+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-16774 | ||||||||||||||||||
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Title | in situ Subtomogram average of Immature Rotavirus TLP spike | ||||||||||||||||||
Map data | main map | ||||||||||||||||||
Sample |
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Function / homology | Function and homology information viral intermediate capsid / host cell endoplasmic reticulum lumen / T=13 icosahedral viral capsid / host cell rough endoplasmic reticulum / host cytoskeleton / viral outer capsid / permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner membrane / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell surface receptor binding ...viral intermediate capsid / host cell endoplasmic reticulum lumen / T=13 icosahedral viral capsid / host cell rough endoplasmic reticulum / host cytoskeleton / viral outer capsid / permeabilization of host organelle membrane involved in viral entry into host cell / symbiont entry into host cell via permeabilization of inner membrane / host cell endoplasmic reticulum-Golgi intermediate compartment / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / viral envelope / virion attachment to host cell / host cell plasma membrane / structural molecule activity / membrane / metal ion binding Similarity search - Function | ||||||||||||||||||
Biological species | Rotavirus A | ||||||||||||||||||
Method | subtomogram averaging / cryo EM / Resolution: 4.5 Å | ||||||||||||||||||
Authors | Shah PNM / Stuart DI | ||||||||||||||||||
Funding support | United Kingdom, 5 items
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Citation | Journal: Cell Host Microbe / Year: 2023 Title: Characterization of the rotavirus assembly pathway in situ using cryoelectron tomography. Authors: Pranav N M Shah / James B Gilchrist / Björn O Forsberg / Alister Burt / Andrew Howe / Shyamal Mosalaganti / William Wan / Julika Radecke / Yuriy Chaban / Geoff Sutton / David I Stuart / Mark Boyce / Abstract: Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and ...Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and visualization of the assembly process has been hampered by the inaccessibility of unstable intermediates. We characterize the assembly pathway of group A rotaviruses observed in situ within cryo-preserved infected cells through the use of cryoelectron tomography of cellular lamellae. Our findings demonstrate that the viral polymerase VP1 recruits viral genomes during particle assembly, as revealed by infecting with a conditionally lethal mutant. Additionally, pharmacological inhibition to arrest the transiently enveloped stage uncovered a unique conformation of the VP4 spike. Subtomogram averaging provided atomic models of four intermediate states, including a pre-packaging single-layered intermediate, the double-layered particle, the transiently enveloped double-layered particle, and the fully assembled triple-layered virus particle. In summary, these complementary approaches enable us to elucidate the discrete steps involved in forming an intracellular rotavirus particle. | ||||||||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_16774.map.gz | 7.6 MB | EMDB map data format | |
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Header (meta data) | emd-16774-v30.xml emd-16774.xml | 19.4 KB 19.4 KB | Display Display | EMDB header |
Images | emd_16774.png | 116 KB | ||
Others | emd_16774_half_map_1.map.gz emd_16774_half_map_2.map.gz | 7.9 MB 7.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-16774 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-16774 | HTTPS FTP |
-Validation report
Summary document | emd_16774_validation.pdf.gz | 882.4 KB | Display | EMDB validaton report |
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Full document | emd_16774_full_validation.pdf.gz | 882 KB | Display | |
Data in XML | emd_16774_validation.xml.gz | 9.3 KB | Display | |
Data in CIF | emd_16774_validation.cif.gz | 10.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-16774 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-16774 | HTTPS FTP |
-Related structure data
Related structure data | 8coaMC 8bp8C 8co6C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_16774.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | main map | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.97 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: half1
File | emd_16774_half_map_1.map | ||||||||||||
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Annotation | half1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half2
File | emd_16774_half_map_2.map | ||||||||||||
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Annotation | half2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Rotavirus A
Entire | Name: Rotavirus A |
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Components |
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-Supramolecule #1: Rotavirus A
Supramolecule | Name: Rotavirus A / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 28875 / Sci species name: Rotavirus A / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No |
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-Macromolecule #1: Outer capsid protein VP4
Macromolecule | Name: Outer capsid protein VP4 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Rotavirus A |
Molecular weight | Theoretical: 86.767953 KDa |
Recombinant expression | Organism: Chlorocebus aethiops aethiops (mammal) |
Sequence | String: MASLIYRQLL TNSYTVDLSD EIQEIGSTKS QNVTINPGPF AQTGYAPVNW GPGEINDSTT VGPLLDGPYQ PTTFNPPVDY WMLLAPTTP GVIVEGTNNT DRWLATILIE PNVQSENRTY TIFGIQEQLT VSNTSQDQWK FIDVVKTTAN GSIGQYGPLL S SPKLYAVM ...String: MASLIYRQLL TNSYTVDLSD EIQEIGSTKS QNVTINPGPF AQTGYAPVNW GPGEINDSTT VGPLLDGPYQ PTTFNPPVDY WMLLAPTTP GVIVEGTNNT DRWLATILIE PNVQSENRTY TIFGIQEQLT VSNTSQDQWK FIDVVKTTAN GSIGQYGPLL S SPKLYAVM KHNEKLYTYE GQTPNATTAH YSTTNYDSVN MTAFCDFYII PRSEESKCTE YINNGLPPIQ NTRNVVPLSL TA RDVIHYR AQANEDIVIS KTSLWKEMQY NRDITIRFKF ANTIIKSGGL GYKWSEISFK PANYQYTYTR DGEEVTAHTT CSV NGMNDF SFNGGYLPTD FVVSKFEVIK ENSYVYIDYW DDSQAFRNVV YVRSLAANLN SVMCTGGSYN FSLPVGQWPV LTGG AVSLH SAGVTLSTQF TDFVSLNSLR FRFRLAVEEP HFKLTRTRLD RLYGLPAADP NNGKEYYEIA GRFSLISLVP SNDDY QTPI ANSVTVRQDL ERQLGELREE FNALSQEIAM SQLIDLALLP LDMFSMFSGI KSTIDAAKSM ATNVMKKFKK SGLANS VST LTDSLSDAAS SISRGSSIRS IGSSASAWTD VSTQITDISS SVSSVSTQTS TISRRLRLKE MATQTEGMNF DDISAAV LK TKIDKSTQIS PNTIPDIVTE ASEKFIPNRA YRVINNDDVF EAGIDGKFFA YKVDTFEEIP FDVQKFADLV TDSPVISA I IDFKTLKNLN DNYGISRQQA FNLLRSDPRV LREFINQDNP IIRNRIEQLI MQCRL |
-Macromolecule #2: Outer capsid glycoprotein VP7
Macromolecule | Name: Outer capsid glycoprotein VP7 / type: protein_or_peptide / ID: 2 / Number of copies: 13 / Enantiomer: LEVO |
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Source (natural) | Organism: Rotavirus A |
Molecular weight | Theoretical: 37.230664 KDa |
Recombinant expression | Organism: Chlorocebus aethiops aethiops (mammal) |
Sequence | String: MYGIEYTTVL TFLISIILLN YILKSLTRIM DFIIYRFLFI IVILSPFLRA QNYGINLPIT GSMDIAYANS TQEEPFLTST LCLYYPTEA ATEINDNSWK DTLSQLFLTK GWPTGSVYFK EYTNIASFSV DPQLYCDYNV VLMKYDATLQ LDMSELADLI L NEWLCNPM ...String: MYGIEYTTVL TFLISIILLN YILKSLTRIM DFIIYRFLFI IVILSPFLRA QNYGINLPIT GSMDIAYANS TQEEPFLTST LCLYYPTEA ATEINDNSWK DTLSQLFLTK GWPTGSVYFK EYTNIASFSV DPQLYCDYNV VLMKYDATLQ LDMSELADLI L NEWLCNPM DITLYYYQQT DEANKWISMG SSCTIKVCPL NTQTLGIGCL TTDATTFEEV ATAEKLVITD VVDGVNHKLD VT TATCTIR NCKKLGPREN VAVIQVGGSD ILDITADPTT APQTERMMRI NWKKWWQVFY TVVDYVDQII QVMSKRSRSL NSA AFYYRV |
-Macromolecule #3: Intermediate capsid protein VP6
Macromolecule | Name: Intermediate capsid protein VP6 / type: protein_or_peptide / ID: 3 / Number of copies: 13 / Enantiomer: LEVO |
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Source (natural) | Organism: Rotavirus A |
Molecular weight | Theoretical: 44.910738 KDa |
Recombinant expression | Organism: Chlorocebus aethiops aethiops (mammal) |
Sequence | String: MDVLYSLSKT LKDARDKIVE GTLYSNVSDL IQQFNQMIIT MNGNEFQTGG IGNLPIRNWN FNFGLLGTTL LNLDANYVET ARNTIDYFV DFVDNVCMDE MVRESQRNGI APQSDSLRKL SAIKFKRINF DNSSEYIENW NLQNRRQRTG FTFHKPNIFP Y SASFTLNR ...String: MDVLYSLSKT LKDARDKIVE GTLYSNVSDL IQQFNQMIIT MNGNEFQTGG IGNLPIRNWN FNFGLLGTTL LNLDANYVET ARNTIDYFV DFVDNVCMDE MVRESQRNGI APQSDSLRKL SAIKFKRINF DNSSEYIENW NLQNRRQRTG FTFHKPNIFP Y SASFTLNR SQPAHDNLMG TMWLNAGSEI QVAGFDYSCA INAPANIQQF EHIVPLRRVL TTATITLLPD AERFSFPRVI NS ADGATTW FFNPVILRPN NVEVEFLLNG QIINTYQARF GTIVARNFDT IRLSFQLMRP PNMTPAVAVL FPNAQPFEHH ATV GLTLRI ESAVCESVLA DASETLLANV TSVRQEYAIP VGPVFPPGMN WTDLITNYSP SREDNLQRVF TVASIRSMLI K |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | cell |
-Sample preparation
Buffer | pH: 7.5 |
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Grid | Model: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY |
Vitrification | Cryogen name: ETHANE |
Details | MA104 cells infected with Rotavirus |
-Electron microscopy
Microscope | TFS KRIOS |
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Image recording | Film or detector model: FEI FALCON IV (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.19 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Warp (ver. 1.0.9) / Number subtomograms used: 16740 |
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Extraction | Number tomograms: 85 / Number images used: 16740 / Software - Name: crYOLO |
Final 3D classification | Software - Name: RELION (ver. 3.0) |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) |
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
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Output model | PDB-8coa: |