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Yorodumi- EMDB-11053: Cryo-EM density map of nanobody-4C8 bound, human CD9 in complex w... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-11053 | |||||||||
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Title | Cryo-EM density map of nanobody-4C8 bound, human CD9 in complex with EWI-F-deltaIg1-5 | |||||||||
Map data | Local-resolution filtered, masked cryo-EM density map of the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex, sharpened with B = -1200 A2. | |||||||||
Sample |
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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 | |||||||||
Biological species | Homo sapiens (human) / Lama glama (llama) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 8.6 Å | |||||||||
Authors | Oosterheert W / Gros P | |||||||||
Funding support | Netherlands, 2 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_11053.map.gz | 2.4 MB | EMDB map data format | |
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Header (meta data) | emd-11053-v30.xml emd-11053.xml | 40.7 KB 40.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_11053_fsc.xml | 3.7 KB | Display | FSC data file |
Images | emd_11053.png | 56.9 KB | ||
Masks | emd_11053_msk_1.map | 3.8 MB | Mask map | |
Others | emd_11053_additional_1.map.gz emd_11053_additional_2.map.gz emd_11053_additional_3.map.gz emd_11053_additional_4.map.gz emd_11053_additional_5.map.gz emd_11053_additional_6.map.gz emd_11053_additional_7.map.gz emd_11053_half_map_1.map.gz emd_11053_half_map_2.map.gz | 2.7 MB 2.4 MB 411 KB 1.1 MB 1.1 MB 1.1 MB 1.1 MB 2.8 MB 2.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-11053 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-11053 | HTTPS FTP |
-Validation report
Summary document | emd_11053_validation.pdf.gz | 307.4 KB | Display | EMDB validaton report |
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Full document | emd_11053_full_validation.pdf.gz | 306.5 KB | Display | |
Data in XML | emd_11053_validation.xml.gz | 9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-11053 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-11053 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_11053.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Local-resolution filtered, masked cryo-EM density map of the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex, sharpened with B = -1200 A2. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.0855 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
+Mask #1
+Additional map: Refined, unsharpened cryo-EM density map of the EWI-F%u0394Ig1-5...
+Additional map: Local-resolution filtered, masked cryo-EM density map of the...
+Additional map: Unsharpened cryo-EM density map of the EWI-F%u0394Ig1-5 -...
+Additional map: Class #1 of a 3D classification of the...
+Additional map: Class #2 of a 3D classification of the...
+Additional map: Class #3 of a 3D classification of the...
+Additional map: Class #4 of a 3D classification of the...
+Half map: Unfiltered half map 1 the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex.
+Half map: Unfiltered half map 2 the EWI-F%u0394Ig1-5 - CD9 - 4C8 complex.
-Sample components
-Entire : Nanobody-4C8 bound, human CD9 in complex with EWI-F-deltaIg1-5
Entire | Name: Nanobody-4C8 bound, human CD9 in complex with EWI-F-deltaIg1-5 |
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Components |
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-Supramolecule #1: Nanobody-4C8 bound, human CD9 in complex with EWI-F-deltaIg1-5
Supramolecule | Name: 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 |
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Molecular weight | Theoretical: 188 KDa |
-Supramolecule #2: CD9 - EWI-F-deltaIg1-5 complex
Supramolecule | Name: 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. |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK293 GNT1- |
Molecular weight | Theoretical: 158 KDa |
-Supramolecule #3: Nanobody 4C8 molecule 2
Supramolecule | Name: Nanobody 4C8 molecule 2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3 Details: The nanobody was purified from a bacterial expression system. |
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Source (natural) | Organism: Lama glama (llama) |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant cell: BL-21 Codon Plus (DE3)-RIL / Recombinant plasmid: pHEN6 |
Molecular weight | Theoretical: 30 KDa |
-Macromolecule #1: CD9
Macromolecule | Name: CD9 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: 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 |
-Macromolecule #2: EWI-F / CD9-partner-1 / FPRP
Macromolecule | Name: EWI-F / CD9-partner-1 / FPRP / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: 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 |
-Macromolecule #3: Nanobody 4C8
Macromolecule | Name: Nanobody 4C8 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Lama glama (llama) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: EVQLVESGGG LVQAGGSLRL SCAASGRTFS DYVMGWFRQA PGKERTFVAR IGWSGDLTYY ADSVKGRFTI SRDNAKNTVY LQMNSLKPED TAIYYCAADE RWGTGGKFDY WGQGTQVTVS SHGSGLVPRG SGGGHHHHHH |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 3.0 mg/mL | ||||||||||||
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Buffer | pH: 7.8 Component:
Details: 50 mM Tris pH 7.8, 150 mM NaCl, 0.08 % (w/v) digitonin. | ||||||||||||
Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE | ||||||||||||
Vitrification | Cryogen 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.. | ||||||||||||
Details | The CD9 - EWI-F-deltaIg1-5 was purified from HEK293 GNT1- cells. Nanobody 4C8 was added to the thew complex prior to size-exclusion chromatography. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV |
Image recording | Film 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 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model |
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Details | Models were rigid-body fitted into the map using UCSF Chimera and not refined. |