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- EMDB-11053: Cryo-EM density map of nanobody-4C8 bound, human CD9 in complex w... -

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Basic information

Entry
Database: EMDB / ID: EMD-11053
TitleCryo-EM density map of nanobody-4C8 bound, human CD9 in complex with EWI-F-deltaIg1-5
Map dataLocal-resolution filtered, masked cryo-EM density map of the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex, sharpened with B = -1200 A2.
Sample
  • Complex: Nanobody-4C8 bound, human CD9 in complex with EWI-F-deltaIg1-5
    • Complex: CD9 - EWI-F-deltaIg1-5 complex
      • Protein or peptide: CD9
      • Protein or peptide: EWI-F / CD9-partner-1 / FPRP
    • Complex: Nanobody 4C8 molecule 2
      • Protein or peptide: Nanobody 4C8
Function / homology
Function and homology information


Acrosome Reaction and Sperm:Oocyte Membrane Binding / myoblast fusion involved in skeletal muscle regeneration / sperm-egg recognition / fusion of sperm to egg plasma membrane involved in single fertilization / regulation of macrophage migration / paranodal junction assembly / glial cell migration / platelet alpha granule membrane / negative regulation of platelet aggregation / Uptake and function of diphtheria toxin ...Acrosome Reaction and Sperm:Oocyte Membrane Binding / myoblast fusion involved in skeletal muscle regeneration / sperm-egg recognition / fusion of sperm to egg plasma membrane involved in single fertilization / regulation of macrophage migration / paranodal junction assembly / glial cell migration / platelet alpha granule membrane / negative regulation of platelet aggregation / Uptake and function of diphtheria toxin / cellular response to low-density lipoprotein particle stimulus / clathrin-coated endocytic vesicle membrane / receptor internalization / platelet activation / endocytic vesicle membrane / extracellular vesicle / integrin binding / Platelet degranulation / cell population proliferation / cell adhesion / negative regulation of cell population proliferation / external side of plasma membrane / focal adhesion / protein-containing complex / extracellular space / extracellular exosome / membrane / plasma membrane
Similarity search - Function
CD9, extracellular domain / Tetraspanin, conserved site / Transmembrane 4 family signature. / Tetraspanin, animals / Tetraspanin, EC2 domain superfamily / Tetraspanin/Peripherin / Tetraspanin family
Similarity search - Domain/homology
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.6 Å
AuthorsOosterheert W / Gros P
Funding support Netherlands, 2 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)731.015.201 Netherlands
Netherlands Organisation for Scientific Research (NWO)024.002.009 Netherlands
CitationJournal: Life Sci Alliance / Year: 2020
Title: Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F.
Authors: Wout Oosterheert / Katerina T Xenaki / Viviana Neviani / Wouter Pos / Sofia Doulkeridou / Jip Manshande / Nicholas M Pearce / Loes Mj Kroon-Batenburg / Martin Lutz / Paul Mp van Bergen En ...Authors: Wout Oosterheert / Katerina T Xenaki / Viviana Neviani / Wouter Pos / Sofia Doulkeridou / Jip Manshande / Nicholas M Pearce / Loes Mj Kroon-Batenburg / Martin Lutz / Paul Mp van Bergen En Henegouwen / Piet Gros /
Abstract: Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster ...Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into tetraspanin-enriched microdomains is unknown. Here, we present crystal structures of the large extracellular loop of CD9 bound to nanobodies 4C8 and 4E8 and, the cryo-EM structure of 4C8-bound CD9 in complex with its partner EWI-F. CD9-EWI-F displays a tetrameric arrangement with two central EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at loops C and D, which is in agreement with the 4C8 conformation in the CD9-EWI-F complex. The complex varies from nearly twofold symmetric (with the two CD9 copies nearly anti-parallel) to ca. 50° bent arrangements. This flexible arrangement of CD9-EWI-F with potential CD9 homo-dimerization at either end provides a "concatenation model" for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.
History
DepositionMay 19, 2020-
Header (metadata) releaseSep 23, 2020-
Map releaseSep 23, 2020-
UpdateSep 30, 2020-
Current statusSep 30, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.268
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.268
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11053.png

Downloads & links

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Map

FileDownload / File: emd_11053.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal-resolution filtered, masked cryo-EM density map of the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex, sharpened with B = -1200 A2.
Voxel sizeX=Y=Z: 3.0855 Å
Density
Contour LevelBy AUTHOR: 0.268 / Movie #1: 0.268
Minimum - Maximum-0.5952537 - 0.9095872
Average (Standard dev.)0.0033312985 (±0.032967992)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 308.55 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.08553.08553.0855
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z308.550308.550308.550
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-0.5950.9100.003

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Supplemental data

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Mask #1

Fileemd_11053_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Refined, unsharpened cryo-EM density map of the EWI-F%u0394Ig1-5...

Fileemd_11053_additional_1.map
AnnotationRefined, unsharpened cryo-EM density map of the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Local-resolution filtered, masked cryo-EM density map of the...

Fileemd_11053_additional_2.map
AnnotationLocal-resolution filtered, masked cryo-EM density map of the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex, sharpened with B = -750 A2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened cryo-EM density map of the EWI-F%u0394Ig1-5 -...

Fileemd_11053_additional_3.map
AnnotationUnsharpened cryo-EM density map of the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex, subjected to a postprocessing step in Relion.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Class #1 of a 3D classification of the...

Fileemd_11053_additional_4.map
AnnotationClass #1 of a 3D classification of the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex, performed in Relion3.1beta.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Class #2 of a 3D classification of the...

Fileemd_11053_additional_5.map
AnnotationClass #2 of a 3D classification of the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex, performed in Relion3.1beta.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Class #3 of a 3D classification of the...

Fileemd_11053_additional_6.map
AnnotationClass #3 of a 3D classification of the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex, performed in Relion3.1beta.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Class #4 of a 3D classification of the...

Fileemd_11053_additional_7.map
AnnotationClass #4 of a 3D classification of the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex, performed in Relion3.1beta.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map 1 the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex.

Fileemd_11053_half_map_1.map
AnnotationUnfiltered half map 1 the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half map 2 the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex.

Fileemd_11053_half_map_2.map
AnnotationUnfiltered half map 2 the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Nanobody-4C8 bound, human CD9 in complex with EWI-F-deltaIg1-5

EntireName: Nanobody-4C8 bound, human CD9 in complex with EWI-F-deltaIg1-5
Components
  • Complex: Nanobody-4C8 bound, human CD9 in complex with EWI-F-deltaIg1-5
    • Complex: CD9 - EWI-F-deltaIg1-5 complex
      • Protein or peptide: CD9
      • Protein or peptide: EWI-F / CD9-partner-1 / FPRP
    • Complex: Nanobody 4C8 molecule 2
      • Protein or peptide: Nanobody 4C8

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Supramolecule #1: Nanobody-4C8 bound, human CD9 in complex with EWI-F-deltaIg1-5

SupramoleculeName: Nanobody-4C8 bound, human CD9 in complex with EWI-F-deltaIg1-5
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The complex is arranged as a hetero-hexamer: 4C8 - CD9 - EWI-F - EWI-F - CD9 - 4C8
Molecular weightTheoretical: 188 KDa

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Supramolecule #2: CD9 - EWI-F-deltaIg1-5 complex

SupramoleculeName: CD9 - EWI-F-deltaIg1-5 complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Details: The complex was purified from HEK293 GNT1- cells as a hetero-tetramer, arranged as CD9 - EWI-F - EWI-F - CD9.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293 GNT1-
Molecular weightTheoretical: 158 KDa

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Supramolecule #3: Nanobody 4C8 molecule 2

SupramoleculeName: Nanobody 4C8 molecule 2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3
Details: The nanobody was purified from a bacterial expression system.
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant cell: BL-21 Codon Plus (DE3)-RIL / Recombinant plasmid: pHEN6
Molecular weightTheoretical: 30 KDa

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Macromolecule #1: CD9

MacromoleculeName: CD9 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GSWSHPQFEK GSWSHPQFEK GSPVKGGTKC IKYLLFGFNF IFWLAGIAVL AIGLWLRFDS QTKSIFEQET NNNNSSFYTG VYILIGAGAL MMLVGFLGCC GAVQESQCML GLFFGFLLVI FAIEIAAAIW GYSHKDEVIK EVQEFYKDTY NKLKTKDEPQ RETLKAIHYA ...String:
GSWSHPQFEK GSWSHPQFEK GSPVKGGTKC IKYLLFGFNF IFWLAGIAVL AIGLWLRFDS QTKSIFEQET NNNNSSFYTG VYILIGAGAL MMLVGFLGCC GAVQESQCML GLFFGFLLVI FAIEIAAAIW GYSHKDEVIK EVQEFYKDTY NKLKTKDEPQ RETLKAIHYA LNCCGLAGGV EQFISDICPK KDVLETFTVK SCPDAIKEVF DNKFHIIGAV GIGIAVVMIF GMIFSMILCC AIRRNREMVA AA

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Macromolecule #2: EWI-F / CD9-partner-1 / FPRP

MacromoleculeName: EWI-F / CD9-partner-1 / FPRP / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGSTSGPIFN ASVHSDTPSV IRGDLIKLFC IITVEGAALD PDDMAFDVSW FAVHSFGLDK APVLLSSLDR KGIVTTSRRD WKSDLSLERV SVLEFLLQVH GSEDQDFGNY YCSVTPWVKS PTGSWQKEAE IHSKPVFITV KMDVLNAFKY PLLIGVGLST VIGLLSCLIG ...String:
MGSTSGPIFN ASVHSDTPSV IRGDLIKLFC IITVEGAALD PDDMAFDVSW FAVHSFGLDK APVLLSSLDR KGIVTTSRRD WKSDLSLERV SVLEFLLQVH GSEDQDFGNY YCSVTPWVKS PTGSWQKEAE IHSKPVFITV KMDVLNAFKY PLLIGVGLST VIGLLSCLIG YCSSHWCCKK EVQETRRERR RLMSMEMDAA AHHHHHH

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Macromolecule #3: Nanobody 4C8

MacromoleculeName: Nanobody 4C8 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
EVQLVESGGG LVQAGGSLRL SCAASGRTFS DYVMGWFRQA PGKERTFVAR IGWSGDLTYY ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAIYYCAADE RWGTGGKFDY WGQGTQVTVS SHGSGLVPRG SGGGHHHHHH

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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

Concentration3.0 mg/mL
BufferpH: 7.8
Component:
ConcentrationNameFormula
50.0 mMTris
150.0 mMsodium chlorideNaClSodium chloride
0.08 % (w/v)digitonin

Details: 50 mM Tris pH 7.8, 150 mM NaCl, 0.08 % (w/v) digitonin.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
Details: The blotting was performed at 20 degrees Celsius for four seconds with blot force 1..
DetailsThe CD9 - EWI-F-deltaIg1-5 was purified from HEK293 GNT1- cells. Nanobody 4C8 was added to the thew complex prior to size-exclusion chromatography.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-36 / Number grids imaged: 1 / Number real images: 10724 / Average exposure time: 7.2 sec. / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1148718 / Details: particles were picked in EMAN2
CTF correctionSoftware - Name: Gctf (ver. 1.18)
Startup modelType of model: OTHER / Details: Original model was obtained in Relion.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1beta) / Details: Performed in Relion
Final 3D classificationNumber classes: 20 / Avg.num./class: 34400 / Software - Name: RELION (ver. 3.1beta)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1beta)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1beta)
Details: 354272 particles were 3D-auto refined using a mask and solvent flattening fourier shell correlations (FSCs), which yielded a map at a global resolution of 8.6 Angstrom based on the gold- ...Details: 354272 particles were 3D-auto refined using a mask and solvent flattening fourier shell correlations (FSCs), which yielded a map at a global resolution of 8.6 Angstrom based on the gold-standard FSC = 0.143 criterion. This map was sharpened with a B-factor of -1200 A2 and filtered based on local-resolution in Relion.
Number images used: 354272
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6z20

chain_id: A

6z20

chain_id: B

5tcx

chain_id: A, residue_range: 6-116

5tcx

chain_id: A, residue_range: 201-232

1f3r

chain_id: B, residue_range: 9-122

1f3r

chain_id: B, residue_range: 9-122

5eh4

chain_id: A, residue_range: 70-98

5eh4

chain_id: A, residue_range: 70-98
DetailsModels were rigid-body fitted into the map using UCSF Chimera and not refined.

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