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- EMDB-22911: SARS-CoV-2 Spike bound to Nb11 in open conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-22911
TitleSARS-CoV-2 Spike bound to Nb11 in open conformation
Map data
SampleComplex of Spike S2P glycoprotein with synthetic nanobody Nb11:
Spike S2P glycoprotein / synthetic nanobody Nb11
Biological speciesSevere acute respiratory syndrome coronavirus 2 / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.74 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 / Liu Y / 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 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 ...The 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. Cryogenic electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) 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-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.012
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_22911.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 512 pix.
= 427.008 Å
0.83 Å/pix.
x 512 pix.
= 427.008 Å
0.83 Å/pix.
x 512 pix.
= 427.008 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.834 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.012
Minimum - Maximum-0.023753475 - 0.059988003
Average (Standard dev.)0.0000294524 (±0.0017544556)
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
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-0.0240.0600.000

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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
Number of components: 3

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Component #1: protein, Complex of Spike S2P glycoprotein with synthetic nanobod...

ProteinName: Complex of Spike S2P glycoprotein with synthetic nanobody Nb11
Recombinant expression: No

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Component #2: protein, Spike S2P glycoprotein

ProteinName: Spike S2P glycoprotein / Recombinant expression: No
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Cricetulus griseus (Chinese hamster) / Cell of expression system: ExpiCHO

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Component #3: protein, synthetic nanobody Nb11

ProteinName: synthetic nanobody Nb11 / Recombinant expression: No
SourceSpecies: synthetic construct (others)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 66 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 105000 X (nominal) / Imaging mode: BRIGHT FIELD / Energy filter: GIF Bioquantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: OTHER

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Image acquisition

Image acquisitionNumber of digital images: 4103

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 21570
3D reconstructionSoftware: RELION
CTF correction: Performed at reconstruction, as is standard for cryoSPARC
Resolution: 3.74 Å / Resolution method: FSC 0.143 CUT-OFF

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