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- PDB-8e14: Cryo-EM structure of Rous sarcoma virus strand transfer complex -

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Basic information

Entry
Database: PDB / ID: 8.0E+14
TitleCryo-EM structure of Rous sarcoma virus strand transfer complex
Components
  • DNA (42-MER)
  • DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*TP*AP*CP*TP*C)-3')
  • DNA (5'-D(*AP*GP*TP*GP*TP*CP*TP*TP*CP*TP*TP*CP*TP*TP*TP*C)-3')
  • integrase
KeywordsVIRAL PROTEIN/DNA / intasome / integrase-viral DNA complex / strand transfer complex / VIRAL PROTEIN-DNA complex
Function / homology
Function and homology information


Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / viral nucleocapsid ...Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / viral nucleocapsid / DNA recombination / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / proteolysis / DNA binding / RNA binding / zinc ion binding
Similarity search - Function
Retroviral Gag polyprotein, M / Retroviral M domain / gag protein p24 N-terminal domain / Reverse transcriptase thumb / Reverse transcriptase thumb domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / Integrase DNA binding domain / Integrase, C-terminal domain superfamily, retroviral ...Retroviral Gag polyprotein, M / Retroviral M domain / gag protein p24 N-terminal domain / Reverse transcriptase thumb / Reverse transcriptase thumb domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / Integrase DNA binding domain / Integrase, C-terminal domain superfamily, retroviral / Integrase, N-terminal zinc-binding domain / Integrase, C-terminal, retroviral / Integrase DNA binding domain profile. / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Ribonuclease H domain / RNase H type-1 domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Retrovirus capsid, C-terminal / Retroviral matrix protein / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / Gag-Pol polyprotein
Similarity search - Component
Biological speciesRous sarcoma virus - Prague C
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsPandey, K.K. / Bera, S. / Shi, K. / Aihara, H. / Grandgenett, D.P.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI165081 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118047 United States
CitationJournal: J Biol Chem / Year: 2023
Title: Molecular determinants for Rous sarcoma virus intasome assemblies involved in retroviral integration.
Authors: Sibes Bera / Ke Shi / Hideki Aihara / Duane P Grandgenett / Krishan K Pandey /
Abstract: Integration of retroviral DNA into the host genome involves the formation of integrase (IN)-DNA complexes termed intasomes. Further characterization of these complexes is needed to understand their ...Integration of retroviral DNA into the host genome involves the formation of integrase (IN)-DNA complexes termed intasomes. Further characterization of these complexes is needed to understand their assembly process. Here, we report the single-particle cryo-EM structure of the Rous sarcoma virus (RSV) strand transfer complex (STC) intasome produced with IN and a preassembled viral/target DNA substrate at 3.36 Å resolution. The conserved intasome core region consisting of IN subunits contributing active sites interacting with viral/target DNA has a resolution of 3 Å. Our structure demonstrated the flexibility of the distal IN subunits relative to the IN subunits in the conserved intasome core, similar to results previously shown with the RSV octameric cleaved synaptic complex intasome produced with IN and viral DNA only. An extensive analysis of higher resolution STC structure helped in the identification of nucleoprotein interactions important for intasome assembly. Using structure-function studies, we determined the mechanisms of several IN-DNA interactions critical for assembly of both RSV intasomes. We determined the role of IN residues R244, Y246, and S124 in cleaved synaptic complex and STC intasome assemblies and their catalytic activities, demonstrating differential effects. Taken together, these studies advance our understanding of different RSV intasome structures and molecular determinants involved in their assembly.
History
DepositionAug 9, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 26, 2023Provider: repository / Type: Initial release
Revision 2.0May 31, 2023Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Database references / Derived calculations / Refinement description / Structure summary
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / citation / citation_author / database_PDB_caveat / em_3d_fitting_list / em_software / ndb_struct_na_base_pair / ndb_struct_na_base_pair_step / pdbx_audit_support / pdbx_contact_author / pdbx_validate_rmsd_angle / pdbx_validate_rmsd_bond / pdbx_validate_torsion / refine_ls_restr / software
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _ndb_struct_na_base_pair.buckle / _ndb_struct_na_base_pair.opening / _ndb_struct_na_base_pair.propeller / _ndb_struct_na_base_pair.shear / _ndb_struct_na_base_pair.stagger / _ndb_struct_na_base_pair.stretch / _ndb_struct_na_base_pair_step.helical_rise / _ndb_struct_na_base_pair_step.helical_twist / _ndb_struct_na_base_pair_step.inclination / _ndb_struct_na_base_pair_step.rise / _ndb_struct_na_base_pair_step.roll / _ndb_struct_na_base_pair_step.shift / _ndb_struct_na_base_pair_step.slide / _ndb_struct_na_base_pair_step.tilt / _ndb_struct_na_base_pair_step.tip / _ndb_struct_na_base_pair_step.twist / _ndb_struct_na_base_pair_step.x_displacement / _ndb_struct_na_base_pair_step.y_displacement / _pdbx_validate_torsion.phi / _pdbx_validate_torsion.psi
Description: Missing anisotropic B-factor / Details: updated model / Provider: author / Type: Coordinate replacement
Revision 2.1Aug 16, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Revision 2.2Oct 18, 2023Group: Refinement description / Source and taxonomy
Category: em_3d_fitting_list / em_entity_assembly_naturalsource ...em_3d_fitting_list / em_entity_assembly_naturalsource / entity_src_gen / pdbx_entity_src_syn
Item: _em_3d_fitting_list.initial_refinement_model_id / _em_entity_assembly_naturalsource.ncbi_tax_id ..._em_3d_fitting_list.initial_refinement_model_id / _em_entity_assembly_naturalsource.ncbi_tax_id / _em_entity_assembly_naturalsource.organism / _entity_src_gen.gene_src_strain / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name / _pdbx_entity_src_syn.ncbi_taxonomy_id / _pdbx_entity_src_syn.organism_scientific

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: integrase
B: integrase
C: integrase
D: integrase
E: integrase
F: integrase
G: integrase
H: integrase
I: DNA (42-MER)
J: DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*TP*AP*CP*TP*C)-3')
K: DNA (5'-D(*AP*GP*TP*GP*TP*CP*TP*TP*CP*TP*TP*CP*TP*TP*TP*C)-3')
L: DNA (42-MER)
M: DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*TP*AP*CP*TP*C)-3')
N: DNA (5'-D(*AP*GP*TP*GP*TP*CP*TP*TP*CP*TP*TP*CP*TP*TP*TP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)296,71316
Polymers296,58314
Non-polymers1312
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
integrase /


Mass: 30926.582 Da / Num. of mol.: 8 / Fragment: UNP residues 1281-1558
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rous sarcoma virus - Prague C / Strain: Prague C / Plasmid: pET11a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21DE pLysS
References: UniProt: P03354, Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases, RNA-directed DNA polymerase, DNA-directed DNA polymerase, ribonuclease H, Transferases; ...References: UniProt: P03354, Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases, RNA-directed DNA polymerase, DNA-directed DNA polymerase, ribonuclease H, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, Hydrolases; Acting on ester bonds
#2: DNA chain DNA (42-MER)


Mass: 13047.456 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Rous sarcoma virus - Prague C
#3: DNA chain DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*TP*AP*CP*TP*C)-3')


Mass: 6716.363 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Rous sarcoma virus - Prague C
#4: DNA chain DNA (5'-D(*AP*GP*TP*GP*TP*CP*TP*TP*CP*TP*TP*CP*TP*TP*TP*C)-3')


Mass: 4821.123 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Rous sarcoma virus - Prague C
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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

ComponentName: RSV strand transfer complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.257 MDa / Experimental value: YES
Source (natural)Organism: Rous sarcoma virus - Prague C
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Cs: 0.01 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 3000
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
7PHENIXmodel fitting
9PHENIXmodel refinement
12cryoSPARCclassification
13cryoSPARC3.1.13D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1811357
3D reconstructionResolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141000 / Symmetry type: POINT
Atomic model buildingB value: 30 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: correlation coefficient
Atomic model buildingPDB-ID: 5EJK

5ejk


Accession code: 5EJK / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00713072
ELECTRON MICROSCOPYf_angle_d1.12418426
ELECTRON MICROSCOPYf_dihedral_angle_d27.2622829
ELECTRON MICROSCOPYf_chiral_restr0.0682036
ELECTRON MICROSCOPYf_plane_restr0.0061774

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