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- EMDB-22922: ACE2-RBD Focused Refinement Using Symmetry Expansion of Applied C... -

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Basic information

Entry
Database: EMDB / ID: EMD-22922
TitleACE2-RBD Focused Refinement Using Symmetry Expansion of Applied C3 for Triple ACE2-bound SARS-CoV-2 Trimer Spike at pH 7.4
Map dataPrimary sharpened map
Sample
  • Complex: ACE2-RBD pH 7.4
    • Protein or peptide: Angiotensin-converting enzyme 2
    • Protein or peptide: Spike glycoproteinSpike protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


positive regulation of amino acid transport / angiotensin-converting enzyme 2 / positive regulation of L-proline import across plasma membrane / Hydrolases; Acting on peptide bonds (peptidases); Metallocarboxypeptidases / angiotensin-mediated drinking behavior / tryptophan transport / positive regulation of gap junction assembly / regulation of systemic arterial blood pressure by renin-angiotensin / regulation of vasoconstriction / regulation of cardiac conduction ...positive regulation of amino acid transport / angiotensin-converting enzyme 2 / positive regulation of L-proline import across plasma membrane / Hydrolases; Acting on peptide bonds (peptidases); Metallocarboxypeptidases / angiotensin-mediated drinking behavior / tryptophan transport / positive regulation of gap junction assembly / regulation of systemic arterial blood pressure by renin-angiotensin / regulation of vasoconstriction / regulation of cardiac conduction / peptidyl-dipeptidase activity / angiotensin maturation / maternal process involved in female pregnancy / Metabolism of Angiotensinogen to Angiotensins / metallocarboxypeptidase activity / Attachment and Entry / carboxypeptidase activity / negative regulation of signaling receptor activity / viral life cycle / regulation of cytokine production / positive regulation of cardiac muscle contraction / blood vessel diameter maintenance / negative regulation of smooth muscle cell proliferation / regulation of transmembrane transporter activity / brush border membrane / cilium / negative regulation of ERK1 and ERK2 cascade / endocytic vesicle membrane / metallopeptidase activity / positive regulation of reactive oxygen species metabolic process / virus receptor activity / regulation of cell population proliferation / regulation of inflammatory response / Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / endopeptidase activity / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / Potential therapeutics for SARS / 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 / symbiont entry into host cell / membrane raft / apical plasma membrane / fusion of virus membrane with host plasma membrane / endoplasmic reticulum lumen / 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 / cell surface / extracellular space / extracellular exosome / zinc ion binding / extracellular region / membrane / identical protein binding / plasma membrane
Similarity search - Function
Collectrin-like domain profile. / Collectrin domain / Renal amino acid transporter / Peptidase family M2 domain profile. / Peptidase M2, peptidyl-dipeptidase A / Angiotensin-converting enzyme / Neutral zinc metallopeptidases, zinc-binding region signature. / 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. ...Collectrin-like domain profile. / Collectrin domain / Renal amino acid transporter / Peptidase family M2 domain profile. / Peptidase M2, peptidyl-dipeptidase A / Angiotensin-converting enzyme / Neutral zinc metallopeptidases, zinc-binding region signature. / 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
Spike glycoprotein / Angiotensin-converting enzyme 2
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsGorman J / Kwong PD / Shapiro L
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM129539 United States
Other privateSF349247 (Simons Foundation) 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.
History
DepositionNov 2, 2020-
Header (metadata) releaseDec 9, 2020-
Map releaseDec 9, 2020-
UpdateDec 15, 2021-
Current statusDec 15, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.4
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  • Surface view colored by cylindrical radius
  • Surface level: 0.4
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  • Surface view with fitted model
  • Atomic models: PDB-7kmb
  • Surface level: 0.4
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7kmb
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22922.png

Downloads & links

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Map

FileDownload / File: emd_22922.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary sharpened map
Voxel sizeX=Y=Z: 1.058 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.4
Minimum - Maximum-1.1036049 - 4.789007
Average (Standard dev.)0.00090746034 (±0.031100543)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 444.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0581.0581.058
M x/y/z420420420
origin x/y/z0.0000.0000.000
length x/y/z444.360444.360444.360
α/β/γ90.00090.00090.000
start NX/NY/NZ937643
NX/NY/NZ114126230
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS420420420
D min/max/mean-1.1044.7890.001

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Supplemental data

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Mask #1

Fileemd_22922_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: C3 unmasked map

Fileemd_22922_additional_1.map
AnnotationC3 unmasked map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map

Fileemd_22922_additional_2.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened C3 unmasked map

Fileemd_22922_additional_3.map
AnnotationSharpened C3 unmasked map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_22922_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_22922_half_map_2.map
AnnotationHalf map B
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : ACE2-RBD pH 7.4

EntireName: ACE2-RBD pH 7.4
Components
  • Complex: ACE2-RBD pH 7.4
    • Protein or peptide: Angiotensin-converting enzyme 2
    • Protein or peptide: Spike glycoproteinSpike protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: ACE2-RBD pH 7.4

SupramoleculeName: ACE2-RBD pH 7.4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Angiotensin-converting enzyme 2

MacromoleculeName: Angiotensin-converting enzyme 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: angiotensin-converting enzyme 2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 69.153664 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: STIEEQAKTF LDKFNHEAED LFYQSSLASW NYNTNITEEN VQNMNNAGDK WSAFLKEQST LAQMYPLQEI QNLTVKLQLQ ALQQNGSSV LSEDKSKRLN TILNTMSTIY STGKVCNPDN PQECLLLEPG LNEIMANSLD YNERLWAWES WRSEVGKQLR P LYEEYVVL ...String:
STIEEQAKTF LDKFNHEAED LFYQSSLASW NYNTNITEEN VQNMNNAGDK WSAFLKEQST LAQMYPLQEI QNLTVKLQLQ ALQQNGSSV LSEDKSKRLN TILNTMSTIY STGKVCNPDN PQECLLLEPG LNEIMANSLD YNERLWAWES WRSEVGKQLR P LYEEYVVL KNEMARANHY EDYGDYWRGD YEVNGVDGYD YSRGQLIEDV EHTFEEIKPL YEHLHAYVRA KLMNAYPSYI SP IGCLPAH LLGDMWGRFW TNLYSLTVPF GQKPNIDVTD AMVDQAWDAQ RIFKEAEKFF VSVGLPNMTQ GFWENSMLTD PGN VQKAVC HPTAWDLGKG DFRILMCTKV TMDDFLTAHH EMGHIQYDMA YAAQPFLLRN GANEGFHEAV GEIMSLSAAT PKHL KSIGL LSPDFQEDNE TEINFLLKQA LTIVGTLPFT YMLEKWRWMV FKGEIPKDQW MKKWWEMKRE IVGVVEPVPH DETYC DPAS LFHVSNDYSF IRYYTRTLYQ FQFQEALCQA AKHEGPLHKC DISNSTEAGQ KLFNMLRLGK SEPWTLALEN VVGAKN MNV RPLLNYFEPL FTWLKDQNKN SFVGWSTDWS PYAD

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Macromolecule #2: Spike glycoprotein

MacromoleculeName: Spike glycoprotein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 140.721266 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: VNLTTRTQLP PAYTNSFTRG VYYPDKVFRS SVLHSTQDLF LPFFSNVTWF HAIHVSGTNG TKRFDNPVLP FNDGVYFAST EKSNIIRGW IFGTTLDSKT QSLLIVNNAT NVVIKVCEFQ FCNDPFLGVY YHKNNKSWME SEFRVYSSAN NCTFEYVSQP F LMDLEGKQ ...String:
VNLTTRTQLP PAYTNSFTRG VYYPDKVFRS SVLHSTQDLF LPFFSNVTWF HAIHVSGTNG TKRFDNPVLP FNDGVYFAST EKSNIIRGW IFGTTLDSKT QSLLIVNNAT NVVIKVCEFQ FCNDPFLGVY YHKNNKSWME SEFRVYSSAN NCTFEYVSQP F LMDLEGKQ GNFKNLREFV FKNIDGYFKI YSKHTPINLV RDLPQGFSAL EPLVDLPIGI NITRFQTLLA LHRSYLTPGD SS SGWTAGA AAYYVGYLQP RTFLLKYNEN GTITDAVDCA LDPLSETKCT LKSFTVEKGI YQTSNFRVQP TESIVRFPNI TNL CPFGEV FNATRFASVY AWNRKRISNC VADYSVLYNS ASFSTFKCYG VSPTKLNDLC FTNVYADSFV IRGDEVRQIA PGQT GKIAD YNYKLPDDFT GCVIAWNSNN LDSKVGGNYN YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSY GFQP TNGVGYQPYR VVVLSFELLH APATVCGPKK STNLVKNKCV NFNFNGLTGT GVLTESNKKF LPFQQFGRDI ADTTDA VRD PQTLEILDIT PCSFGGVSVI TPGTNTSNQV AVLYQDVNCT EVPVAIHADQ LTPTWRVYST GSNVFQTRAG CLIGAEH VN NSYECDIPIG AGICASYQTQ TNSPGSASSV ASQSIIAYTM SLGAENSVAY SNNSIAIPTN FTISVTTEIL PVSMTKTS V DCTMYICGDS TECSNLLLQY GSFCTQLNRA LTGIAVEQDK NTQEVFAQVK QIYKTPPIKD FGGFNFSQIL PDPSKPSKR SFIEDLLFNK VTLADAGFIK QYGDCLGDIA ARDLICAQKF NGLTVLPPLL TDEMIAQYTS ALLAGTITSG WTFGAGAALQ IPFAMQMAY RFNGIGVTQN VLYENQKLIA NQFNSAIGKI QDSLSSTASA LGKLQDVVNQ NAQALNTLVK QLSSNFGAIS S VLNDILSR LDPPEAEVQI DRLITGRLQS LQTYVTQQLI RAAEIRASAN LAATKMSECV LGQSKRVDFC GKGYHLMSFP QS APHGVVF LHVTYVPAQE KNFTTAPAIC HDGKAHFPRE GVFVSNGTHW FVTQRNFYEP QIITTDNTFV SGNCDVVIGI VNN TVYDPL QPELDSFKEE LDKYFKNHTS PDVDLGDISG INASVVNIQK EIDRLNEVAK NLNESLIDLQ ELGKYEQGSG YIPE APRDG QAYVRKDGEW VLLSTFLGRS LEVLFQGPGH HHHHHHHSAW SHPQFEKGGG SGGGGSGGSA WSHPQFEK

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 5 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration1.0 mg/mL
BufferpH: 7.4 / Component - Formula: PBS
GridModel: C-flat-1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3104 / Average exposure time: 2.0 sec. / Average electron dose: 53.49 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 541541
CTF correctionSoftware - Name: cryoSPARC
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationSoftware - Name: cryoSPARC (ver. 2.12)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 128841

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Atomic model buiding 1

Initial model(PDB ID:

6vxx

,

6m0j

)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7kmb:
ACE2-RBD Focused Refinement Using Symmetry Expansion of Applied C3 for Triple ACE2-bound SARS-CoV-2 Trimer Spike at pH 7.4

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