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- PDB-8t23: Cryo-EM structure of the RBD-ACE2 interface of the SARS-CoV-2 tri... -

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Basic information

Entry
Database: PDB / ID: 8t23
TitleCryo-EM structure of the RBD-ACE2 interface of the SARS-CoV-2 trimeric spike protein bound to ACE2 receptor after local refinement at upRBD conformation
Components
  • Angiotensin-converting enzyme
  • Spike glycoprotein
KeywordsVIRAL PROTEIN / Coronavirus / Spike / ACE2 / Mink / PROTEIN BINDING
Function / homology
Function and homology information


Hydrolases; Acting on peptide bonds (peptidases) / peptidyl-dipeptidase activity / carboxypeptidase activity / metallopeptidase activity / symbiont-mediated disruption of host tissue / Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space ...Hydrolases; Acting on peptide bonds (peptidases) / peptidyl-dipeptidase activity / carboxypeptidase activity / metallopeptidase activity / symbiont-mediated disruption of host tissue / Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / viral translation / 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 / Attachment and Entry / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / host cell surface receptor binding / cilium / symbiont-mediated suppression of host innate immune response / apical plasma membrane / 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 / symbiont entry into host cell / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / proteolysis / extracellular space / metal ion binding / identical protein binding / membrane / plasma membrane / cytoplasm
Similarity search - Function
Collectrin domain / Renal amino acid transporter / Collectrin-like domain profile. / Peptidase M2, peptidyl-dipeptidase A / Angiotensin-converting enzyme / Peptidase family M2 domain profile. / 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 ...Collectrin domain / Renal amino acid transporter / Collectrin-like domain profile. / Peptidase M2, peptidyl-dipeptidase A / Angiotensin-converting enzyme / Peptidase family M2 domain profile. / 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, N-terminal domain superfamily / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein, betacoronavirus / 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 / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Spike glycoprotein S2 superfamily, coronavirus / 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, coronavirus / Coronavirus spike glycoprotein S2
Similarity search - Domain/homology
Angiotensin-converting enzyme / Spike glycoprotein
Similarity search - Component
Biological speciesNeovison vison (American mink)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.87 Å
AuthorsAhn, H.M. / Calderon, B. / Fan, X. / Gao, Y. / Horgan, N. / Zhou, B. / Liang, B.
Funding support United States, 1items
OrganizationGrant numberCountry
Other government United States
CitationJournal: J Med Virol / Year: 2023
Title: Structural basis of the American mink ACE2 binding by Y453F trimeric spike glycoproteins of SARS-CoV-2.
Authors: Hyunjun Ahn / Brenda M Calderon / Xiaoyu Fan / Yunrong Gao / Natalie L Horgan / Nannan Jiang / Dylan S Blohm / Jaber Hossain / Nicole Wedad K Rayyan / Sarah H Osman / Xudong Lin / Michael ...Authors: Hyunjun Ahn / Brenda M Calderon / Xiaoyu Fan / Yunrong Gao / Natalie L Horgan / Nannan Jiang / Dylan S Blohm / Jaber Hossain / Nicole Wedad K Rayyan / Sarah H Osman / Xudong Lin / Michael Currier / John Steel / David E Wentworth / Bin Zhou / Bo Liang /
Abstract: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) enters the host cell by binding to angiotensin-converting enzyme 2 (ACE2). While evolutionarily conserved, ACE2 receptors differ across ...Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) enters the host cell by binding to angiotensin-converting enzyme 2 (ACE2). While evolutionarily conserved, ACE2 receptors differ across various species and differential interactions with Spike (S) glycoproteins of SARS-CoV-2 viruses impact species specificity. Reverse zoonoses led to SARS-CoV-2 outbreaks on multiple American mink (Mustela vison) farms during the pandemic and gave rise to mink-associated S substitutions known for transmissibility between mink and zoonotic transmission to humans. In this study, we used bio-layer interferometry (BLI) to discern the differences in binding affinity between multiple human and mink-derived S glycoproteins of SARS-CoV-2 and their respective ACE2 receptors. Further, we conducted a structural analysis of a mink variant S glycoprotein and American mink ACE2 (mvACE2) using cryo-electron microscopy (cryo-EM), revealing four distinct conformations. We discovered a novel intermediary conformation where the mvACE2 receptor is bound to the receptor-binding domain (RBD) of the S glycoprotein in a "down" position, approximately 34° lower than previously reported "up" RBD. Finally, we compared residue interactions in the S-ACE2 complex interface of S glycoprotein conformations with varying RBD orientations. These findings provide valuable insights into the molecular mechanisms of SARS-CoV-2 entry.
History
DepositionJun 5, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 25, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Angiotensin-converting enzyme
B: Spike glycoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)113,9806
Polymers112,7462
Non-polymers1,2344
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable, gel filtration, assay for oligomerization, bio-layer interferometry
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Angiotensin-converting enzyme


Mass: 89375.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neovison vison (American mink) / Production host: Homo sapiens (human) / References: UniProt: A0A7T0Q2W2
#2: Protein Spike glycoprotein


Mass: 23370.246 Da / Num. of mol.: 1 / Fragment: RBD
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: S, 2 / Production host: Homo sapiens (human) / References: UniProt: P0DTC2
#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.: 1
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: Polysaccharide alpha-L-fucopyranose-(1-6)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 367.349 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
LFucpa1-6DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,2,1/[a2122h-1b_1-5_2*NCC/3=O][a1221m-1a_1-5]/1-2/a6-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(6+1)][a-L-Fucp]{}}LINUCSPDB-CARE
#5: 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


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
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 interestN
Has protein modificationY

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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: mink variant spike RBD-ACE2 interface after local refinement
Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Severe acute respiratory syndrome coronavirus 22697049
31Neovison vison (American mink)452646
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8 / Details: 20 mM Tris pH 8.0, 200 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris BufferTris1
2200 mMSodium ChlorideNaCl1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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: OTHER
Electron lensMode: OTHER / Nominal magnification: 81000 X / Nominal defocus max: 1750 nm / Nominal defocus min: 750 nm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 49.98 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 11392

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 512722 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 152.24 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00416635
ELECTRON MICROSCOPYf_angle_d0.91959012
ELECTRON MICROSCOPYf_chiral_restr0.0499946
ELECTRON MICROSCOPYf_plane_restr0.00861164
ELECTRON MICROSCOPYf_dihedral_angle_d8.9311896

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