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- EMDB-35930: CryoEM structure of SARS CoV-2 RBD and Aptamer complex -

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Entry
Database: EMDB / ID: EMD-35930
TitleCryoEM structure of SARS CoV-2 RBD and Aptamer complex
Map dataProtein:RBD_Fab_AM032_AM047, EM: Titan krios 300keV, Dose:50, Defocus:-0.8 to -2.0, Magnification: 105k, movies: 11743 plus 9597, Map:from 3DFlex reconstruction
Sample
  • Complex: RBD_Fab_AM032_AM047 complex
    • DNA: AM032-0
    • DNA: AM047-0
    • Protein or peptide: Fab Light chain (REGN10987)
    • Protein or peptide: Fab heavy chain (REGN10987)
    • Protein or peptide: Spike protein S1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsAptamer / SARS CoV-2 RBD / Inhibitor / NapdU / VIRAL PROTEIN / VIRAL PROTEIN-IMMUNE SYSTEM-DNA complex
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 / host cell endoplasmic reticulum-Golgi intermediate compartment membrane ...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 / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / entry receptor-mediated virion attachment to host cell / 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 / 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 / synthetic construct (others) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsRahman MS / Jang SK / Lee JO
Funding support Korea, Republic Of, 1 items
OrganizationGrant numberCountry
National Research Foundation (NRF, Korea)NRF-2019M3E5D6063871 Korea, Republic Of
CitationJournal: Molecules / Year: 2023
Title: Structure-Guided Development of Bivalent Aptamers Blocking SARS-CoV-2 Infection.
Authors: Md Shafiqur Rahman / Min Jung Han / Sang Won Kim / Seong Mu Kang / Bo Ri Kim / Heesun Kim / Chang Jun Lee / Jung Eun Noh / Hanseong Kim / Jie-Oh Lee / Sung Key Jang /
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused devastation to human society through its high virulence, infectivity, and genomic mutations, which reduced the efficacy of ...Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused devastation to human society through its high virulence, infectivity, and genomic mutations, which reduced the efficacy of vaccines. Here, we report the development of aptamers that effectively interfere with SARS-CoV-2 infection by targeting its spike protein, which plays a pivotal role in host cell entry of the virus through interaction with the viral receptor angiotensin-converting enzyme 2 (ACE2). To develop highly effective aptamers and to understand their mechanism in inhibiting viral infection, we determined the three-dimensional (3D) structures of aptamer/receptor-binding domain (RBD) complexes using cryogenic electron microscopy (cryo-EM). Moreover, we developed bivalent aptamers targeting two distinct regions of the RBD in the spike protein that directly interact with ACE2. One aptamer interferes with the binding of ACE2 by blocking the ACE2-binding site in RBD, and the other aptamer allosterically inhibits ACE2 by binding to a distinct face of RBD. Using the 3D structures of aptamer-RBD complexes, we minimized and optimized these aptamers. By combining the optimized aptamers, we developed a bivalent aptamer that showed a stronger inhibitory effect on virus infection than the component aptamers. This study confirms that the structure-based aptamer-design approach has a high potential in developing antiviral drugs against SARS-CoV-2 and other viruses.
History
DepositionApr 13, 2023-
Header (metadata) releaseJun 21, 2023-
Map releaseJun 21, 2023-
UpdateJul 12, 2023-
Current statusJul 12, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35930.png

Downloads & links

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Map

FileDownload / File: emd_35930.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationProtein:RBD_Fab_AM032_AM047, EM: Titan krios 300keV, Dose:50, Defocus:-0.8 to -2.0, Magnification: 105k, movies: 11743 plus 9597, Map:from 3DFlex reconstruction
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.00454
Minimum - Maximum-0.0091261305 - 0.019595627
Average (Standard dev.)0.000060948805 (±0.00059930043)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 209.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_35930_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_35930_half_map_2.map
AnnotationHalf map B
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_Fab_AM032_AM047 complex

EntireName: RBD_Fab_AM032_AM047 complex
Components
  • Complex: RBD_Fab_AM032_AM047 complex
    • DNA: AM032-0
    • DNA: AM047-0
    • Protein or peptide: Fab Light chain (REGN10987)
    • Protein or peptide: Fab heavy chain (REGN10987)
    • Protein or peptide: Spike protein S1
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: RBD_Fab_AM032_AM047 complex

SupramoleculeName: RBD_Fab_AM032_AM047 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2 / Strain: Wuhan
Molecular weightTheoretical: 124 KDa

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Macromolecule #1: AM032-0

MacromoleculeName: AM032-0 / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 26.51775 KDa
SequenceString: (DT)(DA)(DT)(DC)(DG)(DA)(DG)(DC)(DG)(DT) (DC)(DC)(DT)(DG)(DC)(DC)(DT)(DT)(DT)(DG) (DC)(DG)(DG)(DA)(DG)(DG)(85Y)(DG)(DA) (DA)(DC)(DC)(85Y)(85Y)(DA)(DC)(DG)(85Y) (85Y)(DC)(DA)(DA)(DC)(85Y)(DG) ...String:
(DT)(DA)(DT)(DC)(DG)(DA)(DG)(DC)(DG)(DT) (DC)(DC)(DT)(DG)(DC)(DC)(DT)(DT)(DT)(DG) (DC)(DG)(DG)(DA)(DG)(DG)(85Y)(DG)(DA) (DA)(DC)(DC)(85Y)(85Y)(DA)(DC)(DG)(85Y) (85Y)(DC)(DA)(DA)(DC)(85Y)(DG)(DG)(DA)(85Y) (DC)(DG)(85Y)(DG)(85Y)(DG)(85Y)(DA) (DG) (DG)(85Y)(DA)(DC)(DA)(DC)(DC)(DG)(DA)(DC) (DA)(DG)(DC)(DC)(DA)(DC)(DC)(DC) (DA) (DG)(DA)(DA)(DA)

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Macromolecule #2: AM047-0

MacromoleculeName: AM047-0 / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.034312 KDa
SequenceString: (DC)(DT)(DG)(85Y)(DC)(DG)(DG)(85Y)(DA)(DA) (DG)(DG)(DG)(DA)(DG)(DC)(85Y)(DA)(DG) (85Y)(DG)(85Y)(DA)(85Y)(DG)(DC)(DC)(DC)(DC) (85Y)(DG)(DA)(DG)(85Y)(85Y)(DG)(DG) (DG) (DG)(DA)(85Y)(DA)(85Y) ...String:
(DC)(DT)(DG)(85Y)(DC)(DG)(DG)(85Y)(DA)(DA) (DG)(DG)(DG)(DA)(DG)(DC)(85Y)(DA)(DG) (85Y)(DG)(85Y)(DA)(85Y)(DG)(DC)(DC)(DC)(DC) (85Y)(DG)(DA)(DG)(85Y)(85Y)(DG)(DG) (DG) (DG)(DA)(85Y)(DA)(85Y)(DC)(DG)(DC)(DC)(DG) (DA)(DC)(DA)(DG)

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Macromolecule #3: Fab Light chain (REGN10987)

MacromoleculeName: Fab Light chain (REGN10987) / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.90933 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMI YDVSKRPSGV SNRFSGSKSG NTASLTISGL QSEDEADYY CNSLTSISTW VFGGGTKLTV LGQPKAAPSV TLFPPSSEEL QANKATLVCL ISDFYPGAVT VAWKADSSPV K AGVETTTP ...String:
QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMI YDVSKRPSGV SNRFSGSKSG NTASLTISGL QSEDEADYY CNSLTSISTW VFGGGTKLTV LGQPKAAPSV TLFPPSSEEL QANKATLVCL ISDFYPGAVT VAWKADSSPV K AGVETTTP SKQSNNKYAA SSYLSLTPEQ WKSHRSYSCQ VTHEGSTVEK TVAPTECSHH HHHHHH

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Macromolecule #4: Fab heavy chain (REGN10987)

MacromoleculeName: Fab heavy chain (REGN10987) / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 26.705879 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: QVQLVESGGG VVQPGRSLRL SCAASGFTFS NYAMYWVRQA PGKGLEWVAV ISYDGSNKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRTE DTAVYYCASG SDYGDYLLVY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS ...String:
QVQLVESGGG VVQPGRSLRL SCAASGFTFS NYAMYWVRQA PGKGLEWVAV ISYDGSNKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRTE DTAVYYCASG SDYGDYLLVY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCDKSNS LVPRGSPSRL EE ELRRRLT E

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Macromolecule #5: Spike protein S1

MacromoleculeName: Spike protein S1 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2 / Strain: Wuhan
Molecular weightTheoretical: 28.415693 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: RVQPTESIVR FPNITNLCPF GEVFNATRFA SVYAWNRKRI SNCVADYSVL YNSASFSTFK CYGVSPTKLN DLCFTNVYAD SFVIRGDEV RQIAPGQTGK IADYNYKLPD DFTGCVIAWN SNNLDSKVGG NYNYLYRLFR KSNLKPFERD ISTEIYQAGS T PCNGVEGF ...String:
RVQPTESIVR FPNITNLCPF GEVFNATRFA SVYAWNRKRI SNCVADYSVL YNSASFSTFK CYGVSPTKLN DLCFTNVYAD SFVIRGDEV RQIAPGQTGK IADYNYKLPD DFTGCVIAWN SNNLDSKVGG NYNYLYRLFR KSNLKPFERD ISTEIYQAGS T PCNGVEGF NCYFPLQSYG FQPTNGVGYQ PYRVVVLSFE LLHAPATVCG PKKSTNLVKN KCVNFENLYF QGAAAGGSHH HH HHGGSDY KDDDDK

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration0.74 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloride
25.0 mMTris-HClTris-Hydrogen Chloride

Details: 1mM MgCl2, 0.15% amphipol A8-35 and 0.003% cymal-6 additive added during sample preparation
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.034 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: 2.5 ul of samples placed on grid before plunge frozen with 5s blot time.
DetailsAptamer AM032 and AM047 mixed with RBD_Fab complex at 1:1.2 molar ratio and incubate 1hr on ice before preparing grids

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 2 / Number real images: 21340 / Average exposure time: 4.56 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.73 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5448100 / Details: Particles picked using TOPAZ train
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1.0 and 4.2.1)
Details: Final map generated from 3D flex reconstruction following non-uniform refinement.
Number images used: 387000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final 3D classificationNumber classes: 4 / Avg.num./class: 271253 / Software - Name: cryoSPARC (ver. 4.0.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(Chain: PDB, experimental model, PDB, experimental model, PDB, experimental model)
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8j1q:
CryoEM structure of SARS CoV-2 RBD and Aptamer complex

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