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Yorodumi- PDB-7ku7: Cryo-EM structure of Rous sarcoma virus cleaved synaptic complex ... -
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-Basic information
Entry | Database: PDB / ID: 7ku7 | |||||||||
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Title | Cryo-EM structure of Rous sarcoma virus cleaved synaptic complex (CSC) with HIV-1 integrase strand transfer inhibitor MK-2048. Cluster identified by 3-dimensional variability analysis in cryoSPARC. | |||||||||
Components |
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Keywords | HYDROLASE/DNA/INHIBITOR / intasome / integrase-viral DNA complex / HYDROLASE-DNA-INHIBITOR complex | |||||||||
Function / homology | Function and homology information ribonuclease H / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / RNA stem-loop binding / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases ...ribonuclease H / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / RNA stem-loop binding / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / viral nucleocapsid / DNA recombination / DNA-directed DNA polymerase / Hydrolases; Acting on ester bonds / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / proteolysis / DNA binding / zinc ion binding Similarity search - Function | |||||||||
Biological species | Rous sarcoma virus | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å | |||||||||
Authors | Pandey, K.K. / Bera, S. / Shi, K. / Aihara, H. / Grandgenett, D.P. | |||||||||
Funding support | United States, 2items
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Citation | Journal: Commun Biol / Year: 2021 Title: Cryo-EM structure of the Rous sarcoma virus octameric cleaved synaptic complex intasome. Authors: Krishan K Pandey / Sibes Bera / Ke Shi / Michael J Rau / Amarachi V Oleru / James A J Fitzpatrick / Alan N Engelman / Hideki Aihara / Duane P Grandgenett / Abstract: Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate ...Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous sarcoma virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7ku7.cif.gz | 351.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7ku7.ent.gz | 280.7 KB | Display | PDB format |
PDBx/mmJSON format | 7ku7.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7ku7_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 7ku7_full_validation.pdf.gz | 1.7 MB | Display | |
Data in XML | 7ku7_validation.xml.gz | 74.4 KB | Display | |
Data in CIF | 7ku7_validation.cif.gz | 109.2 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ku/7ku7 ftp://data.pdbj.org/pub/pdb/validation_reports/ku/7ku7 | HTTPS FTP |
-Related structure data
Related structure data | 23035MC 7jn3C 7kuiC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 1 types, 8 molecules ABCDEFGH
#1: Protein | Mass: 30926.582 Da / Num. of mol.: 8 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Rous sarcoma virus (strain Schmidt-Ruppin A) Strain: Schmidt-Ruppin A / Gene: gag-pol / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): pLysS References: UniProt: P03354, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases |
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-DNA chain , 2 types, 4 molecules IKJL
#2: DNA chain | Mass: 5520.600 Da / Num. of mol.: 2 / Source method: obtained synthetically Source: (synth.) Rous sarcoma virus (strain Schmidt-Ruppin A) #3: DNA chain | Mass: 4899.232 Da / Num. of mol.: 2 / Source method: obtained synthetically Source: (synth.) Rous sarcoma virus (strain Schmidt-Ruppin A) |
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-Non-polymers , 3 types, 8 molecules
#4: Chemical | #5: Chemical | ChemComp-MG / #6: Chemical | |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Cleaved synaptic complex (CSC) formed with Rous sarcoma virus integrase and viral DNA in presence of HIV-1 integrase strand inhibitor MK-2048 Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT |
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Molecular weight | Value: 0.257 MDa / Experimental value: NO |
Source (natural) | Organism: Rous sarcoma virus (strain Schmidt-Ruppin A) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Cs: 0.01 mm / Alignment procedure: BASIC |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 66 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 5187 Details: Images were collected in movie mode at 0.2 seconds per frame. |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Movie frames/image: 40 |
-Processing
Software | Name: PHENIX / Version: 1.19rc3_4028: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1811357 | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 64449 Details: Refined map is for one cluster identified by 3-dimensional variability analysis done in cryoSPARC. Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | B value: 30 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: correlation coefficient | ||||||||||||||||||||||||
Atomic model building | PDB-ID: 7JN3 Accession code: 7JN3 / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||
Refine LS restraints |
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