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Basic information

Entry
Database: PDB / ID: 3jb7
TitleIn situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus
Components
  • CPV RNA-dependent RNA polymerase
  • RNA (5'-R(P*CP*CP*CP*CP*C)-3')
  • RNA (5'-R(P*GP*GP*GP*GP*GP*G)-3')
  • VP1 CSP
  • Viral structural protein 4
KeywordsTRANSFERASE/VIRAL PROTEIN/RNA / dsRNA genome organization / viral polymerase / TRANSFERASE-VIRAL PROTEIN-RNA complex
Function / homology
Function and homology information


viral genome replication / RNA-dependent RNA polymerase activity / RNA binding
Similarity search - Function
: / : / CPV Capsid shell protein VP1, small protrusion domain / Inner layer core protein VP1-like, C-terminal / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile.
Similarity search - Domain/homology
CYTIDINE-5'-TRIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / RNA / RNA-dependent RNA polymerase / VP1 / Viral structural protein 4
Similarity search - Component
Biological speciesBombyx mori cypovirus 1
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsZhang, X. / Ding, K. / Yu, X.K. / Chang, W. / Sun, J.C. / Zhou, Z.H.
CitationJournal: Nature / Year: 2015
Title: In situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus.
Authors: Xing Zhang / Ke Ding / Xuekui Yu / Winston Chang / Jingchen Sun / Z Hong Zhou /
Abstract: Viruses in the Reoviridae, like the triple-shelled human rotavirus and the single-shelled insect cytoplasmic polyhedrosis virus (CPV), all package a genome of segmented double-stranded RNAs (dsRNAs) ...Viruses in the Reoviridae, like the triple-shelled human rotavirus and the single-shelled insect cytoplasmic polyhedrosis virus (CPV), all package a genome of segmented double-stranded RNAs (dsRNAs) inside the viral capsid and carry out endogenous messenger RNA synthesis through a transcriptional enzyme complex (TEC). By direct electron-counting cryoelectron microscopy and asymmetric reconstruction, we have determined the organization of the dsRNA genome inside quiescent CPV (q-CPV) and the in situ atomic structures of TEC within CPV in both quiescent and transcribing (t-CPV) states. We show that the ten segmented dsRNAs in CPV are organized with ten TECs in a specific, non-symmetric manner, with each dsRNA segment attached directly to a TEC. The TEC consists of two extensively interacting subunits: an RNA-dependent RNA polymerase (RdRP) and an NTPase VP4. We find that the bracelet domain of RdRP undergoes marked conformational change when q-CPV is converted to t-CPV, leading to formation of the RNA template entry channel and access to the polymerase active site. An amino-terminal helix from each of two subunits of the capsid shell protein (CSP) interacts with VP4 and RdRP. These findings establish the link between sensing of environmental cues by the external proteins and activation of endogenous RNA transcription by the TEC inside the virus.
History
DepositionAug 3, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 28, 2015Provider: repository / Type: Initial release
Revision 1.1Nov 11, 2015Group: Database references
Revision 1.2Dec 2, 2015Group: Database references
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_imaging_optics / em_software
Item: _em_imaging_optics.energyfilter_name / _em_software.image_processing_id / _em_software.name
Revision 1.4Apr 5, 2023Group: Database references / Derived calculations / Category: database_2 / pdbx_database_related / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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Assembly

Deposited unit
A: CPV RNA-dependent RNA polymerase
B: Viral structural protein 4
C: VP1 CSP
t: RNA (5'-R(P*GP*GP*GP*GP*GP*G)-3')
m: RNA (5'-R(P*CP*CP*CP*CP*C)-3')
D: VP1 CSP
hetero molecules


Theoretical massNumber of molelcules
Total (without water)212,9999
Polymers211,4706
Non-polymers1,5303
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein CPV RNA-dependent RNA polymerase


Mass: 138810.859 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bombyx mori cypovirus 1 / References: UniProt: D0EZK6, RNA-directed RNA polymerase
#2: Protein Viral structural protein 4


Mass: 63683.738 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bombyx mori cypovirus 1 / References: UniProt: Q9IR43

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Protein/peptide , 1 types, 2 molecules CD

#3: Protein/peptide VP1 CSP


Mass: 2734.002 Da / Num. of mol.: 2 / Fragment: UNP residues 111-134 / Source method: isolated from a natural source / Source: (natural) Bombyx mori cypovirus 1 / References: UniProt: D3JWE6

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RNA chain , 2 types, 2 molecules tm

#4: RNA chain RNA (5'-R(P*GP*GP*GP*GP*GP*G)-3')


Mass: 2026.277 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: genomic RNA / Source: (natural) Bombyx mori cypovirus 1
#5: RNA chain RNA (5'-R(P*CP*CP*CP*CP*C)-3')


Mass: 1480.952 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: genomic RNA / Source: (natural) Bombyx mori cypovirus 1

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Non-polymers , 2 types, 3 molecules

#6: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#7: Chemical ChemComp-CTP / CYTIDINE-5'-TRIPHOSPHATE / Cytidine triphosphate


Mass: 483.156 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H16N3O14P3

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeParent-ID
1CPV VP4 + polymerase complex (TEC)COMPLEX0
2RNA polymerase1
3VP41
Buffer solutionName: 70 mM Tris-Cl, 10 mM MgCl2, 100 mM NaCl, 2 mM GTP / pH: 8 / Details: 70 mM Tris-Cl, 10 mM MgCl2, 100 mM NaCl, 2 mM GTP
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 200 mesh Quantifoil holey carbon film
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Details: Plunged into liquid ethane (FEI VITROBOT MARK II).

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Mar 7, 2015
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM / Electron beam tilt params: 0
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Calibrated magnification: 36765 X / Nominal defocus max: 3400 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 80 K
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum
Image scansNum. digital images: 4907

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Processing

EM softwareName: FREALIGN / Category: 3D reconstruction
CTF correctionDetails: Each particle
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: Frealign / Resolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81887 / Nominal pixel size: 1.36 Å / Actual pixel size: 1.36 Å / Details: (Single particle--Applied symmetry: C1) / Num. of class averages: 1 / Symmetry type: POINT
RefinementResolution: 4.001→49.737 Å / SU ML: 0.51 / σ(F): 2 / Phase error: 28.46 / Stereochemistry target values: MLHL
RfactorNum. reflection% reflection
Rfree0.1965 456 0.03 %
Rwork0.2164 1378767 -
obs0.2164 1379223 99.93 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 434.16 Å2 / Biso mean: 128.2658 Å2 / Biso min: 20 Å2
Refinement stepCycle: LAST / Resolution: 4.001→49.737 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13845 238 93 0 14176
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01514199
ELECTRON MICROSCOPYf_angle_d1.94819297
ELECTRON MICROSCOPYf_chiral_restr0.0772163
ELECTRON MICROSCOPYf_plane_restr0.0092405
ELECTRON MICROSCOPYf_dihedral_angle_d22.1645263
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Total num. of bins used: 3 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all
4.0005-4.57910.28521680.2839459656459824
4.5791-5.76770.18451320.2059460134460266
5.7677-49.7410.17451560.1974458977459133
Refinement TLS params.Method: refined / Origin x: 111.6318 Å / Origin y: -30.761 Å / Origin z: 179.7434 Å
111213212223313233
T0.7164 Å20.0619 Å20.0174 Å2-0.7516 Å2-0.0168 Å2--0.7923 Å2
L0.302 °20.5098 °2-0.0504 °2-0.5491 °2-0.1817 °2--0.2169 °2
S-0.0417 Å °0.0015 Å °0.1135 Å °0.0279 Å °0.0251 Å °0.168 Å °-0.0234 Å °-0.0957 Å °0.0194 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1ELECTRON MICROSCOPY1allA5 - 1211
2ELECTRON MICROSCOPY1allA1 - 1301
3ELECTRON MICROSCOPY1allB2 - 560
4ELECTRON MICROSCOPY1allB562 - 601
5ELECTRON MICROSCOPY1allt0 - 5
6ELECTRON MICROSCOPY1allm1 - 5
7ELECTRON MICROSCOPY1allA1 - 1302
8ELECTRON MICROSCOPY1allC114 - 134
9ELECTRON MICROSCOPY1allD111 - 126

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