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- EMDB-24105: CryoEm structure of SARS-CoV-2 spike protein (S-6P, 1-up) in comp... -

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Basic information

Entry
Database: EMDB / ID: EMD-24105
TitleCryoEm structure of SARS-CoV-2 spike protein (S-6P, 1-up) in complex with sybodies (Sb45)
Map dataCryoEM map of SARS-CoV-2 Spike protein (S-6P) in complex with Sybodies (Sb45)
Sample
  • Complex: Spike protein (S-6P) in complex with Synthetic nanobody (Sb45)
    • Protein or peptide: Spike glycoproteinSpike protein
    • Protein or peptide: Synthetic nanobody (sybody), Sb45
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
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 / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / host cell endoplasmic reticulum-Golgi intermediate compartment membrane ...Maturation of spike protein / viral translation / Translation of Structural Proteins / Virion Assembly and Release / host cell surface / host extracellular space / suppression by virus of host tetherin activity / Induction of Cell-Cell Fusion / structural constituent of virion / 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 / fusion of virus membrane with host plasma membrane / 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 / membrane / identical protein binding / 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 / 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 ...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
Biological speciessynthetic construct (others) / Severe acute respiratory syndrome coronavirus 2
Methodsingle particle reconstruction / cryo EM / Resolution: 3.02 Å
AuthorsJiang J / Huang R / Margulies D
CitationJournal: J Biol Chem / Year: 2021
Title: Structures of synthetic nanobody-SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction.
Authors: Javeed Ahmad / Jiansheng Jiang / Lisa F Boyd / Allison Zeher / Rick Huang / Di Xia / Kannan Natarajan / David H Margulies /
Abstract: Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of ...Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16-RBD, Sb45-RBD, Sb14-RBD-Sb68, and Sb45-RBD-Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD-angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both "up" and "down" configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.
History
DepositionMay 25, 2021-
Header (metadata) releaseJun 2, 2021-
Map releaseJun 2, 2021-
UpdateOct 20, 2021-
Current statusOct 20, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7n0g
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24105.png

Downloads & links

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Map

FileDownload / File: emd_24105.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM map of SARS-CoV-2 Spike protein (S-6P) in complex with Sybodies (Sb45)
Voxel sizeX=Y=Z: 1.052 Å
Density
Contour LevelBy AUTHOR: 0.0429 / Movie #1: 0.07
Minimum - Maximum-0.3285856 - 0.97729623
Average (Standard dev.)-0.0005494764 (±0.018468276)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 420.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0521.0521.052
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z420.800420.800420.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ401401401
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.3290.977-0.001

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Supplemental data

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Mask #1

Fileemd_24105_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Local resolution map

Fileemd_24105_additional_1.map
AnnotationLocal resolution map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Spike protein (S-6P) in complex with Synthetic nanobody (Sb45)

EntireName: Spike protein (S-6P) in complex with Synthetic nanobody (Sb45)
Components
  • Complex: Spike protein (S-6P) in complex with Synthetic nanobody (Sb45)
    • Protein or peptide: Spike glycoproteinSpike protein
    • Protein or peptide: Synthetic nanobody (sybody), Sb45
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Spike protein (S-6P) in complex with Synthetic nanobody (Sb45)

SupramoleculeName: Spike protein (S-6P) in complex with Synthetic nanobody (Sb45)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Synthetic nanobody Sb45 was mixed with freshly purified S-6P in the mole ratio of 3:1, incubated, and subjected to Negative stain and frozen grids for cryoEM data collection.
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
Molecular weightTheoretical: 600 KDa

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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: 142.427438 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDG VYFASTEKSN IIRGWIFGTT LDSKTQSLLI VNNATNVVIK VCEFQFCNDP FLGVYYHKNN KSWMESEFRV Y SSANNCTF ...String:
MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDG VYFASTEKSN IIRGWIFGTT LDSKTQSLLI VNNATNVVIK VCEFQFCNDP FLGVYYHKNN KSWMESEFRV Y SSANNCTF EYVSQPFLMD LEGKQGNFKN LREFVFKNID GYFKIYSKHT PINLVRDLPQ GFSALEPLVD LPIGINITRF QT LLALHRS YLTPGDSSSG WTAGAAAYYV GYLQPRTFLL KYNENGTITD AVDCALDPLS ETKCTLKSFT VEKGIYQTSN FRV QPTESI VRFPNITNLC PFGEVFNATR FASVYAWNRK RISNCVADYS VLYNSASFST FKCYGVSPTK LNDLCFTNVY ADSF VIRGD EVRQIAPGQT GKIADYNYKL PDDFTGCVIA WNSNNLDSKV GGNYNYLYRL FRKSNLKPFE RDISTEIYQA GSTPC NGVE GFNCYFPLQS YGFQPTNGVG YQPYRVVVLS FELLHAPATV CGPKKSTNLV KNKCVNFNFN GLTGTGVLTE SNKKFL PFQ QFGRDIADTT DAVRDPQTLE ILDITPCSFG GVSVITPGTN TSNQVAVLYQ DVNCTEVPVA IHADQLTPTW RVYSTGS NV FQTRAGCLIG AEHVNNSYEC DIPIGAGICA SYQTQTNSPG SASSVASQSI IAYTMSLGAE NSVAYSNNSI AIPTNFTI S VTTEILPVSM TKTSVDCTMY ICGDSTECSN LLLQYGSFCT QLNRALTGIA VEQDKNTQEV FAQVKQIYKT PPIKDFGGF NFSQILPDPS KPSKRSPIED LLFNKVTLAD AGFIKQYGDC LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFG AGPALQIPFP MQMAYRFNGI GVTQNVLYEN QKLIANQFNS AIGKIQDSLS STPSALGKLQ DVVNQNAQAL N TLVKQLSS NFGAISSVLN DILSRLDPPE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI RASANLAATK MSECVLGQSK RV DFCGKGY HLMSFPQSAP HGVVFLHVTY VPAQEKNFTT APAICHDGKA HFPREGVFVS NGTHWFVTQR NFYEPQIITT DNT FVSGNC DVVIGIVNNT VYDPLQPELD SFKEELDKYF KNHTSPDVDL GDISGINASV VNIQKEIDRL NEVAKNLNES LIDL QELGK YEQGSGYIPE APRDGQAYVR KDGEWVLLST FLGRSLEVLF QGPGHHHHHH HHSAWSHPQF EKGGGSGGGG SGGSA WSHP QFEK

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Macromolecule #2: Synthetic nanobody (sybody), Sb45

MacromoleculeName: Synthetic nanobody (sybody), Sb45 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.574048 KDa
Recombinant expressionOrganism: Escherichia coli MC1061 (bacteria)
SequenceString:
QVQLVESGGG LVQAGGSLRL SCAASGFPVY RDRMAWYRQA PGKEREWVAA IYSAGQQTRY ADSVKGRFTI SRDNAKNTVY LQMNSLKPE DTAVYYCNVK DVGHHYEYYD YWGQGTQVTV SA

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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: 35 / 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

Concentration0.6 mg/mL
BufferpH: 8 / Component - Concentration: 0.25 mg/mL / Component - Name: TBS
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 120.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 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: 100.0 µm / Calibrated defocus max: 2.431 µm / Calibrated defocus min: 0.535 µm / Calibrated magnification: 47529 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 78.0 K / Max: 80.0 K
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Sampling interval: 5.001 µm / Number grids imaged: 2 / Number real images: 9725 / Average exposure time: 8.0 sec. / Average electron dose: 56.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1433963
Details: A total of 9,725 micrographs were imported into cryoSPARC. Following patch Motion correction, patch CTF estimation, and curation, the number of micrographs was reduced to 9,237. The blob ...Details: A total of 9,725 micrographs were imported into cryoSPARC. Following patch Motion correction, patch CTF estimation, and curation, the number of micrographs was reduced to 9,237. The blob picker with the particle diameter between 128 and 256 angstroms was used for picking up particles. We used the box size of 400 pixels and extracted 1,433,963 particles initially.
CTF correctionSoftware - Name: cryoSPARC (ver. v3.2.0) / Details: patch CTF estimation
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6xkl


Details: The initial model was built based on 6XKL, but RBD and Sb45 are from 7GKJ and dock on S-6P (6XKL). NTD was replaced by 7B32 with a full sequence.
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 100 / Software - Name: cryoSPARC (ver. 3.2.0)
Details: The best 18 classes were selected with 662,994 particles. 3D classifications further identify two conformations of S-6P: 1-up RBD with 214,171 particles, 2-up RBD with 61,026 particles
Final angle assignmentType: NOT APPLICABLE
Final reconstructionNumber classes used: 18 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Resolution.type: BY AUTHOR / Resolution: 3.02 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. V3.2.0)
Details: A series of "Ab-initio 3D reconstruction" dividing 2 or 4 subclasses to identify two forms of configurations of S-6P, i.e. one RBD up or two RBD up.
Number images used: 214171
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6xkl

chain_id: A, residue_range: 529-1146

6xkl

chain_id: B, residue_range: 529-1146

6xkl

chain_id: C, residue_range: 529-1146

7kgj

chain_id: A, residue_range: 334-528

7kgj

chain_id: B, residue_range: 1-121

7b32

chain_id: A, residue_range: 14-289
DetailsAn initial model for S-6P was generated using PDB 6XKL and rigid body fitted into the map using Chimera. The RBD domain (334-528) and Sb45 are taken from 7KGJ which was superimposed onto the S-6P model in PyMol. The NTD domain (14-289) is taken from 7B32 with full sequence and replace that of 6XKL. We have rebuilt and added more glycans (NAGs). We used the real-space refinement in PHENIX including rigid-body refinement. RBD and NTD are subjected to rigid-body refinement. Simulate annealing (SA) runs once at the initial micro-step, local grid search and ADP refinements were included, using the secondary structure restraints.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 157 / Target criteria: Correlation Coefficient
Output model

PDB-7n0g:
CryoEm structure of SARS-CoV-2 spike protein (S-6P, 1-up) in complex with sybodies (Sb45)

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