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- EMDB-14576: CRYO-EM STRUCTURE OF SARS-COV-2 SPIKE : H11-H4 Q98R H100E nanobod... -

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Basic information

Entry
Database: EMDB / ID: EMD-14576
TitleCRYO-EM STRUCTURE OF SARS-COV-2 SPIKE : H11-H4 Q98R H100E nanobody complex in 2Up1Down conformation
Map dataSharpened map, output from postprocessing.
Sample
  • Complex: Complex between Spike and nanobody H11-H4 Q98R H100E
    • Complex: Spike glycoprotein trimer with fibritin tag
      • Protein or peptide: Spike glycoprotein,Fibritin
    • Complex: Nanobody H11-H4 Q98R H100E
      • Protein or peptide: Nanobody H11-H4 Q98R H100E
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


virion component / 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 ...virion component / 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
Fibritin C-terminal / Fibritin C-terminal region / 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 ...Fibritin C-terminal / Fibritin C-terminal region / 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 / Fibritin
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2 / Lama glama (llama) / Escherichia virus T4
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsWeckener M / Naismith JH
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Engineering and Physical Sciences Research CouncilP2019-0006 United Kingdom
Wellcome Trust100209/Z/12/Z United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Correlation between the binding affinity and the conformational entropy of nanobody SARS-CoV-2 spike protein complexes.
Authors: Halina Mikolajek / Miriam Weckener / Z Faidon Brotzakis / Jiandong Huo / Evmorfia V Dalietou / Audrey Le Bas / Pietro Sormanni / Peter J Harrison / Philip N Ward / Steven Truong / Lucile ...Authors: Halina Mikolajek / Miriam Weckener / Z Faidon Brotzakis / Jiandong Huo / Evmorfia V Dalietou / Audrey Le Bas / Pietro Sormanni / Peter J Harrison / Philip N Ward / Steven Truong / Lucile Moynie / Daniel K Clare / Maud Dumoux / Joshua Dormon / Chelsea Norman / Naveed Hussain / Vinod Vogirala / Raymond J Owens / Michele Vendruscolo / James H Naismith /
Abstract: Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. ...Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure-activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein-nanobody complex are associated with tighter binding. To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.
History
DepositionMar 21, 2022-
Header (metadata) releaseJul 13, 2022-
Map releaseJul 13, 2022-
UpdateOct 5, 2022-
Current statusOct 5, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14576.png

Downloads & links

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Map

FileDownload / File: emd_14576.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map, output from postprocessing.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 300 pix.
= 321.6 Å		1.07 Å/pix.
x 300 pix.
= 321.6 Å		1.07 Å/pix.
x 300 pix.
= 321.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.072 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.012
Minimum - Maximum-0.04936798 - 0.097599305
Average (Standard dev.)0.00027588467 (±0.0025593287)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 321.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14576_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Map from final 3D refinement.

Fileemd_14576_additional_1.map
AnnotationMap from final 3D refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened map, output from LocScale.

Fileemd_14576_additional_2.map
AnnotationSharpened map, output from LocScale.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map from final 3D refinement.

Fileemd_14576_half_map_1.map
AnnotationHalf map from final 3D refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map from final 3D refinement.

Fileemd_14576_half_map_2.map
AnnotationHalf map from final 3D refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex between Spike and nanobody H11-H4 Q98R H100E

EntireName: Complex between Spike and nanobody H11-H4 Q98R H100E
Components
  • Complex: Complex between Spike and nanobody H11-H4 Q98R H100E
    • Complex: Spike glycoprotein trimer with fibritin tag
      • Protein or peptide: Spike glycoprotein,Fibritin
    • Complex: Nanobody H11-H4 Q98R H100E
      • Protein or peptide: Nanobody H11-H4 Q98R H100E
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Complex between Spike and nanobody H11-H4 Q98R H100E

SupramoleculeName: Complex between Spike and nanobody H11-H4 Q98R H100E / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: The Spike protein is found in the 2Up1Down conformation.
Molecular weightTheoretical: 15 KDa

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Supramolecule #2: Spike glycoprotein trimer with fibritin tag

SupramoleculeName: Spike glycoprotein trimer with fibritin tag / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Severe acute respiratory syndrome coronavirus 2
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK 293T

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Supramolecule #3: Nanobody H11-H4 Q98R H100E

SupramoleculeName: Nanobody H11-H4 Q98R H100E / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Lama glama (llama)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: WK6

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Macromolecule #1: Spike glycoprotein,Fibritin

MacromoleculeName: Spike glycoprotein,Fibritin / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia virus T4
Molecular weightTheoretical: 139.877906 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 KPSKRSFIED LLFNKVTLAD AGFIKQYGDC LGDIAARDLI CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFG AGAALQIPFA MQMAYRFNGI GVTQNVLYEN QKLIANQFNS AIGKIQDSLS STASALGKLQ 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 FLSLLNDIFE AQKIEWHEKH HHHHH

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Macromolecule #2: Nanobody H11-H4 Q98R H100E

MacromoleculeName: Nanobody H11-H4 Q98R H100E / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 14.107723 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVESGGG LMQAGGSLRL SCAVSGRTFS TAAMGWFRQA PGKEREFVAA IRWSGGSAYY ADSVKGRFTI SRDKAKNTVY LQMNSLKYE DTAVYYCART EYVSYLLSDY ATWPYDYWGQ GTQVTVSS

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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: 26 / 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 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
10.0 mMHEPESHEPES
150.0 mMNaClSodium chlorideSodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: Grids were glow discharged twice, 60s each.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 289.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS 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 / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsEPU auto-function coma free correction
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Average exposure time: 3.85 sec. / Average electron dose: 50.0 e/Å2 / Details: The images were collected as 50 frame movies
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 909790
CTF correctionSoftware - Name: Gctf
Startup modelType of model: EMDB MAP
EMDB ID:

21374

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.08)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.08)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.08)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.08) / Number images used: 110410
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6zhd

chain_id: A, residue_range: 27-1147

6zhd

chain_id: B, residue_range: 27-1147
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT
Output model

PDB-7z9r:
CRYO-EM STRUCTURE OF SARS-COV-2 SPIKE : H11-H4 Q98R H100E nanobody complex in 2Up1Down conformation

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