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- EMDB-13105: SARS-CoV-2 S (Spike) protein in complex with VHH Re5D06 (multi-bo... -

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Basic information

Entry
Database: EMDB / ID: EMD-13105
TitleSARS-CoV-2 S (Spike) protein in complex with VHH Re5D06 (multi-body-refined RBD-Re5D06 body)
Map datasharpened map
Sample
  • Complex: SARS-CoV-2 HexaPro S (Spike) glycoprotein in complex with neutralizing VHH Re5D06
    • Complex: SARS-CoV-2 HexaPro S (Spike) glycoprotein
      • Protein or peptide: SARS-CoV-2 HexaPro S (Spike) glycoprotein
    • Complex: VHH Re5D06
      • Protein or peptide: VHH Re5D06
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 / membrane fusion / Attachment and Entry / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / 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
Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like ...Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsGuttler T / Aksu M / Dienemann C / Gorlich D
Funding support Germany, 1 items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: 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
DepositionJun 23, 2021-
Header (metadata) releaseAug 11, 2021-
Map releaseAug 11, 2021-
UpdateOct 13, 2021-
Current statusOct 13, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13105.png

Downloads & links

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Map

FileDownload / File: emd_13105.map.gz / Format: CCP4 / Size: 6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.25148052 - 0.4333297
Average (Standard dev.)0.0011576124 (±0.012395878)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions116116116
Spacing116116116
CellA=B=C: 121.799995 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z116116116
origin x/y/z0.0000.0000.000
length x/y/z121.800121.800121.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS116116116
D min/max/mean-0.2510.4330.001

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Supplemental data

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Mask #1

Fileemd_13105_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: unsharpened map

Fileemd_13105_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_13105_half_map_1.map
Annotationhalf map 2
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_13105_half_map_2.map
Annotationhalf map 1
Projections & Slices
AxesZYX

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Sample components

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Entire : SARS-CoV-2 HexaPro S (Spike) glycoprotein in complex with neutral...

EntireName: SARS-CoV-2 HexaPro S (Spike) glycoprotein in complex with neutralizing VHH Re5D06
Components
  • Complex: SARS-CoV-2 HexaPro S (Spike) glycoprotein in complex with neutralizing VHH Re5D06
    • Complex: SARS-CoV-2 HexaPro S (Spike) glycoprotein
      • Protein or peptide: SARS-CoV-2 HexaPro S (Spike) glycoprotein
    • Complex: VHH Re5D06
      • Protein or peptide: VHH Re5D06

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Supramolecule #1: SARS-CoV-2 HexaPro S (Spike) glycoprotein in complex with neutral...

SupramoleculeName: 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

SupramoleculeName: SARS-CoV-2 HexaPro S (Spike) glycoprotein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2 / Location in cell: viral surface
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: Expi293F / Recombinant plasmid: Addgene #154745
Molecular weightTheoretical: 600 KDa

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Supramolecule #3: VHH Re5D06

SupramoleculeName: VHH Re5D06 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Vicugna pacos (alpaca)
Recombinant expressionOrganism: Komagataella pastoris (fungus)
Molecular weightTheoretical: 14.4 kDa/nm

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Macromolecule #1: SARS-CoV-2 HexaPro S (Spike) glycoprotein

MacromoleculeName: 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
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: 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

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Macromolecule #2: VHH Re5D06

MacromoleculeName: VHH Re5D06 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString:
EGSQVQLVES GGGLVQPGGS LRLSCAASGF TLDYYAIGWF RQAPGKEREG VSRIRSSDGS TDYADSVKGR FTISRDNAKN TVYLQMNSLK PEDTAVYYCA YGPLTKYGSS WYWPYEYDYW GQGTQVTVSS

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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

Concentration1.0 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
2.0 mMTris/HCl
150.0 mMsodium chlorideNaCl
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.04 kPa / Details: 15 mA
VitrificationCryogen 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.
DetailsThe 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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm 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 beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 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 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: 4700000
CTF correctionSoftware - Name: Warp (ver. 1.0.9)
Startup modelType of model: OTHER
Details: An ab-initio 3D model was generated from particles of good 2D classes using cryoSPARC (version 2.15).
Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 652015
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.15)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 3.1)
Details: The RBD-nanobody sub-volume (for the RBD-VHH Re5D06 module in the down conformation) was masked for focused 3D classification, which yielded 5 classes. Three classes were selected for ...Details: The RBD-nanobody sub-volume (for the RBD-VHH Re5D06 module in the down conformation) was masked for focused 3D classification, which yielded 5 classes. Three classes were selected for subsequent global and multi-body refinement runs.
FSC plot (resolution estimation)

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