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Yorodumi- EMDB-13107: SARS-CoV-2 S (Spike) protein in complex with VHH Re5D06 (all RBDs up) -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13107 | |||||||||
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Title | SARS-CoV-2 S (Spike) protein in complex with VHH Re5D06 (all RBDs up) | |||||||||
Map data | unsharpened map | |||||||||
Sample |
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Function / homology | Function and homology information Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell ...Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated endocytosis of virus by host cell / Attachment and Entry / membrane fusion / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / symbiont-mediated suppression of host innate immune response / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / identical protein binding / membrane / plasma membrane Similarity search - Function | |||||||||
Biological species | Severe acute respiratory syndrome coronavirus 2 / Vicugna pacos (alpaca) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.8 Å | |||||||||
Authors | Guttler T / Aksu M / Dienemann C / Gorlich D | |||||||||
Funding support | Germany, 1 items
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Citation | Journal: EMBO J / Year: 2021 Title: Neutralization of SARS-CoV-2 by highly potent, hyperthermostable, and mutation-tolerant nanobodies. Authors: Thomas Güttler / Metin Aksu / Antje Dickmanns / Kim M Stegmann / Kathrin Gregor / Renate Rees / Waltraud Taxer / Oleh Rymarenko / Jürgen Schünemann / Christian Dienemann / Philip Gunkel / ...Authors: Thomas Güttler / Metin Aksu / Antje Dickmanns / Kim M Stegmann / Kathrin Gregor / Renate Rees / Waltraud Taxer / Oleh Rymarenko / Jürgen Schünemann / Christian Dienemann / Philip Gunkel / Bianka Mussil / Jens Krull / Ulrike Teichmann / Uwe Groß / Volker C Cordes / Matthias Dobbelstein / Dirk Görlich / Abstract: Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) ...Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17-50 pM concentration (0.2-0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low-picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such "fold-promoting" nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations. | |||||||||
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_13107.map.gz | 232.5 MB | EMDB map data format | |
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Header (meta data) | emd-13107-v30.xml emd-13107.xml | 23 KB 23 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_13107_fsc.xml | 15 KB | Display | FSC data file |
Images | emd_13107.png | 114.3 KB | ||
Masks | emd_13107_msk_1.map | 290.8 MB | Mask map | |
Others | emd_13107_half_map_1.map.gz emd_13107_half_map_2.map.gz | 233.9 MB 233.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13107 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13107 | HTTPS FTP |
-Validation report
Summary document | emd_13107_validation.pdf.gz | 447.6 KB | Display | EMDB validaton report |
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Full document | emd_13107_full_validation.pdf.gz | 447.1 KB | Display | |
Data in XML | emd_13107_validation.xml.gz | 23 KB | Display | |
Data in CIF | emd_13107_validation.cif.gz | 30 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13107 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13107 | 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_13107.map.gz / Format: CCP4 / Size: 290.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | unsharpened map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.05 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_13107_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: half map 1
File | emd_13107_half_map_1.map | ||||||||||||
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Annotation | half map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map 2
File | emd_13107_half_map_2.map | ||||||||||||
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Annotation | half map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : SARS-CoV-2 HexaPro S (Spike) glycoprotein in complex with neutral...
Entire | Name: SARS-CoV-2 HexaPro S (Spike) glycoprotein in complex with neutralizing VHH Re5D06 |
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Components |
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-Supramolecule #1: SARS-CoV-2 HexaPro S (Spike) glycoprotein in complex with neutral...
Supramolecule | Name: SARS-CoV-2 HexaPro S (Spike) glycoprotein in complex with neutralizing VHH Re5D06 type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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-Supramolecule #2: SARS-CoV-2 HexaPro S (Spike) glycoprotein
Supramolecule | Name: SARS-CoV-2 HexaPro S (Spike) glycoprotein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 / Location in cell: viral surface |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant strain: Expi293F / Recombinant plasmid: Addgene #154745 |
Molecular weight | Theoretical: 600 KDa |
-Supramolecule #3: VHH Re5D06
Supramolecule | Name: VHH Re5D06 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
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Source (natural) | Organism: Vicugna pacos (alpaca) |
Recombinant expression | Organism: Komagataella pastoris (fungus) |
Molecular weight | Theoretical: 14.4 kDa/nm |
-Macromolecule #1: SARS-CoV-2 HexaPro S (Spike) glycoprotein
Macromolecule | Name: SARS-CoV-2 HexaPro S (Spike) glycoprotein / type: protein_or_peptide / ID: 1 Details: Spike ectodomain (1-1208) with six exchanges to proline (F817P, A892P, A899P, A942P, K986P, V986P), a GSAS substitution at the furin cleavage site (residues 682-685), a C-terminal T4 foldon ...Details: Spike ectodomain (1-1208) with six exchanges to proline (F817P, A892P, A899P, A942P, K986P, V986P), a GSAS substitution at the furin cleavage site (residues 682-685), a C-terminal T4 foldon trimerization domain, followed by a HRV 3C protease site, a His8-tag and a Twin-Strep tag Enantiomer: LEVO |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY ...String: MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY VSQPFLMDLE GKQGNFKNLR EFVFKNIDGY FKIYSKHTPI NLVRDLPQGF SALEPLVDLP IGINITRFQT LLALHRSYLT PGDSSSGWTA GAAAYYVGYL QPRTFLLKYN ENGTITDAVD CALDPLSETK CTLKSFTVEK GIYQTSNFRV QPTESIVRFP NITNLCPFGE VFNATRFASV YAWNRKRISN CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV VVLSFELLHA PATVCGPKKS TNLVKNKCVN FNFNGLTGTG VLTESNKKFL PFQQFGRDIA DTTDAVRDPQ TLEILDITPC SFGGVSVITP GTNTSNQVAV LYQDVNCTEV PVAIHADQLT PTWRVYSTGS NVFQTRAGCL IGAEHVNNSY ECDIPIGAGI CASYQTQTNS PGSASSVASQ SIIAYTMSLG AENSVAYSNN SIAIPTNFTI SVTTEILPVS MTKTSVDCTM YICGDSTECS NLLLQYGSFC TQLNRALTGI AVEQDKNTQE VFAQVKQIYK TPPIKDFGGF NFSQILPDPS KPSKRSPIED LLFNKVTLAD AGFIKQYGDC LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFGA GPALQIPFPM QMAYRFNGIG VTQNVLYENQ KLIANQFNSA IGKIQDSLSS TPSALGKLQD VVNQNAQALN TLVKQLSSNF GAISSVLNDI LSRLDPPEAE VQIDRLITGR LQSLQTYVTQ QLIRAAEIRA SANLAATKMS ECVLGQSKRV DFCGKGYHLM SFPQSAPHGV VFLHVTYVPA QEKNFTTAPA ICHDGKAHFP REGVFVSNGT HWFVTQRNFY EPQIITTDNT FVSGNCDVVI GIVNNTVYDP LQPELDSFKE ELDKYFKNHT SPDVDLGDIS GINASVVNIQ KEIDRLNEVA KNLNESLIDL QELGKYEQGS GYIPEAPRDG QAYVRKDGEW VLLSTFLGRS LEVLFQGPGH HHHHHHHSAW SHPQFEKGGG SGGGGSGGSA WSHPQFEK |
-Macromolecule #2: VHH Re5D06
Macromolecule | Name: VHH Re5D06 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Vicugna pacos (alpaca) |
Recombinant expression | Organism: Komagataella pastoris (fungus) |
Sequence | String: EGSQVQLVES GGGLVQPGGS LRLSCAASGF TLDYYAIGWF RQAPGKEREG VSRIRSSDGS TDYADSVKGR FTISRDNAKN TVYLQMNSLK PEDTAVYYCA YGPLTKYGSS WYWPYEYDYW GQGTQVTVSS |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.0 mg/mL | |||||||||
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Buffer | pH: 8 Component:
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Grid | Model: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.04 kPa / Details: 15 mA | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV Details: sample volume: 3.0 microliters blotting time: 4 s blot force setting: 5. | |||||||||
Details | The Spike protein was mixed with a 9-fold molar excess of VHH Re5D06 and purified by size exclusion chromatography (Superose 6 Increase 3.2/300, Cytiva). The peak eluate fraction (1 mg/ml) was immediately applied to a freshly glow-discharged grid. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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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 K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Digitization - Sampling interval: 5.0 µm / Number grids imaged: 1 / Number real images: 15636 / Average exposure time: 2.0 sec. / Average electron dose: 39.91 e/Å2 Details: counting mode (non-super-resolution) 4 images per hole (beam-image shift) |
Electron beam | Acceleration voltage: 300 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: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |