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- PDB-7n0h: CryoEM structure of SARS-CoV-2 spike protein (S-6P, 2-up) in comp... -

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Basic information

Entry
Database: PDB / ID: 7n0h
TitleCryoEM structure of SARS-CoV-2 spike protein (S-6P, 2-up) in complex with sybodies (Sb45)
Components
  • Spike glycoproteinSpike protein
  • Synthetic nanobody (Sb45)
KeywordsVIRAL PROTEIN / SARS-CoV-2 / Spike Protein / S-6P (HexaPro) / nanobody / sybody / Sb45 / neutralization
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 / 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
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.34 Å
AuthorsJiang, J. / Huang, R. / Margulies, D.
CitationJournal: J Biol Chem / Year: 2021
Title: Structures of synthetic nanobody-SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction.
Authors: Javeed Ahmad / Jiansheng Jiang / Lisa F Boyd / Allison Zeher / Rick Huang / Di Xia / Kannan Natarajan / David H Margulies /
Abstract: Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of ...Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16-RBD, Sb45-RBD, Sb14-RBD-Sb68, and Sb45-RBD-Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD-angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both "up" and "down" configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.
History
DepositionMay 25, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 29, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Oct 20, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.title

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-24106
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Spike glycoprotein
B: Spike glycoprotein
C: Spike glycoprotein
X: Synthetic nanobody (Sb45)
Z: Synthetic nanobody (Sb45)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)468,28152
Polymers454,4305
Non-polymers13,85147
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Spike glycoprotein / Spike protein / S glycoprotein / E2 / Peplomer protein


Mass: 142427.438 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: S, 2 / Cell (production host): HEK293F / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P0DTC2
#2: Antibody Synthetic nanobody (Sb45)


Mass: 13574.048 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pET21b / Production host: Escherichia coli (E. coli)
#3: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 17
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Sugar...
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 30 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Spike protein (S-6P, 2-up) in complex with Synthetic nanobody (Sb45)
Type: COMPLEX
Details: Synthetic nanobody Sb45 was mixed with freshly purified S-6P in the mole ratio of 3:1, incubated, and subjected to Negative stain and frozen grids for cryoEM data collection.
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.6 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21synthetic construct (others)32630
31Severe acute respiratory syndrome coronavirus 22697049
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
31Escherichia coli (E. coli)562
Buffer solutionpH: 8
Buffer componentConc.: 0.25 mg/mL / Name: TBS
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Calibrated magnification: 47529 X / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm / Calibrated defocus min: 535 nm / Calibrated defocus max: 2431 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 78 K
Image recordingAverage exposure time: 8 sec. / Electron dose: 56 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 9725
Image scansSampling size: 5.001 µm / Movie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv3.2.0particle selection
2SerialEM3.8.0image acquisition
4cryoSPARCv3.2.0CTF correction
7UCSF Chimera2021model fitting
9PHENIX1.19.2model refinement
12cryoSPARC3.2.0classification
13cryoSPARCV3.2.03D reconstruction
CTF correctionDetails: patch CTF estimation / Type: NONE
Particle selectionNum. of particles selected: 1433963
Details: A total of 9,725 micrographs were imported into cryoSPARC. Following patch Motion correction, patch CTF estimation, and curation, the number of micrographs was reduced to 9,237. The blob ...Details: A total of 9,725 micrographs were imported into cryoSPARC. Following patch Motion correction, patch CTF estimation, and curation, the number of micrographs was reduced to 9,237. The blob picker with the particle diameter between 128 and 256 angstroms was used for picking up particles. We used the box size of 400 pixels and extracted 1,433,963 particles initially.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61026 / Algorithm: SIMULTANEOUS ITERATIVE (SIRT)
Details: A series of Ab-initio 3D reconstruction (classification)dividing 2 or 4 subclasses to identify two forms of S-6P, i.e. one RBD up, or two RBD up.
Num. of class averages: 18 / Symmetry type: POINT
Atomic model buildingB value: 157 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation Coefficient
Details: An initial model for S-6P was generated using PDB 6XKL and rigid body fitted into the map using Chimera. The RBD domain (334-528) and Sb45 are taken from 7KGJ which was superimposed onto the ...Details: An initial model for S-6P was generated using PDB 6XKL and rigid body fitted into the map using Chimera. The RBD domain (334-528) and Sb45 are taken from 7KGJ which was superimposed onto the S-6P model in PyMol. The NTD domain (14-289) is taken from 7B32 with full sequence and replace that of 6XKL. We have rebuilt and added more glycans (NAGs). We used the real-space refinement in PHENIX including rigid-body refinement. RBD and NTD are subjected to rigid-body refinement. Simulate annealing (SA) runs once at the initial micro-step, local grid search and ADP refinements were included, using the secondary structure restraints.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
16XKL

6xkl

A1529-1146
26XKL

6xkl

B1529-1146
36XKL

6xkl

C1529-1146
47KGJ

7kgj

A1334-528
57KGJ

7kgj

B11-121
67B32

7b32

A114-289

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