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- EMDB-22947: Negative stain electron microscopy reconstruction of non-neutrali... -

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Entry
Database: EMDB / ID: EMD-22947
TitleNegative stain electron microscopy reconstruction of non-neutralizing NTD-directed antibody Fab in complex with SARS-CoV-2 spike ectodomain in the 1-RBD-up state
Map dataFinal sharpened map of non-neutralizing NTD-directed antibody Fab in complex with SARS-CoV-2 spike ectodomain in the 1-RBD-up state
Sample
  • Complex: Complex of RBD-directed Fab DH1042 with hexapro SARS-CoV-2 spike ectodomain
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / negative staining / Resolution: 17.5 Å
AuthorsEdwards RJ / Mansouri K
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI145687 United States
CitationJournal: bioRxiv / Year: 2021
Title: The functions of SARS-CoV-2 neutralizing and infection-enhancing antibodies in vitro and in mice and nonhuman primates.
Authors: Dapeng Li / Robert J Edwards / Kartik Manne / David R Martinez / Alexandra Schäfer / S Munir Alam / Kevin Wiehe / Xiaozhi Lu / Robert Parks / Laura L Sutherland / Thomas H Oguin / Charlene ...Authors: Dapeng Li / Robert J Edwards / Kartik Manne / David R Martinez / Alexandra Schäfer / S Munir Alam / Kevin Wiehe / Xiaozhi Lu / Robert Parks / Laura L Sutherland / Thomas H Oguin / Charlene McDanal / Lautaro G Perez / Katayoun Mansouri / Sophie M C Gobeil / Katarzyna Janowska / Victoria Stalls / Megan Kopp / Fangping Cai / Esther Lee / Andrew Foulger / Giovanna E Hernandez / Aja Sanzone / Kedamawit Tilahun / Chuancang Jiang / Longping V Tse / Kevin W Bock / Mahnaz Minai / Bianca M Nagata / Kenneth Cronin / Victoria Gee-Lai / Margaret Deyton / Maggie Barr / Tarra Von Holle / Andrew N Macintyre / Erica Stover / Jared Feldman / Blake M Hauser / Timothy M Caradonna / Trevor D Scobey / Wes Rountree / Yunfei Wang / M Anthony Moody / Derek W Cain / C Todd DeMarco / ThomasN Denny / Christopher W Woods / Elizabeth W Petzold / Aaron G Schmidt / I-Ting Teng / Tongqing Zhou / Peter D Kwong / John R Mascola / Barney S Graham / Ian N Moore / Robert Seder / Hanne Andersen / Mark G Lewis / David C Montefiori / Gregory D Sempowski / Ralph S Baric / Priyamvada Acharya / Barton F Haynes / Kevin O Saunders
Abstract: SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor- ...SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection , while five non-neutralizing NTD antibodies mediated FcγR-independent infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Nonetheless, three of 31 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while antibody-enhanced infection does not necessarily herald enhanced infection , increased lung inflammation can occur in SARS-CoV-2 antibody-infused macaques.
History
DepositionNov 4, 2020-
Header (metadata) releaseNov 18, 2020-
Map releaseNov 18, 2020-
UpdateSep 1, 2021-
Current statusSep 1, 2021Processing site: RCSB / 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
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22947.png

Downloads & links

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Map

FileDownload / File: emd_22947.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal sharpened map of non-neutralizing NTD-directed antibody Fab in complex with SARS-CoV-2 spike ectodomain in the 1-RBD-up state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.02 Å/pix.
x 96 pix.
= 385.92 Å		4.02 Å/pix.
x 96 pix.
= 385.92 Å		4.02 Å/pix.
x 96 pix.
= 385.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.02 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.07278391 - 0.14631686
Average (Standard dev.)0.00080358644 (±0.013131316)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 385.91998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.024.024.02
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z385.920385.920385.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS969696
D min/max/mean-0.0730.1460.001

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Supplemental data

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Mask #1

Fileemd_22947_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map of non-neutralizing NTD-directed antibody Fab in...

Fileemd_22947_additional_1.map
AnnotationUnsharpened map of non-neutralizing NTD-directed antibody Fab in complex with SARS-CoV-2 spike ectodomain in the 1-RBD-up state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1 of non-neutralizing NTD-directed antibody Fab in...

Fileemd_22947_half_map_1.map
AnnotationHalf-map 1 of non-neutralizing NTD-directed antibody Fab in complex with SARS-CoV-2 spike ectodomain in the 1-RBD-up state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2 of non-neutralizing NTD-directed antibody Fab in...

Fileemd_22947_half_map_2.map
AnnotationHalf-map 2 of non-neutralizing NTD-directed antibody Fab in complex with SARS-CoV-2 spike ectodomain in the 1-RBD-up state
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 RBD-directed Fab DH1042 with hexapro SARS-CoV-2 spike ...

EntireName: Complex of RBD-directed Fab DH1042 with hexapro SARS-CoV-2 spike ectodomain
Components
  • Complex: Complex of RBD-directed Fab DH1042 with hexapro SARS-CoV-2 spike ectodomain

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Supramolecule #1: Complex of RBD-directed Fab DH1042 with hexapro SARS-CoV-2 spike ...

SupramoleculeName: Complex of RBD-directed Fab DH1042 with hexapro SARS-CoV-2 spike ectodomain
type: complex / ID: 1 / Parent: 0
Details: Fab fragment generated by proteolytic cleavage of IgG antibody.
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: 293F / Recombinant plasmid: p-alpha-H
Molecular weightTheoretical: 481.8 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMHEPES buffer
5.0 g/dLglycerol
StainingType: NEGATIVE / Material: Uranyl Formate
Details: Samples were diluted to 0.1 mg/mL in 20 mM HEPES buffer, pH 7.4, with 5% glycerol, 150 mM NaCl, and 7.5 mM glutaraldehyde. After 5 minute incubation at room temperature, sufficient 1 M Tris ...Details: Samples were diluted to 0.1 mg/mL in 20 mM HEPES buffer, pH 7.4, with 5% glycerol, 150 mM NaCl, and 7.5 mM glutaraldehyde. After 5 minute incubation at room temperature, sufficient 1 M Tris stock, pH 7.4, was added to a final concentration of 75 mM Tris to quench unreacted glutaraldehyde, and was then incubated 5 minutes. Sample was then applied to a carbon film over 400 mesh copper EM grids that had been glow-discharged, incubated 1 minute, and then stained with 2% uranyl formate.
GridModel: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 5.0 nm / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeFEI/PHILIPS EM420
Image recordingFilm or detector model: OTHER / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Digitization - Sampling interval: 33.0 µm / Number grids imaged: 1 / Number real images: 222 / Average exposure time: 0.5 sec. / Average electron dose: 32.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 82000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC

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Image processing

Particle selectionNumber selected: 202197
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.8)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6vsb


Details: Map of PDB 6VSB was generated within Chimera using the molmap function at 15 Angstrom resolution and 4.02 Angstrom grid spacing. This map was low-pass filtered to 60 Angstrom before initiating refinements.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 17.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 14021
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 9 / Avg.num./class: 16346 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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