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Yorodumi- EMDB-0141: Cryo-EM structure of in vitro assembled Measles virus N into nucl... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-0141 | ||||||||||||||||||
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Title | Cryo-EM structure of in vitro assembled Measles virus N into nucleocapsid-like particles (NCLPs) bound to polyA RNA hexamers. | ||||||||||||||||||
Map data | Cryo-EM map of in vitro assembled Measles virus N into nucleocapsid-like particles (NCLPs) bound to polyA RNA hexamers. | ||||||||||||||||||
Sample |
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Keywords | Measles / Nucleocapsid / Helical / RNA / VIRAL PROTEIN | ||||||||||||||||||
Function / homology | Paramyxovirus nucleocapsid protein / Paramyxovirus nucleocapsid protein / helical viral capsid / viral nucleocapsid / host cell cytoplasm / ribonucleoprotein complex / structural molecule activity / RNA binding / Nucleocapsid Function and homology information | ||||||||||||||||||
Biological species | Measles virus / synthetic construct (others) / Measles morbillivirus | ||||||||||||||||||
Method | helical reconstruction / cryo EM / Resolution: 3.3 Å | ||||||||||||||||||
Authors | Desfosses A / Milles S | ||||||||||||||||||
Funding support | France, 5 items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2019 Title: Assembly and cryo-EM structures of RNA-specific measles virus nucleocapsids provide mechanistic insight into paramyxoviral replication. Authors: Ambroise Desfosses / Sigrid Milles / Malene Ringkjøbing Jensen / Serafima Guseva / Jacques-Philippe Colletier / Damien Maurin / Guy Schoehn / Irina Gutsche / Rob W H Ruigrok / Martin Blackledge / Abstract: Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control ...Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5') in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5' and 3' binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3' end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly. | ||||||||||||||||||
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_0141.map.gz | 25.8 MB | EMDB map data format | |
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Header (meta data) | emd-0141-v30.xml emd-0141.xml | 15 KB 15 KB | Display Display | EMDB header |
Images | emd_0141.png | 173.7 KB | ||
Filedesc metadata | emd-0141.cif.gz | 6.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-0141 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-0141 | HTTPS FTP |
-Validation report
Summary document | emd_0141_validation.pdf.gz | 278 KB | Display | EMDB validaton report |
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Full document | emd_0141_full_validation.pdf.gz | 277.1 KB | Display | |
Data in XML | emd_0141_validation.xml.gz | 6.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-0141 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-0141 | HTTPS FTP |
-Related structure data
Related structure data | 6h5qMC 0142C 6h5sC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_0141.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Cryo-EM map of in vitro assembled Measles virus N into nucleocapsid-like particles (NCLPs) bound to polyA RNA hexamers. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.816 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 : Measles virus Nucleoprotein assembled into nucleocapsid-like part...
Entire | Name: Measles virus Nucleoprotein assembled into nucleocapsid-like particles (NCLPs) bound to polyA RNA hexamers. |
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Components |
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-Supramolecule #1: Measles virus Nucleoprotein assembled into nucleocapsid-like part...
Supramolecule | Name: Measles virus Nucleoprotein assembled into nucleocapsid-like particles (NCLPs) bound to polyA RNA hexamers. type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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-Supramolecule #2: Measles virus Nucleoprotein
Supramolecule | Name: Measles virus Nucleoprotein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: Measles virus |
-Supramolecule #3: polyA RNA
Supramolecule | Name: polyA RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
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Source (natural) | Organism: synthetic construct (others) |
-Macromolecule #1: Nucleocapsid
Macromolecule | Name: Nucleocapsid / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Measles morbillivirus |
Molecular weight | Theoretical: 46.425863 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MATLLRSLAL FKRNKDKPPI TSGSGGAIRG IKHIIIVPIP GDSSITTRSR LLDRLVRLIG NPDVSGPKLT GALIGILSLF VESPGQLIQ RITDDPDVSI RLLEVVQSDQ SQSGLTFASR GTNMEDEADQ YFSHDDPISS DQSRFGWFEN KEISDIEVQD P EGFNMILG ...String: MATLLRSLAL FKRNKDKPPI TSGSGGAIRG IKHIIIVPIP GDSSITTRSR LLDRLVRLIG NPDVSGPKLT GALIGILSLF VESPGQLIQ RITDDPDVSI RLLEVVQSDQ SQSGLTFASR GTNMEDEADQ YFSHDDPISS DQSRFGWFEN KEISDIEVQD P EGFNMILG TILAQIWVLL AKAVTAPDTA ADSELRRWIK YTQQRRVVGE FRLERKWLDV VRNRIAEDLS LRRFMVALIL DI KRTPGNK PRIAEMICDI DTYIVEAGLA SFILTIKFGI ETMYPALGLH EFAGELSTLE SLMNLYQQMG ETAPYMVILE NSI QNKFSA GSYPLLWSYA MGVGVELENS MGGLNFGRSY FDPAYFRLGQ EMVRRSAGKV SSTLASELGI TAEDARLVSE IAMH TTEDK VEHHHHHHHH UniProtKB: Nucleocapsid |
-Macromolecule #2: RNA (5'-R(*AP*AP*AP*AP*AP*A)-3')
Macromolecule | Name: RNA (5'-R(*AP*AP*AP*AP*AP*A)-3') / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 1.930277 KDa |
Sequence | String: AAAAAA |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | helical array |
-Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 6 / Details: 50 mM Na-phosphate pH 6, 150 mM NaCl, 2 mM DTT |
Grid | Model: Quantifoil R2/1 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293.15 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI POLARA 300 |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 4000 pixel / Digitization - Dimensions - Height: 4000 pixel / Digitization - Frames/image: 2-20 / Number grids imaged: 1 / Number real images: 186 / Average exposure time: 4.0 sec. / Average electron dose: 30.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 0.003 µm / Nominal defocus min: 0.0008 µm / Nominal magnification: 23000 |
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Applied symmetry - Helical parameters - Δz: 3.920265781 Å Applied symmetry - Helical parameters - Δ&Phi: -29.17105583 ° Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric) Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 111582 |
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Segment selection | Number selected: 111582 / Software - Name: RELION (ver. 2.1) |
Startup model | Type of model: EMDB MAP EMDB ID: |
Final angle assignment | Type: NOT APPLICABLE / Software - Name: RELION (ver. 2.1) |