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- EMDB-22909: SARS-CoV-2 Spike bound to Nb11 in closed conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-22909
TitleSARS-CoV-2 Spike bound to Nb11 in closed conformation
Map data
Sample
  • Complex: Complex of Spike S2P glycoprotein with synthetic nanobody Nb11
    • Complex: Spike S2P glycoprotein
    • Complex: synthetic nanobody Nb11
Biological speciesSevere acute respiratory syndrome coronavirus 2 / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.18 Å
AuthorsSchoof MS / Faust BF / Saunders RA / Sangwan S / Rezelj V / Hoppe N / Boone M / Billesboelle CB / Puchades C / Azumaya CM ...Schoof MS / Faust BF / Saunders RA / Sangwan S / Rezelj V / Hoppe N / Boone M / Billesboelle CB / Puchades C / Azumaya CM / Kratochvil HT / Zimanyi M / Desphande I / Liang J / Dickinson S / Nguyen HC / Chio CM / Merz GE / Thompson MC / Diwanji D / Schaefer K / Anand AA / Dobzinski N / Zha BS / Simoneau CR / Leon K / White KM / Chio US / Gupta M / Jin M / Li F / Liu Y / Zhang K / Bulkley D / Sun M / Smith AM / Rizo AN / Moss F / Brilot AF / Pourmal S / Trenker R / Pospiech T / Gupta S / Barsi-Rhyne B / Belyy V / Barile-Hill AW / Nock S / Krogan NJ / Ralston CY / Swaney DL / Garcia-Sastre A / Ott M / Vignuzzi M / Walter P / Manglik A / QCRG Structural Biology Consortium
Funding support United States, 1 items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Science / Year: 2020
Title: An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike.
Authors: Michael Schoof / Bryan Faust / Reuben A Saunders / Smriti Sangwan / Veronica Rezelj / Nick Hoppe / Morgane Boone / Christian B Billesbølle / Cristina Puchades / Caleigh M Azumaya / Huong T ...Authors: Michael Schoof / Bryan Faust / Reuben A Saunders / Smriti Sangwan / Veronica Rezelj / Nick Hoppe / Morgane Boone / Christian B Billesbølle / Cristina Puchades / Caleigh M Azumaya / Huong T Kratochvil / Marcell Zimanyi / Ishan Deshpande / Jiahao Liang / Sasha Dickinson / Henry C Nguyen / Cynthia M Chio / Gregory E Merz / Michael C Thompson / Devan Diwanji / Kaitlin Schaefer / Aditya A Anand / Niv Dobzinski / Beth Shoshana Zha / Camille R Simoneau / Kristoffer Leon / Kris M White / Un Seng Chio / Meghna Gupta / Mingliang Jin / Fei Li / Yanxin Liu / Kaihua Zhang / David Bulkley / Ming Sun / Amber M Smith / Alexandrea N Rizo / Frank Moss / Axel F Brilot / Sergei Pourmal / Raphael Trenker / Thomas Pospiech / Sayan Gupta / Benjamin Barsi-Rhyne / Vladislav Belyy / Andrew W Barile-Hill / Silke Nock / Yuwei Liu / Nevan J Krogan / Corie Y Ralston / Danielle L Swaney / Adolfo García-Sastre / Melanie Ott / Marco Vignuzzi / / Peter Walter / Aashish Manglik /
Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). ...The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.
History
DepositionOct 27, 2020-
Header (metadata) releaseNov 11, 2020-
Map releaseNov 11, 2020-
UpdateApr 21, 2021-
Current statusApr 21, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22909.png

Downloads & links

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Map

FileDownload / File: emd_22909.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.834 Å
Density
Contour LevelBy AUTHOR: 0.009 / Movie #1: 0.01
Minimum - Maximum-0.01607592 - 0.046804536
Average (Standard dev.)5.9433463e-05 (±0.0015558585)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 427.008 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8340.8340.834
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z427.008427.008427.008
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-0.0160.0470.000

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Supplemental data

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Sample components

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Entire : Complex of Spike S2P glycoprotein with synthetic nanobody Nb11

EntireName: Complex of Spike S2P glycoprotein with synthetic nanobody Nb11
Components
  • Complex: Complex of Spike S2P glycoprotein with synthetic nanobody Nb11
    • Complex: Spike S2P glycoprotein
    • Complex: synthetic nanobody Nb11

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Supramolecule #1: Complex of Spike S2P glycoprotein with synthetic nanobody Nb11

SupramoleculeName: Complex of Spike S2P glycoprotein with synthetic nanobody Nb11
type: complex / ID: 1 / Parent: 0

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Supramolecule #2: Spike S2P glycoprotein

SupramoleculeName: Spike S2P glycoprotein / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster) / Recombinant cell: ExpiCHO

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Supramolecule #3: synthetic nanobody Nb11

SupramoleculeName: synthetic nanobody Nb11 / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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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

BufferpH: 8
GridModel: Quantifoil / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 4103 / Average electron dose: 66.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1204855
CTF correctionSoftware - Name: cryoSPARC (ver. 2.15.0)
Details: Performed at reconstruction, as is standard for cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.18 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.0) / Number images used: 27611

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