+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-23035 | |||||||||
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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. | |||||||||
Map data | Map | |||||||||
Sample |
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Keywords | 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 (strain Schmidt-Ruppin A) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.4 Å | |||||||||
Authors | Pandey KK / Bera S | |||||||||
Funding support | United States, 2 items
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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 | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_23035.map.gz | 203.5 MB | EMDB map data format | |
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Header (meta data) | emd-23035-v30.xml emd-23035.xml | 20.9 KB 20.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_23035_fsc.xml | 13.3 KB | Display | FSC data file |
Images | emd_23035.png | 280.6 KB | ||
Filedesc metadata | emd-23035.cif.gz | 6.8 KB | ||
Others | emd_23035_half_map_1.map.gz emd_23035_half_map_2.map.gz | 200.1 MB 200.1 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-23035 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-23035 | HTTPS FTP |
-Validation report
Summary document | emd_23035_validation.pdf.gz | 1.1 MB | Display | EMDB validaton report |
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Full document | emd_23035_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | emd_23035_validation.xml.gz | 21.7 KB | Display | |
Data in CIF | emd_23035_validation.cif.gz | 28.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23035 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23035 | HTTPS FTP |
-Related structure data
Related structure data | 7ku7MC 7kuiMC 7jn3C C: citing same article (ref.) M: atomic model generated by this map |
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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_23035.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.1 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Half map: Half map A
File | emd_23035_half_map_1.map | ||||||||||||
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Annotation | Half map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map B
File | emd_23035_half_map_2.map | ||||||||||||
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Annotation | Half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Cleaved synaptic complex (CSC) formed with Rous sarcoma virus int...
Entire | Name: Cleaved synaptic complex (CSC) formed with Rous sarcoma virus integrase and viral DNA in presence of HIV-1 integrase strand inhibitor MK-2048 |
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Components |
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-Supramolecule #1: Cleaved synaptic complex (CSC) formed with Rous sarcoma virus int...
Supramolecule | 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 / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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Source (natural) | Organism: Rous sarcoma virus (strain Schmidt-Ruppin A) |
Molecular weight | Theoretical: 257 KDa |
-Macromolecule #1: integrase
Macromolecule | Name: integrase / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases |
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Source (natural) | Organism: Rous sarcoma virus (strain Schmidt-Ruppin A) / Strain: Schmidt-Ruppin A |
Molecular weight | Theoretical: 30.926582 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: PLREAKDLHT ALHIGPRALS KACNISMQQA REVVQTCPHC NSAPALEAGV NPRGLGPLQI WQTDFTLEPR MAPRSWLAVT VDTASSAIV VTQHGRVTSV AVQHHWATAI AVLGRPKAIK TDNGSCFTSK STREWLARWG IAHTTGIPGN SQGQAMVERA N RLLKDKIR ...String: PLREAKDLHT ALHIGPRALS KACNISMQQA REVVQTCPHC NSAPALEAGV NPRGLGPLQI WQTDFTLEPR MAPRSWLAVT VDTASSAIV VTQHGRVTSV AVQHHWATAI AVLGRPKAIK TDNGSCFTSK STREWLARWG IAHTTGIPGN SQGQAMVERA N RLLKDKIR VLAEGDGFMK RIPTSKQGEL LAKAMYALNH FERGENTKTP IQKHWRPTVL TEGPPVKIRI ETGEWEKGWN VL VWGRGYA AVKNRDTDKV IWVPSRKVKP DITQKDEVTK K UniProtKB: Gag-Pol polyprotein |
-Macromolecule #2: DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*T)-3')
Macromolecule | Name: DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*T)-3') type: dna / ID: 2 / Number of copies: 2 / Classification: DNA |
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Source (natural) | Organism: Rous sarcoma virus (strain Schmidt-Ruppin A) |
Molecular weight | Theoretical: 5.5206 KDa |
Sequence | String: (DA)(DA)(DT)(DG)(DT)(DT)(DG)(DT)(DC)(DT) (DT)(DA)(DT)(DG)(DC)(DA)(DA)(DT) |
-Macromolecule #3: DNA (5'-D(*AP*TP*TP*GP*CP*AP*TP*AP*AP*GP*AP*CP*AP*AP*CP*A)-3')
Macromolecule | Name: DNA (5'-D(*AP*TP*TP*GP*CP*AP*TP*AP*AP*GP*AP*CP*AP*AP*CP*A)-3') type: dna / ID: 3 / Number of copies: 2 / Classification: DNA |
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Source (natural) | Organism: Rous sarcoma virus (strain Schmidt-Ruppin A) |
Molecular weight | Theoretical: 4.899232 KDa |
Sequence | String: (DA)(DT)(DT)(DG)(DC)(DA)(DT)(DA)(DA)(DG) (DA)(DC)(DA)(DA)(DC)(DA) |
-Macromolecule #4: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Macromolecule #5: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #6: (6S)-2-(3-chloro-4-fluorobenzyl)-8-ethyl-10-hydroxy-N,6-dimethyl-...
Macromolecule | Name: (6S)-2-(3-chloro-4-fluorobenzyl)-8-ethyl-10-hydroxy-N,6-dimethyl-1,9-dioxo-1,2,6,7,8,9-hexahydropyrazino[1',2':1,5]pyrrolo[2,3-d]pyridazine-4-carboxamide type: ligand / ID: 6 / Number of copies: 2 / Formula: ZZX |
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Molecular weight | Theoretical: 461.874 Da |
Chemical component information | ChemComp-ZZX: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.5 mg/mL |
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Buffer | pH: 7.5 |
Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | TFS KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 5187 / Average electron dose: 66.0 e/Å2 Details: Images were collected in movie mode at 0.2 seconds per frame. |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal magnification: 105000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Refinement | Space: REAL / Protocol: RIGID BODY FIT / Overall B value: 30 / Target criteria: correlation coefficient |
Output model | PDB-7ku7: PDB-7kui: |