[English] 日本語
Yorodumi- EMDB-14539: CRYO-EM STRUCTURE OF SARS-COV-2 SPIKE : H11-H6 nanobody complex -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-14539 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | CRYO-EM STRUCTURE OF SARS-COV-2 SPIKE : H11-H6 nanobody complex | |||||||||
Map data | Sharpened map, output from postprocessing | |||||||||
Sample |
| |||||||||
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 / entry receptor-mediated virion attachment to host cell / host cell endoplasmic reticulum-Golgi intermediate compartment membrane / membrane fusion / receptor-mediated endocytosis of virus by host cell / Attachment and Entry / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / receptor ligand activity / symbiont-mediated suppression of host innate immune response / host cell surface receptor binding / 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 | |||||||||
Biological species | Severe acute respiratory syndrome coronavirus 2 / Lama glama (llama) / Escherichia virus T4 | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||
Authors | Weckener M / Naismith JH | |||||||||
Funding support | United Kingdom, 2 items
| |||||||||
Citation | Journal: 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 |
|
-Structure visualization
Supplemental images |
---|
-Downloads & links
-EMDB archive
Map data | emd_14539.map.gz | 12.9 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-14539-v30.xml emd-14539.xml | 29.4 KB 29.4 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_14539_fsc.xml | 10.7 KB | Display | FSC data file |
Images | emd_14539.png | 129.8 KB | ||
Masks | emd_14539_msk_1.map | 103 MB | Mask map | |
Others | emd_14539_additional_1.map.gz emd_14539_additional_2.map.gz emd_14539_half_map_1.map.gz emd_14539_half_map_2.map.gz | 6 MB 80.9 MB 80.9 MB 80.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-14539 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-14539 | HTTPS FTP |
-Validation report
Summary document | emd_14539_validation.pdf.gz | 647.5 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_14539_full_validation.pdf.gz | 647.1 KB | Display | |
Data in XML | emd_14539_validation.xml.gz | 17.8 KB | Display | |
Data in CIF | emd_14539_validation.cif.gz | 23.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-14539 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-14539 | HTTPS FTP |
-Related structure data
Related structure data | 7z7xMC 7z1aC 7z1bC 7z1cC 7z1dC 7z1eC 7z6vC 7z85C 7z86C 7z9qC 7z9rC M: atomic model generated by this map C: citing same article (ref.) |
---|---|
Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_14539.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Sharpened map, output from postprocessing | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.06 Å | ||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
|
-Supplemental data
-Mask #1
File | emd_14539_msk_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Additional map: Sharpened map, output from LocScale
File | emd_14539_additional_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Sharpened map, output from LocScale | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Additional map: Output from final 3D refinement
File | emd_14539_additional_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Output from final 3D refinement | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: Half map from final 3D refinement
File | emd_14539_half_map_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Half map from final 3D refinement | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: Half map from final 3D refinement
File | emd_14539_half_map_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Half map from final 3D refinement | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Sample components
-Entire : Complex between Spike and nanobody H11-H6
Entire | Name: Complex between Spike and nanobody H11-H6 |
---|---|
Components |
|
-Supramolecule #1: Complex between Spike and nanobody H11-H6
Supramolecule | Name: Complex between Spike and nanobody H11-H6 / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 |
---|---|
Molecular weight | Theoretical: 15 KDa |
-Supramolecule #2: Spike glycoprotein trimer with fibritin tag
Supramolecule | Name: 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 expression | Organism: Homo sapiens (human) / Recombinant strain: HEK 293T |
-Supramolecule #3: Nanobody H11-H6
Supramolecule | Name: Nanobody H11-H6 / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2 |
---|---|
Source (natural) | Organism: Lama glama (llama) |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: WK6 |
-Macromolecule #1: Spike glycoprotein,Fibritin
Macromolecule | Name: Spike glycoprotein,Fibritin / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: Escherichia virus T4 |
Molecular weight | Theoretical: 139.877906 KDa |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: 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 |
-Macromolecule #2: Nanobody H11-H6
Macromolecule | Name: Nanobody H11-H6 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: Lama glama (llama) |
Molecular weight | Theoretical: 14.897658 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: QVQLVESGGG LMQAGGSLRL SCAVSGRTFS TAAMGWFRQA PGKEREFVAA IRWSGGSAYY ADSVKGRFTI SRDKAKNTVY LQMNSLKYE DTAVYYCAGS KITRSLLSDY ATWPYDYWGQ GTQVTVSSKH HHHHH |
-Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose
Macromolecule | Name: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 26 / Formula: NAG |
---|---|
Molecular weight | Theoretical: 221.208 Da |
Chemical component information | ChemComp-NAG: |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Buffer | pH: 8 Component:
| |||||||||
Grid | Model: Homemade / Material: COPPER/RHODIUM / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE | |||||||||
Vitrification | Cryogen name: ETHANE / Instrument: SPOTITON Details: SPT Labtech prototype 300 mesh 1.2/2.0 nanowire grids with a highly reproduceable rectangular bar cross-section were used. The grids were glow-discharged on low for 120 s (Plasma Cleaner PDC- ...Details: SPT Labtech prototype 300 mesh 1.2/2.0 nanowire grids with a highly reproduceable rectangular bar cross-section were used. The grids were glow-discharged on low for 120 s (Plasma Cleaner PDC-002-CE, Harrick Plasma) or for 60-80 s, 12 mA (easiGlow, Pelco) to activate the nanowires. Approximately 6 nL of the complex were applied to the grids using a Chameleon EP system (SPT Labtech) at 79-81% relative humidity and ambient temperature.. |
-Electron microscopy
Microscope | TFS KRIOS |
---|---|
Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV |
Details | EPU auto-function coma free correction |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 4 / Average exposure time: 3.0 sec. / Average electron dose: 50.0 e/Å2 / Details: The images were collected as 50 frame movies |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |