:
Cryo-EM structure of synthetic claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain, sFab COP-2, and Nanobody

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