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- EMDB-11728: SARS-CoV-2 spike glycoprotein bound to nanobodies (Ty1) -

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Basic information

Entry
Database: EMDB / ID: EMD-11728
TitleSARS-CoV-2 spike glycoprotein bound to nanobodies (Ty1)
Map datahalf map 2
Sample
  • Complex: SARS-CoV-2 spike glycoprotein with bound nanobodies (Ty1)
    • Protein or peptide: SARS-CoV-2 spike glycoprotein
    • Protein or peptide: Nanobody Ty1
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsHallberg BM / Das H
Citation
Journal: Nat Commun / Year: 2020
Title: An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction.
Authors: Leo Hanke / Laura Vidakovics Perez / Daniel J Sheward / Hrishikesh Das / Tim Schulte / Ainhoa Moliner-Morro / Martin Corcoran / Adnane Achour / Gunilla B Karlsson Hedestam / B Martin ...Authors: Leo Hanke / Laura Vidakovics Perez / Daniel J Sheward / Hrishikesh Das / Tim Schulte / Ainhoa Moliner-Morro / Martin Corcoran / Adnane Achour / Gunilla B Karlsson Hedestam / B Martin Hällberg / Ben Murrell / Gerald M McInerney /
Abstract: SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive ...SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. Ty1 binds the RBD with high affinity, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the 'up' and 'down' conformations, sterically hindering RBD-ACE2 binding. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.
#1: Journal: BiorXiv / Year: 2020
Title: An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction
Authors: Hanke L / Vidakovics Perez L / Sheward DJ / Das H / Schulte T / Moliner-Morro A / Corcoran M / Achour A / Karlsson Hedestam G / Hallberg BM / Murrell B / McInerney GM
History
DepositionSep 15, 2020-
Header (metadata) releaseSep 23, 2020-
Map releaseSep 23, 2020-
UpdateAug 24, 2022-
Current statusAug 24, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11728.png

Downloads & links

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Map

FileDownload / File: emd_11728.map.gz / Format: CCP4 / Size: 299.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhalf map 2
Voxel sizeX=Y=Z: 1.02 Å
Density
Contour LevelBy AUTHOR: 0.27 / Movie #1: 0.2
Minimum - Maximum-0.8010464 - 1.6954437
Average (Standard dev.)-0.00032490134 (±0.030576892)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions428428428
Spacing428428428
CellA=B=C: 436.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.021.021.02
M x/y/z428428428
origin x/y/z0.0000.0000.000
length x/y/z436.560436.560436.560
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS428428428
D min/max/mean-0.8011.695-0.000

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Supplemental data

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Mask #1

Fileemd_11728_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: locally sharpened map

Fileemd_11728_half_map_1.map
Annotationlocally sharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_11728_half_map_2.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SARS-CoV-2 spike glycoprotein with bound nanobodies (Ty1)

EntireName: SARS-CoV-2 spike glycoprotein with bound nanobodies (Ty1)
Components
  • Complex: SARS-CoV-2 spike glycoprotein with bound nanobodies (Ty1)
    • Protein or peptide: SARS-CoV-2 spike glycoprotein
    • Protein or peptide: Nanobody Ty1

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Supramolecule #1: SARS-CoV-2 spike glycoprotein with bound nanobodies (Ty1)

SupramoleculeName: SARS-CoV-2 spike glycoprotein with bound nanobodies (Ty1)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 470 KDa

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Macromolecule #1: SARS-CoV-2 spike glycoprotein

MacromoleculeName: SARS-CoV-2 spike glycoprotein / type: protein_or_peptide / ID: 1 / Details: SARS-CoV-2 spike glycoprotein / 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 KPSKRSFIED LLFNKVTLAD AGFIKQYGDC LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFGA GAALQIPFAM QMAYRFNGIG VTQNVLYENQ KLIANQFNSA IGKIQDSLSS TASALGKLQD 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: Nanobody Ty1

MacromoleculeName: Nanobody Ty1 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAQVQLVETG GGLVQPGGSL RLSCAASGFT FSSVYMNWVR QAPGKGPEWV SRISPNSGNI GYTDSVKGRF TISRDNAKNT LYLQMNNLKP EDTALYYCAI GLNLSSSSVR 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

Concentration0.7 mg/mL
BufferpH: 7
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: A 3-ul aliquot of the sample solution was applied 335to glow-discharged CryoMatrix holey grids with amorphous alloy film (Zhenjiang Lehua Technology) in 336a Vitrobot Mk IV (Thermo Fisher ...Details: A 3-ul aliquot of the sample solution was applied 335to glow-discharged CryoMatrix holey grids with amorphous alloy film (Zhenjiang Lehua Technology) in 336a Vitrobot Mk IV (Thermo Fisher Scientific) at 4 degrees and 100% humidity (blot 10 s, blot force 3)..
DetailsSpike trimer (0.7 mg/ml) and Ty1 (1.3 mg/ml) were mixed 334in a 1:8 molar ratio and incubated on ice for 5 minutes.

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 20.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 0.3 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 38.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: PROJECTION MATCHING
Final 3D classificationDetails: 210832
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 210832
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: OTHER

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