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- PDB-7knh: Cryo-EM Structure of Double ACE2-Bound SARS-CoV-2 Trimer Spike at... -

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Basic information

Entry
Database: PDB / ID: 7knh
TitleCryo-EM Structure of Double ACE2-Bound SARS-CoV-2 Trimer Spike at pH 5.5
Components
  • Angiotensin-converting enzyme 2
  • Spike glycoproteinPeplomer
KeywordsHYDROLASE/VIRAL PROTEIN / COVID / COVID19 / SARS-CoV2 / ACE2 / prefusion / HYDROLASE-VIRAL PROTEIN complex
Function / homology
Function and homology information


positive regulation of L-proline import across plasma membrane / positive regulation of amino acid transport / angiotensin-converting enzyme 2 / Hydrolases; Acting on peptide bonds (peptidases); Metallocarboxypeptidases / tryptophan transport / positive regulation of gap junction assembly / positive regulation of cardiac muscle contraction / angiotensin-mediated drinking behavior / regulation of systemic arterial blood pressure by renin-angiotensin / peptidyl-dipeptidase activity ...positive regulation of L-proline import across plasma membrane / positive regulation of amino acid transport / angiotensin-converting enzyme 2 / Hydrolases; Acting on peptide bonds (peptidases); Metallocarboxypeptidases / tryptophan transport / positive regulation of gap junction assembly / positive regulation of cardiac muscle contraction / angiotensin-mediated drinking behavior / regulation of systemic arterial blood pressure by renin-angiotensin / peptidyl-dipeptidase activity / negative regulation of signaling receptor activity / regulation of vasoconstriction / blood vessel diameter maintenance / angiotensin maturation / carboxypeptidase activity / brush border membrane / metallocarboxypeptidase activity / regulation of cytokine production / regulation of cardiac conduction / suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / receptor-mediated virion attachment to host cell / regulation of transmembrane transporter activity / metallopeptidase activity / host cell surface receptor binding / endocytosis involved in viral entry into host cell / virus receptor activity / positive regulation of reactive oxygen species metabolic process / regulation of inflammatory response / regulation of cell population proliferation / endopeptidase activity / fusion of virus membrane with host plasma membrane / apical plasma membrane / suppression by virus of host type I interferon-mediated signaling pathway / viral entry into host cell / fusion of virus membrane with host endosome membrane / viral envelope / membrane raft / pathogenesis / host cell plasma membrane / virion membrane / cell surface / extracellular space / extracellular exosome / zinc ion binding / integral component of membrane / extracellular region / identical protein binding / plasma membrane / cytoplasm
Angiotensin-converting enzyme / Coronavirus spike glycoprotein S1, C-terminal / Betacoronavirus-like spike glycoprotein S1, N-terminal / Betacoronavirus spike glycoprotein S1, receptor binding / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein S2 superfamily, coronavirus / Spike receptor binding domain superfamily, coronavirus / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Collectrin domain / Spike glycoprotein, heptad repeat 2, coronavirus ...Angiotensin-converting enzyme / Coronavirus spike glycoprotein S1, C-terminal / Betacoronavirus-like spike glycoprotein S1, N-terminal / Betacoronavirus spike glycoprotein S1, receptor binding / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein S2 superfamily, coronavirus / Spike receptor binding domain superfamily, coronavirus / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Collectrin domain / Spike glycoprotein, heptad repeat 2, coronavirus / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike glycoprotein S2, coronavirus / Peptidase M2, peptidyl-dipeptidase A
Spike glycoprotein / Angiotensin-converting enzyme 2 / polysac:dglcpnacb1-4dglcpnacb1-roh:
Biological speciesSevere acute respiratory syndrome coronavirus 2
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.74 Å
AuthorsGorman, J. / Rapp, M. / Kwong, P.D. / Shapiro, L.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM129539 United States
Other privateSF349247 United States
CitationJournal: Cell Host Microbe / Year: 2020
Title: Cryo-EM Structures of SARS-CoV-2 Spike without and with ACE2 Reveal a pH-Dependent Switch to Mediate Endosomal Positioning of Receptor-Binding Domains.
Authors: Tongqing Zhou / Yaroslav Tsybovsky / Jason Gorman / Micah Rapp / Gabriele Cerutti / Gwo-Yu Chuang / Phinikoula S Katsamba / Jared M Sampson / Arne Schön / Jude Bimela / Jeffrey C Boyington ...Authors: Tongqing Zhou / Yaroslav Tsybovsky / Jason Gorman / Micah Rapp / Gabriele Cerutti / Gwo-Yu Chuang / Phinikoula S Katsamba / Jared M Sampson / Arne Schön / Jude Bimela / Jeffrey C Boyington / Alexandra Nazzari / Adam S Olia / Wei Shi / Mallika Sastry / Tyler Stephens / Jonathan Stuckey / I-Ting Teng / Pengfei Wang / Shuishu Wang / Baoshan Zhang / Richard A Friesner / David D Ho / John R Mascola / Lawrence Shapiro / Peter D Kwong /
Abstract: The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the human ACE2 receptor and to facilitate virus entry, which can occur through low-pH-endosomal pathways. To understand ...The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the human ACE2 receptor and to facilitate virus entry, which can occur through low-pH-endosomal pathways. To understand how ACE2 binding and low pH affect spike conformation, we determined cryo-electron microscopy structures-at serological and endosomal pH-delineating spike recognition of up to three ACE2 molecules. RBDs freely adopted "up" conformations required for ACE2 interaction, primarily through RBD movement combined with smaller alterations in neighboring domains. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a solitary all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning through coordinated movements of the entire trimer apex. These structures provide a foundation for understanding prefusion-spike mechanics governing endosomal entry; we suggest that the low pH all-down conformation potentially facilitates immune evasion from RBD-up binding antibody.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 4, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 16, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 23, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.2Jan 27, 2021Group: Structure summary / Category: entity / entity_name_com / Item: _entity.pdbx_description / _entity_name_com.name

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Structure visualization

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Assembly

Deposited unit
C: Spike glycoprotein
A: Spike glycoprotein
E: Angiotensin-converting enzyme 2
B: Spike glycoprotein
D: Angiotensin-converting enzyme 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)577,82157
Polymers565,5055
Non-polymers12,31652
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Spike glycoprotein / Peplomer / S glycoprotein / E2 / Peplomer protein / SARS-CoV-2 spike glycoprotein


Mass: 142399.375 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 / Production host: Homo sapiens (human) / References: UniProt: P0DTC2
#2: Protein Angiotensin-converting enzyme 2 / / ACE-related carboxypeptidase / Angiotensin-converting enzyme homolog / ACEH / Metalloprotease MPROT15


Mass: 69153.664 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ACE2, UNQ868/PRO1885 / Production host: Homo sapiens (human)
References: UniProt: Q9BYF1, angiotensin-converting enzyme 2, Hydrolases; Acting on peptide bonds (peptidases); Metallocarboxypeptidases
#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.: 4
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.: 48 / 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

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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: Double ACE2-bound SARS-CoV-2 Trimer / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 5.5 / Details: PBS with NaAcetate pH 5.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 293 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 / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 51.3 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2Leginonimage acquisition
4cryoSPARCCTF correction
9PHENIXmodel refinement
12cryoSPARC2.12classification
13cryoSPARC2.143D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.74 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55297 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6VXX

6vxx

Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00335267
ELECTRON MICROSCOPYf_angle_d0.64947966
ELECTRON MICROSCOPYf_dihedral_angle_d14.8864920
ELECTRON MICROSCOPYf_chiral_restr0.0465508
ELECTRON MICROSCOPYf_plane_restr0.0046126

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