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- EMDB-22514: SARS CoV2 Spike ectodomain with engineered trimerized VH binder -

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Basic information

Entry
Database: EMDB / ID: EMD-22514
TitleSARS CoV2 Spike ectodomain with engineered trimerized VH binder
Map data
SampleComplex of SARS CoV2 Spike ectodomain with engineered trimerized VH domain
  • engineered trimerized VH domain
  • SARS CoV2 Spike ectodomain
  • autonomous human heavy chain variable domain
  • Spike glycoproteinPeplomer
Function / homology
Function and homology information


suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / host cell surface receptor binding / endocytosis involved in viral entry into host cell / fusion of virus membrane with host plasma membrane / suppression by virus of host type I interferon-mediated signaling pathway / viral entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope ...suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / host cell surface receptor binding / endocytosis involved in viral entry into host cell / fusion of virus membrane with host plasma membrane / suppression by virus of host type I interferon-mediated signaling pathway / viral entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope / go:0009405: / host cell plasma membrane / virion membrane / integral component of membrane / identical protein binding
Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike glycoprotein, heptad repeat 2, coronavirus / Spike glycoprotein S2, coronavirus / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Spike receptor binding domain superfamily, coronavirus / Spike glycoprotein S2 superfamily, coronavirus / Coronavirus spike glycoprotein S1, C-terminal
Spike glycoprotein
Biological speciesHomo sapiens (human) / Severe acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsQCRG Structural Biology Consortium
Funding support United States, 4 items
OrganizationGrant numberCountry
Defense Advanced Research Projects Agency (DARPA)HR0011-19-2-0020 United States
FastGrants United States
Quantitative Biosciences Institute United States
Laboratory for Genomics Research (LGR)Excellence in Research Award (ERA) United States
Citation
Journal: Nat Chem Biol / Year: 2021
Title: Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2.
Authors: Colton J Bracken / Shion A Lim / Paige Solomon / Nicholas J Rettko / Duy P Nguyen / Beth Shoshana Zha / Kaitlin Schaefer / James R Byrnes / Jie Zhou / Irene Lui / Jia Liu / Katarina Pance / ...Authors: Colton J Bracken / Shion A Lim / Paige Solomon / Nicholas J Rettko / Duy P Nguyen / Beth Shoshana Zha / Kaitlin Schaefer / James R Byrnes / Jie Zhou / Irene Lui / Jia Liu / Katarina Pance / / Xin X Zhou / Kevin K Leung / James A Wells /
Abstract: Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy ...Neutralizing agents against SARS-CoV-2 are urgently needed for the treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domains toward neutralizing epitopes. We constructed a VH-phage library and targeted the angiotensin-converting enzyme 2 (ACE2) binding interface of the SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified VH binders to two non-overlapping epitopes and further assembled these into multivalent and bi-paratopic formats. These VH constructs showed increased affinity to Spike (up to 600-fold) and neutralization potency (up to 1,400-fold) on pseudotyped SARS-CoV-2 virus when compared to standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with a half-maximal inhibitory concentration (IC) of 4.0 nM (180 ng ml). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain engaging an RBD at the ACE2 binding site, confirming our original design strategy.
#1: Journal: bioRxiv / Year: 2020
Title: Bi-paratopic and multivalent human VH domains neutralize SARS-CoV-2 by targeting distinct epitopes within the ACE2 binding interface of Spike.
Authors: Colton J Bracken / Shion A Lim / Paige Solomon / Nicholas J Rettko / Duy P Nguyen / Beth Shoshana Zha / Kaitlin Schaefer / James R Byrnes / Jie Zhou / Irene Lui / Jia Liu / Katarina Pance / ...Authors: Colton J Bracken / Shion A Lim / Paige Solomon / Nicholas J Rettko / Duy P Nguyen / Beth Shoshana Zha / Kaitlin Schaefer / James R Byrnes / Jie Zhou / Irene Lui / Jia Liu / Katarina Pance / / Xin X Zhou / Kevin K Leung / James A Wells /
Abstract: Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) ...Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domain binders with high affinity toward neutralizing epitopes without the need for high-resolution structural information. We constructed a VH-phage library and targeted a known neutralizing site, the angiotensin-converting enzyme 2 (ACE2) binding interface of the trimeric SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified 85 unique VH binders to two non-overlapping epitopes within the ACE2 binding site on Spike-RBD. This enabled us to systematically link these VH domains into multivalent and bi-paratopic formats. These multivalent and bi-paratopic VH constructs showed a marked increase in affinity to Spike (up to 600-fold) and neutralization potency (up to 1400-fold) on pseudotyped SARS-CoV-2 virus when compared to the standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with half-minimal inhibitory concentration (IC ) of 4.0 nM (180 ng/mL). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain bound an RBD at the ACE2 binding site, explaining its increased neutralization potency and confirming our original design strategy. Our results demonstrate that targeted selection and engineering campaigns using a VH-phage library can enable rapid assembly of highly avid and potent molecules towards therapeutically important protein interfaces.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionAug 25, 2020-
Header (metadata) releaseOct 7, 2020-
Map releaseOct 7, 2020-
UpdateDec 30, 2020-
Current statusDec 30, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jwb
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22514.png

Downloads & links

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Map

FileDownload / File: emd_22514.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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.1
Minimum - Maximum-0.14129888 - 0.6405934
Average (Standard dev.)0.00042388256 (±0.022483723)
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/NZ304304304
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-0.1410.6410.000

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Supplemental data

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Additional map: High resolution reconstruction (3.2A) filtered by the FSC...

Fileemd_22514_additional_1.map
AnnotationHigh resolution reconstruction (3.2A) filtered by the FSC and sharpened. Shows high resolution features but the VH and RBD regions are diffuse.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire Complex of SARS CoV2 Spike ectodomain with engineered trimerized ...

EntireName: Complex of SARS CoV2 Spike ectodomain with engineered trimerized VH domain
Number of components: 5

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Component #1: protein, Complex of SARS CoV2 Spike ectodomain with engineered tr...

ProteinName: Complex of SARS CoV2 Spike ectodomain with engineered trimerized VH domain
Recombinant expression: No
MassTheoretical: 445 kDa

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Component #2: protein, engineered trimerized VH domain

ProteinName: engineered trimerized VH domain / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli K-12 (bacteria)

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Component #3: protein, SARS CoV2 Spike ectodomain

ProteinName: SARS CoV2 Spike ectodomain / Recombinant expression: No
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Cricetulus griseus (Chinese hamster)

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Component #4: protein, autonomous human heavy chain variable domain

ProteinName: autonomous human heavy chain variable domain / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 44.264402 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli K-12 (bacteria)

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Component #5: protein, Spike glycoprotein

ProteinName: Spike glycoproteinPeplomer / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 133.75325 kDa
SourceSpecies: Severe acute respiratory syndrome coronavirus 2
Source (engineered)Expression System: Cricetulus griseus (Chinese hamster)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.9 mg/mL / Buffer solution: 20 mM HEPES, pH 8, 200 mM NaCl / pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / 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: 78 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: OTHER

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 21000
3D reconstruction #1Resolution: 6 Å / Resolution method: OTHER
Details: This reconstruction is a Gaussian low pass filtered map of a 3.2A gold standard FSC map, to better account for the VH density. The model was fit and relaxed into this map.
3D reconstruction #2Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Same reconstruction as above but filtered based on the gold standard FSC to 3.2A. Shows high resolution features but density for the low occupancy VH is poorly resolved.
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Target criteria: cross correlation
Input PDB model: 6X2B, 4G80

6x2b

4g80


Chain ID: A
Output model

7jwb

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