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- EMDB-22161: Cryo-EM structure of a biotinylated SARS-CoV-2 spike probe in the... -

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Basic information

Entry
Database: EMDB / ID: EMD-22161
TitleCryo-EM structure of a biotinylated SARS-CoV-2 spike probe in the prefusion state (RBDs down)
Map datastructure of a biotinylated SARS-CoV-2 spike probe in the prefusion state
Sample
  • Organelle or cellular component: SARS-CoV-2 spike ectodomain biotinylated probe in the prefusion state
    • Protein or peptide: Spike glycoproteinSpike protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / Attachment and Entry / receptor-mediated virion attachment to host cell / endoplasmic reticulum-Golgi intermediate compartment / host cell surface receptor binding / endocytosis involved in viral entry into host cell ...Maturation of spike protein / Translation of Structural Proteins / Virion Assembly and Release / suppression by virus of host tetherin activity / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / Attachment and Entry / receptor-mediated virion attachment to host cell / endoplasmic reticulum-Golgi intermediate compartment / host cell surface receptor binding / endocytosis involved in viral entry into host cell / endocytic vesicle membrane / fusion of virus membrane with host plasma membrane / suppression by virus of host type I interferon-mediated signaling pathway / fusion of virus membrane with host endosome membrane / viral envelope / viral entry into host cell / endoplasmic reticulum lumen / host cell plasma membrane / virion membrane / integral component of membrane / identical protein binding
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 / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Spike glycoprotein, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein S2, coronavirus, heptad repeat 2 ...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. / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike glycoprotein, N-terminal domain superfamily / Spike glycoprotein, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / 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
Biological speciesSevere acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.45 Å
AuthorsCerutti G / Gorman J / Kwong PD / Shapiro L
CitationJournal: SSRN / Year: 2020
Title: Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes.
Authors: Tongqing Zhou / I-Ting Teng / Adam S Olia / Gabriele Cerutti / Jason Gorman / Alexandra Nazzari / Wei Shi / Yaroslav Tsybovsky / Lingshu Wang / Shuishu Wang / Baoshan Zhang / Yi Zhang / ...Authors: Tongqing Zhou / I-Ting Teng / Adam S Olia / Gabriele Cerutti / Jason Gorman / Alexandra Nazzari / Wei Shi / Yaroslav Tsybovsky / Lingshu Wang / Shuishu Wang / Baoshan Zhang / Yi Zhang / Phinikoula S Katsamba / Yuliya Petrova / Bailey B Banach / Ahmed S Fahad / Lihong Liu / Sheila N Lopez Acevedo / Bharat Madan / Matheus Olivera de Souza / Xiaoli Pan / Pengfei Wang / Jacy R Wolfe / Michael Yin / David D Ho / Emily Phung / Anthony DiPiazza / Lauren Chang / Olubukula Abiona / Kizzmekia S Corbett / Brandon J DeKosky / Barney S Graham / John R Mascola / John Misasi / Tracy Ruckwardt / Nancy J Sullivan / Lawrence Shapiro / Peter D Kwong /
Abstract: Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To ...Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ~0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes. Funding: Support for this work was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID). Support for this work was also provided by COVID-19 Fast Grants, the Jack Ma Foundation, the Self Graduate Fellowship Program, and NIH grants DP5OD023118, R21AI143407, and R21AI144408. Some of this work was performed at the Columbia University Cryo-EM Center at the Zuckerman Institute, and some at the Simons Electron Microscopy Center (SEMC) and National Center for Cryo-EM Access and Training (NCCAT) located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR, and the NIH National Institute of General Medical Sciences (GM103310). Conflict of Interest: The authors declare that they have no conflict of interest. Ethical Approval: Peripheral blood mononuclear cells (PBMCs) for B cell sorting were obtained from a convalescent SARS-CoV-2 patient (collected 75 days post symptom onset under an IRB approved clinical trial protocol, VRC 200 - ClinicalTrials.gov Identifier: NCT00067054) and a healthy control donor from the NIH blood bank pre-SARS-CoV-2 pandemic.
History
DepositionJun 15, 2020-
Header (metadata) releaseSep 2, 2020-
Map releaseSep 2, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xf5
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_22161.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationstructure of a biotinylated SARS-CoV-2 spike probe in the prefusion state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 440 pix.
= 470.8 Å
1.07 Å/pix.
x 440 pix.
= 470.8 Å
1.07 Å/pix.
x 440 pix.
= 470.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.17 / Movie #1: 0.17
Minimum - Maximum-0.24025865 - 0.77692235
Average (Standard dev.)-0.0003268762 (±0.01761238)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 470.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z440440440
origin x/y/z0.0000.0000.000
length x/y/z470.800470.800470.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ512512512
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS440440440
D min/max/mean-0.2400.777-0.000

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Supplemental data

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Additional map: Unsharpened map

Fileemd_22161_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Local refinement map (C1 symmetry applied)

Fileemd_22161_additional_2.map
AnnotationLocal refinement map (C1 symmetry applied)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: structure of a biotinylated SARS-CoV-2 spike probe in...

Fileemd_22161_half_map_1.map
Annotationstructure of a biotinylated SARS-CoV-2 spike probe in the prefusion state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: structure of a biotinylated SARS-CoV-2 spike probe in...

Fileemd_22161_half_map_2.map
Annotationstructure of a biotinylated SARS-CoV-2 spike probe in the prefusion state
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : SARS-CoV-2 spike ectodomain biotinylated probe in the prefusion state

EntireName: SARS-CoV-2 spike ectodomain biotinylated probe in the prefusion state
Components
  • Organelle or cellular component: SARS-CoV-2 spike ectodomain biotinylated probe in the prefusion state
    • Protein or peptide: Spike glycoproteinSpike protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: SARS-CoV-2 spike ectodomain biotinylated probe in the prefusion state

SupramoleculeName: SARS-CoV-2 spike ectodomain biotinylated probe in the prefusion state
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human)

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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: 138.723297 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPQCVNLTTR TQLPPAYTNS FTRGVYYPDK VFRSSVLHST QDLFLPFFSN VTWFHAIHVS GTNGTKRFDN PVLPFNDGVY FASTEKSNI IRGWIFGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY V SQPFLMDL ...String:
GPQCVNLTTR TQLPPAYTNS FTRGVYYPDK VFRSSVLHST QDLFLPFFSN VTWFHAIHVS GTNGTKRFDN PVLPFNDGVY FASTEKSNI IRGWIFGTTL DSKTQSLLIV NNATNVVIKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY V SQPFLMDL EGKQGNFKNL REFVFKNIDG YFKIYSKHTP INLVRDLPQG FSALEPLVDL PIGINITRFQ TLLALHRSYL TP GDSSSGW TAGAAAYYVG YLQPRTFLLK YNENGTITDA VDCALDPLSE TKCTLKSFTV EKGIYQTSNF RVQPTESIVR FPN ITNLCP FGEVFNATRF ASVYAWNRKR ISNCVADYSV LYNSASFSTF KCYGVSPTKL NDLCFTNVYA DSFVIRGDEV RQIA PGQTG KIADYNYKLP DDFTGCVIAW NSNNLDSKVG GNYNYLYRLF RKSNLKPFER DISTEIYQAG STPCNGVEGF NCYFP LQSY GFQPTNGVGY QPYRVVVLSF ELLHAPATVC GPKKSTNLVK NKCVNFNFNG LTGTGVLTES NKKFLPFQQF GRDIAD TTD AVRDPQTLEI LDITPCSFGG VSVITPGTNT SNQVAVLYQD VNCTEVPVAI HADQLTPTWR VYSTGSNVFQ TRAGCLI GA EHVNNSYECD IPIGAGICAS YQTQTNSPGS ASSVASQSII AYTMSLGAEN SVAYSNNSIA IPTNFTISVT TEILPVSM T KTSVDCTMYI CGDSTECSNL LLQYGSFCTQ LNRALTGIAV EQDKNTQEVF AQVKQIYKTP PIKDFGGFNF SQILPDPSK PSKRSFIEDL LFNKVTLADA GFIKQYGDCL GDIAARDLIC AQKFNGLTVL PPLLTDEMIA QYTSALLAGT ITSGWTFGAG AALQIPFAM QMAYRFNGIG VTQNVLYENQ KLIANQFNSA IGKIQDSLSS TASALGKLQD VVNQNAQALN TLVKQLSSNF G AISSVLND ILSRLDPPEA EVQIDRLITG RLQSLQTYVT QQLIRAAEIR ASANLAATKM SECVLGQSKR VDFCGKGYHL MS FPQSAPH GVVFLHVTYV PAQEKNFTTA PAICHDGKAH FPREGVFVSN GTHWFVTQRN FYEPQIITTD NTFVSGNCDV VIG IVNNTV YDPLQPELDS FKEELDKYFK NHTSPDVDLG DISGINASVV NIQKEIDRLN EVAKNLNESL IDLQELGKYE QGSG YIPEA PRDGQAYVRK DGEWVLLSTF LGRSGGGLVP QQSGGLNDIF EAQKIEWHEG

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 36 / 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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 42.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 43176

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Atomic model buiding 1

Initial modelPDB ID:
RefinementProtocol: FLEXIBLE FIT
Output model

PDB-6xf5:
Cryo-EM structure of a biotinylated SARS-CoV-2 spike probe in the prefusion state (RBDs down)

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