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Yorodumi- EMDB-10597: Cryo-EM structure of murine norovirus virions in complex with neu... -
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-Basic information
Entry | Database: EMDB / ID: EMD-10597 | |||||||||
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Title | Cryo-EM structure of murine norovirus virions in complex with neutralizing Nanobody NB-5829 | |||||||||
Map data | Cryo-EM structure of murine norovirus virions in complex with neutralizing Nanobody NB-5829 | |||||||||
Sample |
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Biological species | Murine norovirus | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.7 Å | |||||||||
Authors | Hansman GS / Devant JM | |||||||||
Citation | Journal: J Virol / Year: 2020 Title: Nanobody-Mediated Neutralization Reveals an Achilles Heel for Norovirus. Authors: Anna D Koromyslova / Jessica M Devant / Turgay Kilic / Charles D Sabin / Virginie Malak / Grant S Hansman / Abstract: Human norovirus frequently causes outbreaks of acute gastroenteritis. Although discovered more than five decades ago, antiviral development has, until recently, been hampered by the lack of a ...Human norovirus frequently causes outbreaks of acute gastroenteritis. Although discovered more than five decades ago, antiviral development has, until recently, been hampered by the lack of a reliable human norovirus cell culture system. Nevertheless, a lot of pathogenesis studies were accomplished using murine norovirus (MNV), which can be grown routinely in cell culture. In this study, we analyzed a sizeable library of nanobodies that were raised against the murine norovirus virion with the main purpose of developing nanobody-based inhibitors. We discovered two types of neutralizing nanobodies and analyzed the inhibition mechanisms using X-ray crystallography, cryo-electron microscopy (cryo-EM), and cell culture techniques. The first type bound on the top region of the protruding (P) domain. Interestingly, this nanobody binding region closely overlapped the MNV receptor-binding site and collectively shared numerous P domain-binding residues. In addition, we showed that these nanobodies competed with the soluble receptor, and this action blocked virion attachment to cultured cells. The second type bound at a dimeric interface on the lower side of the P dimer. We discovered that these nanobodies disrupted a structural change in the capsid associated with binding cofactors (i.e., metal cations/bile acid). Indeed, we found that capsids underwent major conformational changes following addition of Mg or Ca Ultimately, these nanobodies directly obstructed a structural modification reserved for a postreceptor attachment stage. Altogether, our new data show that nanobody-based inhibition could occur by blocking functional and structural capsid properties. This research discovered and analyzed two different types of MNV-neutralizing nanobodies. The top-binding nanobodies sterically inhibited the receptor-binding site, whereas the dimeric-binding nanobodies interfered with a structural modification associated with cofactor binding. Moreover, we found that the capsid contained a number of vulnerable regions that were essential for viral replication. In fact, the capsid appeared to be organized in a state of flux, which could be important for cofactor/receptor-binding functions. Blocking these capsid-binding events with nanobodies directly inhibited essential capsid functions. Moreover, a number of MNV-specific nanobody binding epitopes were comparable to human norovirus-specific nanobody inhibitors. Therefore, this additional structural and inhibition information could be further exploited in the development of human norovirus antivirals. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_10597.map.gz | 430.5 MB | EMDB map data format | |
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Header (meta data) | emd-10597-v30.xml emd-10597.xml | 10.7 KB 10.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_10597_fsc.xml | 17.7 KB | Display | FSC data file |
Images | emd_10597.png | 184.2 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-10597 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-10597 | HTTPS FTP |
-Validation report
Summary document | emd_10597_validation.pdf.gz | 298.1 KB | Display | EMDB validaton report |
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Full document | emd_10597_full_validation.pdf.gz | 297.2 KB | Display | |
Data in XML | emd_10597_validation.xml.gz | 15.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-10597 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-10597 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_10597.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Cryo-EM structure of murine norovirus virions in complex with neutralizing Nanobody NB-5829 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.368 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Murine norovirus
Entire | Name: Murine norovirus |
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Components |
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-Supramolecule #1: Murine norovirus
Supramolecule | Name: Murine norovirus / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 357231 / Sci species name: Murine norovirus / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No |
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Host (natural) | Organism: Mus musculus (house mouse) |
Host system | Organism: Mus musculus (house mouse) / Recombinant strain: RAW 264.7 |
Virus shell | Shell ID: 1 / Diameter: 460.0 Å / T number (triangulation number): 3 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 7.4 / Component - Name: PBS |
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER/RHODIUM / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV |
Details | 1 mg/ml virions, incubated with equal amount of Nanobody for 30 min at RT, prior to freezing |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 3599 / Average electron dose: 15.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 64000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |