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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 dataMap filtered to 6A to visualize the low occupancy VH domains. This is the map that was used for VH and RBD model fitting.
Sample
  • Complex: Complex of SARS CoV2 Spike ectodomain with engineered trimerized VH domain
    • Complex: engineered trimerized VH domain
      • Protein or peptide: autonomous human heavy chain variable domain
    • Complex: SARS CoV2 Spike ectodomain
      • Protein or peptide: Spike glycoprotein
KeywordsSpike / VH / SARS / CoV2 / VIRAL PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


Maturation of spike protein / viral translation / Translation of Structural Proteins / host cell surface / Virion Assembly and Release / host extracellular space / symbiont-mediated-mediated suppression 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 / host cell surface / Virion Assembly and Release / host extracellular space / symbiont-mediated-mediated suppression of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / entry receptor-mediated virion attachment to host cell / membrane fusion / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / Attachment and Entry / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / host cell surface receptor binding / symbiont-mediated suppression of host innate immune response / receptor ligand activity / endocytosis involved in viral entry into host cell / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / identical protein binding / membrane / 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 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, betacoronavirus / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus ...Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, betacoronavirus / Spike glycoprotein, N-terminal domain superfamily / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / 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 1 (HR1) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2
Similarity search - Domain/homology
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
Quantitative Biosciences Institute United States
FastGrants United States
Laboratory for Genomics Research (LGR)Excellence in Research Award (ERA) United States
CitationJournal: 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.
History
DepositionAug 25, 2020-
Header (metadata) releaseOct 7, 2020-
Map releaseOct 7, 2020-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing 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)
AnnotationMap filtered to 6A to visualize the low occupancy VH domains. This is the map that was used for VH and RBD model fitting.
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
Components
  • Complex: Complex of SARS CoV2 Spike ectodomain with engineered trimerized VH domain
    • Complex: engineered trimerized VH domain
      • Protein or peptide: autonomous human heavy chain variable domain
    • Complex: SARS CoV2 Spike ectodomain
      • Protein or peptide: Spike glycoprotein

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Supramolecule #1: Complex of SARS CoV2 Spike ectodomain with engineered trimerized ...

SupramoleculeName: Complex of SARS CoV2 Spike ectodomain with engineered trimerized VH domain
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 445 KDa

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Supramolecule #2: engineered trimerized VH domain

SupramoleculeName: engineered trimerized VH domain / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: SARS CoV2 Spike ectodomain

SupramoleculeName: SARS CoV2 Spike ectodomain / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2

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Macromolecule #1: autonomous human heavy chain variable domain

MacromoleculeName: autonomous human heavy chain variable domain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.264402 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: EVQLVESGGG LVQPGGSLRL SCAASGFRIY SYYSYIGWVR RAPGKGEELV ARIYPSSGYT YYADSVKGRF TISADTSKNT AYLQMNSLR AEDTAVYYCA RWDFASPYYP GSSGLDYWGQ GTLVTVSSGG GGTGGGGSGG GGSGGGGSEV QLVESGGGLV Q PGGSLRLS ...String:
EVQLVESGGG LVQPGGSLRL SCAASGFRIY SYYSYIGWVR RAPGKGEELV ARIYPSSGYT YYADSVKGRF TISADTSKNT AYLQMNSLR AEDTAVYYCA RWDFASPYYP GSSGLDYWGQ GTLVTVSSGG GGTGGGGSGG GGSGGGGSEV QLVESGGGLV Q PGGSLRLS CAASGFRIYS YYSYIGWVRR APGKGEELVA RIYPSSGYTY YADSVKGRFT ISADTSKNTA YLQMNSLRAE DT AVYYCAR WDFASPYYPG SSGLDYWGQG TLVTVSSGGG GSGGGGSGGG GSGGGGSEVQ LVESGGGLVQ PGGSLRLSCA ASG FRIYSY YSYIGWVRRA PGKGEELVAR IYPSSGYTYY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARWD FASP YYPGS SGLDYWGQGT LVTVSS

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Macromolecule #2: Spike glycoprotein

MacromoleculeName: Spike glycoprotein / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 133.75325 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDG VYFASTEKSN IIRGWIFGTT LDSKTQSLLI VNNATNVVIK VCEFQFCNDP FLGVYYHKNN KSWMESEFRV Y SSANNCTF ...String:
MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDG VYFASTEKSN IIRGWIFGTT LDSKTQSLLI VNNATNVVIK VCEFQFCNDP FLGVYYHKNN KSWMESEFRV Y SSANNCTF EYVSQPFLMD LEGKQGNFKN LREFVFKNID GYFKIYSKHT PINLVRDLPQ GFSALEPLVD LPIGINITRF QT LLALHRS YLTPGDSSSG WTAGAAAYYV GYLQPRTFLL KYNENGTITD AVDCALDPLS ETKCTLKSFT VEKGIYQTSN FRV QPTESI VRFPNITNLC PFGEVFNATR FASVYAWNRK RISNCVADYS VLYNSASFST FKCYGVSPTK LNDLCFTNVY ADSF VIRGD EVRQIAPGQT GKIADYNYKL PDDFTGCVIA WNSNNLDSKV GGNYNYLYRL FRKSNLKPFE RDISTEIYQA GSTPC NGVE GFNCYFPLQS YGFQPTNGVG YQPYRVVVLS FELLHAPATV CGPKKSTNLV KNKCVNFNFN GLTGTGVLTE SNKKFL PFQ QFGRDIADTT DAVRDPQTLE ILDITPCSFG GVSVITPGTN TSNQVAVLYQ DVNCTEVPVA IHADQLTPTW RVYSTGS NV FQTRAGCLIG AEHVNNSYEC DIPIGAGICA SYQTQTNSPG SASSVASQSI IAYTMSLGAE NSVAYSNNSI AIPTNFTI S VTTEILPVSM TKTSVDCTMY ICGDSTECSN LLLQYGSFCT QLNRALTGIA VEQDKNTQEV FAQVKQIYKT PPIKDFGGF NFSQILPDPS KPSKRSFIED LLFNKVTLAD AGFIKQYGDC LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFG AGAALQIPFA MQMAYRFNGI GVTQNVLYEN QKLIANQFNS AIGKIQDSLS STASALGKLQ DVVNQNAQAL N TLVKQLSS NFGAISSVLN DILSRLDPPE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI RASANLAATK MSECVLGQSK RV DFCGKGY HLMSFPQSAP HGVVFLHVTY VPAQEKNFTT APAICHDGKA HFPREGVFVS NGTHWFVTQR NFYEPQIITT DNT FVSGNC DVVIGIVNNT VYDPLQPELD SFKEELDKYF KNHTSPDVDL GDISGINASV VNIQKEIDRL NEVAKNLNES LIDL QELGK YEQ

UniProtKB: Spike glycoprotein

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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

Concentration0.9 mg/mL
BufferpH: 8 / Details: 20 mM HEPES, pH 8, 200 mM NaCl
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 78.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing #1

Image processing ID1
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.0 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.
Number images used: 21000
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Image processing #2

Image processing ID2
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 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.
Number images used: 21000
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6x2b

source_name: PDB, initial_model_type: experimental model

4g80

chain_id: A, residue_range: 1-124, source_name: PDB, initial_model_type: experimental model
RefinementProtocol: RIGID BODY FIT / Target criteria: cross correlation
Output model

PDB-7jwb:
SARS CoV2 Spike ectodomain with engineered trimerized VH binder

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