[English] 日本語
Yorodumi
- EMDB-44062: SARS CoV-2 Spike protein Ectodomain with internal tag, all RBD-do... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-44062
TitleSARS CoV-2 Spike protein Ectodomain with internal tag, all RBD-down conformation
Map data
Sample
  • Complex: SARS CoV2 Spike protein Ectodomain with Internal tag
    • Protein or peptide: Spike glycoprotein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsSARS CoV-2 Spike protein Ectodomain with internal tag / VIRAL PROTEIN
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 / 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 ...Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / 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 / symbiont-mediated suppression of host innate immune response / 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 / virion attachment to host cell / SARS-CoV-2 activates/modulates innate and adaptive immune responses / host cell plasma membrane / virion membrane / identical protein binding / membrane / 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 / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, betacoronavirus / Spike glycoprotein, N-terminal domain superfamily / 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 / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus ...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, betacoronavirus / Spike glycoprotein, N-terminal domain superfamily / 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 / 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 1 (HR1) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2
Similarity search - Domain/homology
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 2.32 Å
AuthorsSingh S / Hasan SS
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM150187 United States
CitationJournal: J Struct Biol X / Year: 2025
Title: Production and cryo-electron microscopy structure of an internally tagged SARS-CoV-2 spike ecto-domain construct.
Authors: Suruchi Singh / Yi Liu / Meghan Burke / Vamseedhar Rayaprolu / Stephen E Stein / S Saif Hasan /
Abstract: The SARS-CoV-2 spike protein is synthesized in the endoplasmic reticulum of host cells, from where it undergoes export to the Golgi and the plasma membrane or retrieval from the Golgi to the ...The SARS-CoV-2 spike protein is synthesized in the endoplasmic reticulum of host cells, from where it undergoes export to the Golgi and the plasma membrane or retrieval from the Golgi to the endoplasmic reticulum. Elucidating the fundamental principles of this bidirectional secretion are pivotal to understanding virus assembly and designing the next generation of spike genetic vaccine with enhanced export properties. However, the widely used strategy of C-terminal affinity tagging of the spike cytosolic tail interferes with proper bidirectional trafficking. Hence, the structural and biophysical investigations of spike protein trafficking have been hindered by a lack of appropriate spike constructs. Here we describe a strategy for the internal tagging of the spike protein. Using sequence analyses and AlphaFold modeling, we identified a site down-stream of the signal sequence for the insertion of a twin-strep-tag, which facilitates purification of an ecto-domain construct from the extra-cellular medium of mammalian Expi293F cells. Mass spectrometry analyses show that the internal tag has minimal impact on -glycan modifications, which are pivotal for spike-host interactions. Single particle cryo-electron microscopy reconstructions of the spike ecto-domain reveal conformational states compatible for ACE2 receptor interactions, further solidifying the feasibility of the internal tagging strategy. Collectively, these results present a substantial advance towards reagent development for the investigations of spike protein trafficking during coronavirus infection and genetic vaccination.
History
DepositionMar 12, 2024-
Header (metadata) releaseMar 5, 2025-
Map releaseMar 5, 2025-
UpdateMar 19, 2025-
Current statusMar 19, 2025Processing site: RCSB / Status: Released

-
Structure visualization

Supplemental images

emd_44062.png

Downloads & links

-
Map

FileDownload / File: emd_44062.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.99 Å/pix.
x 380 pix.
= 377. Å		0.99 Å/pix.
x 380 pix.
= 377. Å		0.99 Å/pix.
x 380 pix.
= 377. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.99211 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.092
Minimum - Maximum-0.42782125 - 1.0986935
Average (Standard dev.)-0.00008669453 (±0.019557675)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 377.00003 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Half map: #2

Fileemd_44062_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: #1

Fileemd_44062_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : SARS CoV2 Spike protein Ectodomain with Internal tag

EntireName: SARS CoV2 Spike protein Ectodomain with Internal tag
Components
  • Complex: SARS CoV2 Spike protein Ectodomain with Internal tag
    • Protein or peptide: Spike glycoprotein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

-
Supramolecule #1: SARS CoV2 Spike protein Ectodomain with Internal tag

SupramoleculeName: SARS CoV2 Spike protein Ectodomain with Internal tag / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 585 kDa/nm

-
Macromolecule #1: Spike glycoprotein

MacromoleculeName: Spike glycoprotein / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Molecular weightTheoretical: 140.679656 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MFVFLVLLPL VSSQCVNLSA WSHPQFEKGG GSGGGSGGSS AWSHPQFEKS ALVPRGSTTR TQLPPAYTNS FTRGVYYPDK VFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV N NATNVVIK ...String:
MFVFLVLLPL VSSQCVNLSA WSHPQFEKGG GSGGGSGGSS AWSHPQFEKS ALVPRGSTTR TQLPPAYTNS FTRGVYYPDK VFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI IRGWIFGTTL DSKTQSLLIV N NATNVVIK VCEFQFCNDP FLGVYYHKNN KSWMESEFRV YSSANNCTFE YVSQPFLMDL EGKQGNFKNL REFVFKNIDG YF KIYSKHT PINLVRDLPQ GFSALEPLVD LPIGINITRF QTLLALHRSY LTPGDSSSGW TAGAAAYYVG YLQPRTFLLK YNE NGTITD AVDCALDPLS ETKCTLKSFT VEKGIYQTSN FRVQPTESIV RFPNITNLCP FGEVFNATRF ASVYAWNRKR ISNC VADYS VLYNSASFST FKCYGVSPTK LNDLCFTNVY ADSFVIRGDE VRQIAPGQTG KIADYNYKLP DDFTGCVIAW NSNNL DSKV GGNYNYLYRL FRKSNLKPFE RDISTEIYQA GSTPCNGVEG FNCYFPLQSY GFQPTNGVGY QPYRVVVLSF ELLHAP ATV CGPKKSTNLV KNKCVNFNFN GLTGTGVLTE SNKKFLPFQQ FGRDIADTTD AVRDPQTLEI LDITPCSFGG VSVITPG TN TSNQVAVLYQ GVNCTEVPVA IHADQLTPTW RVYSTGSNVF QTRAGCLIGA EHVNNSYECD IPIGAGICAS YQTQTNSP G SASSVASQSI IAYTMSLGAE NSVAYSNNSI AIPTNFTISV TTEILPVSMT KTSVDCTMYI CGDSTECSNL LLQYGSFCT QLNRALTGIA VEQDKNTQEV FAQVKQIYKT PPIKDFGGFN FSQILPDPSK PSKRSFIEDL LFNKVTLADA GFIKQYGDCL GDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFGA GAALQIPFAM QMAYRFNGIG VTQNVLYENQ K LIANQFNS AIGKIQDSLS STASALGKLQ DVVNQNAQAL NTLVKQLSSN FGAISSVLND ILSRLDPPEA EVQIDRLITG RL QSLQTYV TQQLIRAAEI RASANLAATK MSECVLGQSK RVDFCGKGYH LMSFPQSAPH GVVFLHVTYV PAQEKNFTTA PAI CHDGKA HFPREGVFVS NGTHWFVTQR NFYEPQIITT DNTFVSGNCD VVIGIVNNTV YDPLQPELDS FKEELDKYFK NHTS PDVDL GDISGINASV VNIQKEIDRL NEVAKNLNES LIDLQELGKY EQGSGYIPEA PRDGQAYVRK DGEWVLLSTF

UniProtKB: Spike glycoprotein

-
Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 17 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

Concentration2.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloride
25.0 mMTris
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

-
Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average electron dose: 43.97 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 191780 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

+
Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.32 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 157865
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

-
Atomic model buiding 1

Initial modelPDB ID:

7krq


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9b0y:
SARS CoV-2 Spike protein Ectodomain with internal tag, all RBD-down conformation

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more