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- EMDB-41915: Cryo-EM structure of human claudin-4 complex with Clostridium per... -

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Basic information

Entry
Database: EMDB / ID: EMD-41915
TitleCryo-EM structure of human claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain and sFab COP-1
Map data
Sample
  • Complex: Human claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain and sFab COP-1
    • Protein or peptide: Claudin-4
    • Protein or peptide: Heat-labile enterotoxin B chain
    • Protein or peptide: COP-1 sFab Light Chain
    • Protein or peptide: COP-1 sFab Heavy Chain
  • Ligand: Lauryl Maltose Neopentyl Glycol
KeywordsClaudin / Fab / Toxin / MEMBRANE PROTEIN
Function / homology
Function and homology information


paracellular transport / calcium-independent cell-cell adhesion / Tight junction interactions / bicellular tight junction assembly / apicolateral plasma membrane / regulation of cell morphogenesis / tight junction / positive regulation of wound healing / renal absorption / chloride channel activity ...paracellular transport / calcium-independent cell-cell adhesion / Tight junction interactions / bicellular tight junction assembly / apicolateral plasma membrane / regulation of cell morphogenesis / tight junction / positive regulation of wound healing / renal absorption / chloride channel activity / lateral plasma membrane / bicellular tight junction / establishment of skin barrier / chloride channel complex / response to progesterone / basal plasma membrane / female pregnancy / circadian rhythm / cell-cell junction / transmembrane signaling receptor activity / toxin activity / cell adhesion / positive regulation of cell migration / apical plasma membrane / structural molecule activity / extracellular region / identical protein binding / plasma membrane
Similarity search - Function
Claudin-4 / Claudin / Claudin, conserved site / Claudin family signature. / Clostridium enterotoxin / Clostridium enterotoxin / PMP-22/EMP/MP20/Claudin family / PMP-22/EMP/MP20/Claudin superfamily
Similarity search - Domain/homology
Claudin-4 / Heat-labile enterotoxin B chain
Similarity search - Component
Biological speciesHomo sapiens (human) / Clostridium perfringens (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.3 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
Citation
Journal: Commun Biol / Year: 2024
Title: Structural and biophysical insights into targeting of claudin-4 by a synthetic antibody fragment.
Authors: Satchal K Erramilli / Pawel K Dominik / Chinemerem P Ogbu / Anthony A Kossiakoff / Alex J Vecchio /
Abstract: Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As ...Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties-these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes using cryogenic electron microscopy. Structural and biophysical results reveal this sFabs mechanism of select binding to human claudin-4 over other homologous claudins and establish the ability of sFabs to bind hard-to-target claudins to probe tight junction structure and function. The findings provide a framework for tight junction modulation by sFabs for tissue-selective therapies.
#1: Journal: bioRxiv / Year: 2023
Title: Cryo-EM structures of a synthetic antibody against 22 kDa claudin-4 reveal its complex with
Authors: Erramilli SK / Dominik PK / Ogbu CP / Kossiakoff AA / Vecchio AJ
History
DepositionSep 12, 2023-
Header (metadata) releaseSep 27, 2023-
Map releaseSep 27, 2023-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41915.png

Downloads & links

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Map

FileDownload / File: emd_41915.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.51 Å/pix.
x 512 pix.
= 259.584 Å		0.51 Å/pix.
x 512 pix.
= 259.584 Å		0.51 Å/pix.
x 512 pix.
= 259.584 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.507 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.031891707 - 0.08482177
Average (Standard dev.)0.00014671533 (±0.003031392)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 259.584 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41915_msk_1.map
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Half map: #2

Fileemd_41915_half_map_1.map
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Half map: #1

Fileemd_41915_half_map_2.map
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Sample components

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Entire : Human claudin-4 complex with Clostridium perfringens enterotoxin ...

EntireName: Human claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain and sFab COP-1
Components
  • Complex: Human claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain and sFab COP-1
    • Protein or peptide: Claudin-4
    • Protein or peptide: Heat-labile enterotoxin B chain
    • Protein or peptide: COP-1 sFab Light Chain
    • Protein or peptide: COP-1 sFab Heavy Chain
  • Ligand: Lauryl Maltose Neopentyl Glycol

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Supramolecule #1: Human claudin-4 complex with Clostridium perfringens enterotoxin ...

SupramoleculeName: Human claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain and sFab COP-1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: Assembled complex of 4 proteins (Fab is 2 proteins) expressed from insect cells and E coli
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 100 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: 22.613852 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MASMGLQVMG IALAVLGWLA VMLCCALPMW RVTAFIGSNI VTSQTIWEGL WMNCVVQSTG QMQCKVYDSL LALPQDLQAA RALVIISII VAALGVLLSV VGGKCTNCLE DESAKAKTMI VAGVVFLLAG LMVIVPVSWT AHNIIQDFYN PLVASGQKRE M GASLYVGW ...String:
MASMGLQVMG IALAVLGWLA VMLCCALPMW RVTAFIGSNI VTSQTIWEGL WMNCVVQSTG QMQCKVYDSL LALPQDLQAA RALVIISII VAALGVLLSV VGGKCTNCLE DESAKAKTMI VAGVVFLLAG LMVIVPVSWT AHNIIQDFYN PLVASGQKRE M GASLYVGW AASGLLLLGG GLLCCNCPPR TDKPYSAKYS AARSAAASNY VGLVPR

UniProtKB: Claudin-4

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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: 14.044594 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
GSDEILDLAA ATERLNLTDA LNSNPAGNLY DWRSSNSYPW TQKLNLHLTI TATGQKYRIL ASKIVDFNIY SNNFNNLVKL EQSLGDGVK DHYVDISLDA GQYVLVMKAN SSYSGNYPYS ILFQKF

UniProtKB: Heat-labile enterotoxin B chain

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Macromolecule #3: COP-1 sFab Light Chain

MacromoleculeName: COP-1 sFab Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 23.258783 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSSSSLITF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #4: COP-1 sFab Heavy Chain

MacromoleculeName: COP-1 sFab Heavy Chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 28.064498 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKNIAFLLA SMFVFSIATN AYAEISEVQL VESGGGLVQP GGSLRLSCAA SGFNFSSSYI HWVRQAPGKG LEWVASISSS SGSTSYADS VKGRFTISAD TSKNTAYLQM NSLRAEDTAV YYCARWFHPW WWWEYLFRGA IDYWGQGTLV TVSSASTKGP S VFPLAPSS ...String:
MKKNIAFLLA SMFVFSIATN AYAEISEVQL VESGGGLVQP GGSLRLSCAA SGFNFSSSYI HWVRQAPGKG LEWVASISSS SGSTSYADS VKGRFTISAD TSKNTAYLQM NSLRAEDTAV YYCARWFHPW WWWEYLFRGA IDYWGQGTLV TVSSASTKGP S VFPLAPSS KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NH KPSNTKV DKKVEPKSCD KTHT

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Macromolecule #5: Lauryl Maltose Neopentyl Glycol

MacromoleculeName: Lauryl Maltose Neopentyl Glycol / type: ligand / ID: 5 / Number of copies: 1 / Formula: AV0
Molecular weightTheoretical: 1.005188 KDa
Chemical component information

ChemComp-AV0:
Lauryl Maltose Neopentyl Glycol

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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

Concentration6 mg/mL
BufferpH: 7.4 / Details: 20 mM Hepes pH 7.4, 100 mM NaCl, and 0.003% LMNG
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 1073 / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 92000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 37586
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7kp4

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 37296
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 1 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7kp4


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8u5b:
Cryo-EM structure of human claudin-4 complex with Clostridium perfringens enterotoxin C-terminal domain and sFab COP-1

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