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- EMDB-33506: RBD in complex with Fab14 -

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Basic information

Entry
Database: EMDB / ID: EMD-33506
TitleRBD in complex with Fab14
Map datamain map, without mask, cryosparc sharpened
Sample
  • Complex: RBD bound to Fab14
    • Complex: receptor binding domain of wild-type spike
      • Protein or peptide: Spike protein S1
    • Complex: Fab14
      • Protein or peptide: Fab14 light chain
      • Protein or peptide: Fab14 heavy chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell ...Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated endocytosis of virus by host cell / Attachment and Entry / membrane fusion / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / membrane / identical protein binding / plasma membrane
Similarity search - Function
Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like ...Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, betacoronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.3 Å
AuthorsLin JQ / Tan YJE / Wu B / Lescar J
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2022
Title: Engineering SARS-CoV-2 specific cocktail antibodies into a bispecific format improves neutralizing potency and breadth.
Authors: Zhiqiang Ku / Xuping Xie / Jianqing Lin / Peng Gao / Bin Wu / Abbas El Sahili / Hang Su / Yang Liu / Xiaohua Ye / Eddie Yongjun Tan / Xin Li / Xuejun Fan / Boon Chong Goh / Wei Xiong / ...Authors: Zhiqiang Ku / Xuping Xie / Jianqing Lin / Peng Gao / Bin Wu / Abbas El Sahili / Hang Su / Yang Liu / Xiaohua Ye / Eddie Yongjun Tan / Xin Li / Xuejun Fan / Boon Chong Goh / Wei Xiong / Hannah Boyd / Antonio E Muruato / Hui Deng / Hongjie Xia / Jing Zou / Birte K Kalveram / Vineet D Menachery / Ningyan Zhang / Julien Lescar / Pei-Yong Shi / Zhiqiang An /
Abstract: One major limitation of neutralizing antibody-based COVID-19 therapy is the requirement of costly cocktails to reduce emergence of antibody resistance. Here we engineer two bispecific antibodies ...One major limitation of neutralizing antibody-based COVID-19 therapy is the requirement of costly cocktails to reduce emergence of antibody resistance. Here we engineer two bispecific antibodies (bsAbs) using distinct designs and compared them with parental antibodies and their cocktail. Single molecules of both bsAbs block the two epitopes targeted by parental antibodies on the receptor-binding domain (RBD). However, bsAb with the IgG-(scFv) design (14-H-06) but not the CrossMAb design (14-crs-06) shows increased antigen-binding and virus-neutralizing activities against multiple SARS-CoV-2 variants as well as increased breadth of neutralizing activity compared to the cocktail. X-ray crystallography and cryo-EM reveal distinct binding models for individual cocktail antibodies, and computational simulations suggest higher inter-spike crosslinking potentials by 14-H-06 than 14-crs-06. In mouse models of infections by SARS-CoV-2 and multiple variants, 14-H-06 exhibits higher or equivalent therapeutic efficacy than the cocktail. Rationally engineered bsAbs represent a cost-effective alternative to antibody cocktails and a promising strategy to improve potency and breadth.
History
DepositionMay 30, 2022-
Header (metadata) releaseSep 14, 2022-
Map releaseSep 14, 2022-
UpdateOct 5, 2022-
Current statusOct 5, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33506.png

Downloads & links

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Map

FileDownload / File: emd_33506.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map, without mask, cryosparc sharpened
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.6907242 - 1.1427824
Average (Standard dev.)0.00030763148 (±0.02890662)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 281.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: 3D class density, for illustration purposes

Fileemd_33506_additional_1.map
Annotation3D class density, for illustration purposes
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_33506_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33506_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RBD bound to Fab14

EntireName: RBD bound to Fab14
Components
  • Complex: RBD bound to Fab14
    • Complex: receptor binding domain of wild-type spike
      • Protein or peptide: Spike protein S1
    • Complex: Fab14
      • Protein or peptide: Fab14 light chain
      • Protein or peptide: Fab14 heavy chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: RBD bound to Fab14

SupramoleculeName: RBD bound to Fab14 / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: Fab14 was generated by cleaving IgG14 with papain. RBD was obtained by first expressing a MBP-RBD fusion construct, followed by removal of MBP tag using TEV cleavage. A TEV cleavage site was ...Details: Fab14 was generated by cleaving IgG14 with papain. RBD was obtained by first expressing a MBP-RBD fusion construct, followed by removal of MBP tag using TEV cleavage. A TEV cleavage site was present between MBP and RBD. RBD-Fab14 complex was prepared by mixing both in equal molar ratio, followed by purification via SEC.
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #2: receptor binding domain of wild-type spike

SupramoleculeName: receptor binding domain of wild-type spike / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #3: Fab14

SupramoleculeName: Fab14 / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Fab14 light chain

MacromoleculeName: Fab14 light chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.518879 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QAVVTQPASV SGSPGQSITI SCTGTSSDIG AYNYISWYQQ HPGKAPKLII YEVSNRPSGI SYRFSGSKSG NTASLTISGL QAEDEANYY CSSYAGSISF GGGTKVTVLQ PKANPTVTLF PPSSEELQAN KATLVCLISD FYPGAVTVAW KADGSPVKAG V ETTKPSKQ ...String:
QAVVTQPASV SGSPGQSITI SCTGTSSDIG AYNYISWYQQ HPGKAPKLII YEVSNRPSGI SYRFSGSKSG NTASLTISGL QAEDEANYY CSSYAGSISF GGGTKVTVLQ PKANPTVTLF PPSSEELQAN KATLVCLISD FYPGAVTVAW KADGSPVKAG V ETTKPSKQ SNNKYAASSY LSLTPEQWKS HRSYSCQVTH EGSTVEKTVA PTECS

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Macromolecule #2: Fab14 heavy chain

MacromoleculeName: Fab14 heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.583445 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EVQLQQSGPG LVKPSQTLSL TCAISGDSVS SNSAAWNWIR QSPSRGLEWL GRTYYRSKWY NDYAVSVKSR ITINPDTSKN QFSLQLNSV TPEDTAVYYC AREEQQLVHD YYYYGMDVWG QGTMVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD Y FPEPVTVS ...String:
EVQLQQSGPG LVKPSQTLSL TCAISGDSVS SNSAAWNWIR QSPSRGLEWL GRTYYRSKWY NDYAVSVKSR ITINPDTSKN QFSLQLNSV TPEDTAVYYC AREEQQLVHD YYYYGMDVWG QGTMVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD Y FPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKRVEP KSCDKTH

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Macromolecule #3: Spike protein S1

MacromoleculeName: Spike protein S1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 22.819559 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: SANITNLCPF GEVFNATRFA SVYAWNRKRI SNCVADYSVL YNSASFSTFK CYGVSPTKLN DLCFTNVYAD SFVIRGDEVR QIAPGQTGK IADYNYKLPD DFTGCVIAWN SNNLDSKVGG NYNYLYRLFR KSNLKPFERD ISTEIYQAGS TPCNGVEGFN C YFPLQSYG ...String:
SANITNLCPF GEVFNATRFA SVYAWNRKRI SNCVADYSVL YNSASFSTFK CYGVSPTKLN DLCFTNVYAD SFVIRGDEVR QIAPGQTGK IADYNYKLPD DFTGCVIAWN SNNLDSKVGG NYNYLYRLFR KSNLKPFERD ISTEIYQAGS TPCNGVEGFN C YFPLQSYG FQPTNGVGYQ PYRVVVLSFE LLHAPATVCG PKKSTN

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration0.28 mg/mL
BufferpH: 7.2 / Details: PBS pH 7.2
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsSEC was used to purify RBD-Fab14 complex prior to grid preparation.

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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 / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3410 / Average exposure time: 5.3 sec. / Average electron dose: 45.56 e/Å2
Details: Images were collected in movie-mode: 40 frames per 5.3 seconds.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5334997
Details: auto-picked and extracted in REION, with a box size of 256x256 pixels
CTF correctionSoftware - Name: RELION (ver. 3.0)
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Details: Cryosparc autogenerated model based on 2D selected particles
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 117831

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Atomic model buiding 1

Initial model
PDB IDChain

7wpv

chain_id: A

7wpv

chain_id: B

7wph

chain_id: B
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7xxl:
RBD in complex with Fab14

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