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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-4
    • 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: 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, 2024-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.88 Å/pix.
x 392 pix.
= 346.528 Å		0.88 Å/pix.
x 392 pix.
= 346.528 Å		0.88 Å/pix.
x 392 pix.
= 346.528 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.884 Å
Density

Histogram

Histogram (log scale)
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

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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-4
    • 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 mMNaClNaCl

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
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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

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Image processing

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

7dtm

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
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.1)
Final 3D classificationNumber classes: 1 / Software - Name: cryoSPARC (ver. 4.4.1)
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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