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Yorodumi- EMDB-26744: Higher-order assembly of multiple MMTV strand transfer complex in... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-26744 | ||||||||||||
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Title | Higher-order assembly of multiple MMTV strand transfer complex intasomes | ||||||||||||
Map data | sharpened map | ||||||||||||
Sample |
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Keywords | Integrase-DNA complex / hydrolase / VIRAL PROTEIN / HYDROLASE-DNA complex | ||||||||||||
Function / homology | Function and homology information dUTP diphosphatase activity / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA stem-loop binding / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / viral nucleocapsid / DNA recombination / structural constituent of virion ...dUTP diphosphatase activity / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA stem-loop binding / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / viral nucleocapsid / DNA recombination / structural constituent of virion / 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 | Mouse mammary tumor virus | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.8 Å | ||||||||||||
Authors | Jozwik I / Lyumkis D | ||||||||||||
Funding support | United States, 3 items
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Citation | Journal: Nucleic Acids Res / Year: 2022 Title: B-to-A transition in target DNA during retroviral integration. Authors: Ilona K Jóźwik / Wen Li / Da-Wei Zhang / Doris Wong / Julia Grawenhoff / Allison Ballandras-Colas / Sriram Aiyer / Peter Cherepanov / Alan N Engelman / Dmitry Lyumkis / Abstract: Integration into host target DNA (tDNA), a hallmark of retroviral replication, is mediated by the intasome, a multimer of integrase (IN) assembled on viral DNA (vDNA) ends. To ascertain aspects of ...Integration into host target DNA (tDNA), a hallmark of retroviral replication, is mediated by the intasome, a multimer of integrase (IN) assembled on viral DNA (vDNA) ends. To ascertain aspects of tDNA recognition during integration, we have solved the 3.5 Å resolution cryo-EM structure of the mouse mammary tumor virus (MMTV) strand transfer complex (STC) intasome. The tDNA adopts an A-like conformation in the region encompassing the sites of vDNA joining, which exposes the sugar-phosphate backbone for IN-mediated strand transfer. Examination of existing retroviral STC structures revealed conservation of A-form tDNA in the analogous regions of these complexes. Furthermore, analyses of sequence preferences in genomic integration sites selectively targeted by six different retroviruses highlighted consistent propensity for A-philic sequences at the sites of vDNA joining. Our structure additionally revealed several novel MMTV IN-DNA interactions, as well as contacts seen in prior STC structures, including conserved Pro125 and Tyr149 residues interacting with tDNA. In infected cells, Pro125 substitutions impacted the global pattern of MMTV integration without significantly altering local base sequence preferences at vDNA insertion sites. Collectively, these data advance our understanding of retroviral intasome structure and function, as well as factors that influence patterns of vDNA integration in genomic DNA. | ||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_26744.map.gz | 59.3 MB | EMDB map data format | |
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Header (meta data) | emd-26744-v30.xml emd-26744.xml | 22.5 KB 22.5 KB | Display Display | EMDB header |
Images | emd_26744.png | 170.4 KB | ||
Masks | emd_26744_msk_1.map | 64 MB | Mask map | |
Filedesc metadata | emd-26744.cif.gz | 6.8 KB | ||
Others | emd_26744_half_map_1.map.gz emd_26744_half_map_2.map.gz | 59.4 MB 59.4 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-26744 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-26744 | HTTPS FTP |
-Related structure data
Related structure data | 7ut1MC 7usfC C: citing same article (ref.) M: atomic model generated by this map |
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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_26744.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | sharpened map | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.31 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_26744_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: half-map #2
File | emd_26744_half_map_1.map | ||||||||||||
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Annotation | half-map #2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half-map #1
File | emd_26744_half_map_2.map | ||||||||||||
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Annotation | half-map #1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Higher-order assembly of multiple MMTV strand transfer complex in...
Entire | Name: Higher-order assembly of multiple MMTV strand transfer complex intasomes |
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Components |
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-Supramolecule #1: Higher-order assembly of multiple MMTV strand transfer complex in...
Supramolecule | Name: Higher-order assembly of multiple MMTV strand transfer complex intasomes type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 |
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Source (natural) | Organism: Mouse mammary tumor virus |
Molecular weight | Theoretical: 0.6 kDa/nm |
-Macromolecule #1: Integrase
Macromolecule | Name: Integrase / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO |
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Source (natural) | Organism: Mouse mammary tumor virus |
Molecular weight | Theoretical: 35.753332 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: ALESAQESHA LHHQNAAALR FQFHITREQA REIVKLCPNC PDWGHAPQLG VNPRGLKPRV LWQMDVTHVS EFGKLKYVHV TVDTYSHFT FATARTGEAT KDVLQHLAQS FAYMGIPQKI KTDNAPAYVS RSIQEFLARW KISHVTGIPY NPQGQAIVER T HQNIKAQL ...String: ALESAQESHA LHHQNAAALR FQFHITREQA REIVKLCPNC PDWGHAPQLG VNPRGLKPRV LWQMDVTHVS EFGKLKYVHV TVDTYSHFT FATARTGEAT KDVLQHLAQS FAYMGIPQKI KTDNAPAYVS RSIQEFLARW KISHVTGIPY NPQGQAIVER T HQNIKAQL NKLQKAGKYY TPHHLLAHAL FVLNHVNMDN QGHTAAERHW GPISADPKPM VMWKDLLTGS WKGPDVLITA GR GYACVFP QDAESPIWVP DRFIRPFTER KEATPTPGTA EKTPPRDEKD QQESPKNESS PHQREDGLAT SAGVDLRSGG GP UniProtKB: Gag-Pro-Pol polyprotein |
-Macromolecule #2: vDNA strand (non-transferred)
Macromolecule | Name: vDNA strand (non-transferred) / type: dna / ID: 2 / Number of copies: 4 / Classification: DNA |
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Source (natural) | Organism: Mouse mammary tumor virus |
Molecular weight | Theoretical: 6.738343 KDa |
Sequence | String: (DA)(DA)(DT)(DG)(DC)(DC)(DG)(DC)(DA)(DG) (DT)(DC)(DG)(DG)(DC)(DC)(DG)(DA)(DC)(DC) (DT)(DG) |
-Macromolecule #3: vDNA-tDNA strand (transferred)
Macromolecule | Name: vDNA-tDNA strand (transferred) / type: dna / ID: 3 / Number of copies: 4 / Classification: DNA |
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Source (natural) | Organism: Mouse mammary tumor virus |
Molecular weight | Theoretical: 12.875227 KDa |
Sequence | String: (DC)(DA)(DG)(DG)(DT)(DC)(DG)(DG)(DC)(DC) (DG)(DA)(DC)(DT)(DG)(DC)(DG)(DG)(DC)(DA) (DC)(DT)(DC)(DG)(DA)(DG)(DC)(DT)(DA) (DC)(DT)(DT)(DC)(DC)(DC)(DT)(DG)(DT)(DT) (DT) (DA)(DG) |
-Macromolecule #4: tDNA strand
Macromolecule | Name: tDNA strand / type: dna / ID: 4 / Number of copies: 4 / Classification: DNA |
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Source (natural) | Organism: Mouse mammary tumor virus |
Molecular weight | Theoretical: 4.980269 KDa |
Sequence | String: (DC)(DT)(DA)(DA)(DA)(DC)(DA)(DG)(DG)(DG) (DA)(DA)(DG)(DT)(DA)(DG) |
-Macromolecule #5: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 5 / Number of copies: 12 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 6.5 Component:
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Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Instrument: HOMEMADE PLUNGER Details: Cryo-EM grids were prepared by freezing using a manual plunger in cold room at 4C. | ||||||||||||||||||
Details | MMTV STC intasomes were applied onto R1.2/1.3 gold UltrAufoil grids, Au 300 mesh (Quantifoil). Cryo-EM grids were prepared by manually freezing using a manual plunger in cold room at 4C and stored in liquid nitrogen for future data acquisition. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-100 / Number grids imaged: 1 / Number real images: 1578 / Average exposure time: 10.0 sec. / Average electron dose: 67.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Calibrated magnification: 38167 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500 |
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
Details | MMTV STC intasomes were rigid body docked into the EM map in Chimera, and the complex was subjected to a refinement in Phenix. |
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Refinement | Space: REAL / Protocol: FLEXIBLE FIT / Target criteria: CC |
Output model | PDB-7ut1: |