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- EMDB-26679: Subtomogram averaged map of hACE2 dimers on the surface of extrac... -

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Basic information

Entry
Database: EMDB / ID: EMD-26679
TitleSubtomogram averaged map of hACE2 dimers on the surface of extracellular vesicles
Map dataFinal subtomogram averaged map of hACE2 on the surface of extracellular vesicles.
Sample
  • Complex: Dimeric complex consisting of full-length membrane-bound human ACE2 with a LgBiT tag.
    • Protein or peptide: hACE2-LgBiT
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 19.0 Å
AuthorsMarcink TC / Kicmal T / Armbruster E / Zhang Z / Zipursky G / Idris M / Khao J / McGill G / Gallagher T / Porotto M ...Marcink TC / Kicmal T / Armbruster E / Zhang Z / Zipursky G / Idris M / Khao J / McGill G / Gallagher T / Porotto M / des Georges A / Moscona A
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI152275 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI114736 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI160961 United States
CitationJournal: Sci Adv / Year: 2022
Title: Intermediates in SARS-CoV-2 spike-mediated cell entry.
Authors: Tara C Marcink / Thomas Kicmal / Emily Armbruster / Zhening Zhang / Gillian Zipursky / Kate L Golub / Mohab Idris / Jonathan Khao / Jennifer Drew-Bear / Gael McGill / Tom Gallagher / Matteo ...Authors: Tara C Marcink / Thomas Kicmal / Emily Armbruster / Zhening Zhang / Gillian Zipursky / Kate L Golub / Mohab Idris / Jonathan Khao / Jennifer Drew-Bear / Gael McGill / Tom Gallagher / Matteo Porotto / Amédée des Georges / Anne Moscona /
Abstract: SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by ...SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by cryo-electron microscopy and cryo-electron tomography, but the refolding intermediates on the fusion pathway are transient and have not been examined. We used an antiviral lipopeptide entry inhibitor to arrest S protein refolding and thereby capture intermediates as S proteins interact with hACE2 and fusion-activating proteases on cell-derived target membranes. Cryo-electron tomography imaged both extended and partially folded intermediate states of S2, as well as a novel late-stage conformation on the pathway to membrane fusion. The intermediates now identified in this dynamic S protein-directed fusion provide mechanistic insights that may guide the design of CoV entry inhibitors.
History
DepositionApr 18, 2022-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateSep 14, 2022-
Current statusSep 14, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26679.png

Downloads & links

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Map

FileDownload / File: emd_26679.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal subtomogram averaged map of hACE2 on the surface of extracellular vesicles.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.24 Å/pix.
x 96 pix.
= 311.04 Å		3.24 Å/pix.
x 96 pix.
= 311.04 Å		3.24 Å/pix.
x 96 pix.
= 311.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.24 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-4.4522505 - 6.2868614
Average (Standard dev.)0.29135135 (±0.8647251)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 311.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Dimeric complex consisting of full-length membrane-bound human AC...

EntireName: Dimeric complex consisting of full-length membrane-bound human ACE2 with a LgBiT tag.
Components
  • Complex: Dimeric complex consisting of full-length membrane-bound human ACE2 with a LgBiT tag.
    • Protein or peptide: hACE2-LgBiT

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Supramolecule #1: Dimeric complex consisting of full-length membrane-bound human AC...

SupramoleculeName: Dimeric complex consisting of full-length membrane-bound human ACE2 with a LgBiT tag.
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293T / Recombinant plasmid: pcDNA3.1

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Macromolecule #1: hACE2-LgBiT

MacromoleculeName: hACE2-LgBiT / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MSSSSWLLLS LVAVTAAQST IEEQAKTFLD KFNHEAEDLF YQSSLASWNY NTNITEENVQ NMNNAGDKWS AFLKEQSTLA QMYPLQEIQN LTVKLQLQAL QQNGSSVLSE DKSKRLNTIL NTMSTIYSTG KVCNPDNPQE CLLLEPGLNE IMANSLDYNE RLWAWESWRS ...String:
MSSSSWLLLS LVAVTAAQST IEEQAKTFLD KFNHEAEDLF YQSSLASWNY NTNITEENVQ NMNNAGDKWS AFLKEQSTLA QMYPLQEIQN LTVKLQLQAL QQNGSSVLSE DKSKRLNTIL NTMSTIYSTG KVCNPDNPQE CLLLEPGLNE IMANSLDYNE RLWAWESWRS EVGKQLRPLY EEYVVLKNEM ARANHYEDYG DYWRGDYEVN GVDGYDYSRG QLIEDVEHTF EEIKPLYEHL HAYVRAKLMN AYPSYISPIG CLPAHLLGDM WGRFWTNLYS LTVPFGQKPN IDVTDAMVDQ AWDAQRIFKE AEKFFVSVGL PNMTQGFWEN SMLTDPGNVQ KAVCHPTAWD LGKGDFRILM CTKVTMDDFL TAHHEMGHIQ YDMAYAAQPF LLRNGANEGF HEAVGEIMSL SAATPKHLKS IGLLSPDFQE DNETEINFLL KQALTIVGTL PFTYMLEKWR WMVFKGEIPK DQWMKKWWEM KREIVGVVEP VPHDETYCDP ASLFHVSNDY SFIRYYTRTL YQFQFQEALC QAAKHEGPLH KCDISNSTEA GQKLFNMLRL GKSEPWTLAL ENVVGAKNMN VRPLLNYFEP LFTWLKDQNK NSFVGWSTDW SPYADQSIKV RISLKSALGD KAYEWNDNEM YLFRSSVAYA MRQYFLKVKN QMILFGEEDV RVANLKPRIS FNFFVTAPKN VSDIIPRTEV EKAIRMSRSR INDAFRLNDN SLEFLGIQPT LGPPNQPPVS IWLIVFGVVM GVIVVGIVIL IFTGIRDRKK KNKARSGENP YASIDISKGE NNPGFQNTDD VQTSF

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 3.0 nm
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum SE / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 2.7 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 8.0 µm / Nominal defocus min: 4.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Dynamo (ver. 1.1.514) / Number subtomograms used: 315
ExtractionNumber tomograms: 1 / Number images used: 1
CTF correctionSoftware - Name: Warp (ver. 1.0.9)
Final angle assignmentType: NOT APPLICABLE

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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