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Yorodumi- PDB-7ut1: Higher-order assembly of multiple MMTV strand transfer complex in... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7ut1 | ||||||||||||
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Title | Higher-order assembly of multiple MMTV strand transfer complex intasomes | ||||||||||||
Components |
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Keywords | HYDROLASE/DNA / 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 | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å | ||||||||||||
Authors | Jozwik, I. / Lyumkis, D. | ||||||||||||
Funding support | United States, 3items
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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
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7ut1.cif.gz | 706.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7ut1.ent.gz | 562.8 KB | Display | PDB format |
PDBx/mmJSON format | 7ut1.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ut/7ut1 ftp://data.pdbj.org/pub/pdb/validation_reports/ut/7ut1 | HTTPS FTP |
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-Related structure data
Related structure data | 26744MC 7usfC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 35753.332 Da / Num. of mol.: 16 / Mutation: T252S Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mouse mammary tumor virus / Gene: gag-pro-pol / Production host: Escherichia coli (E. coli) / References: UniProt: O56220 #2: DNA chain | Mass: 6738.343 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) Mouse mammary tumor virus #3: DNA chain | Mass: 12875.227 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) Mouse mammary tumor virus #4: DNA chain | Mass: 4980.269 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) Mouse mammary tumor virus #5: Chemical | ChemComp-ZN / Has ligand of interest | N | |
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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: Higher-order assembly of multiple MMTV strand transfer complex intasomes Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT | ||||||||||||||||||||||||||||||
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Molecular weight | Value: 0.6 kDa/nm / Experimental value: NO | ||||||||||||||||||||||||||||||
Source (natural) | Organism: Mouse mammary tumor virus | ||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) | ||||||||||||||||||||||||||||||
Buffer solution | pH: 6.5 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES 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 ...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. | ||||||||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid type: UltrAuFoil R1.2/1.3 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE Details: Cryo-EM grids were prepared by freezing using a manual plunger in cold room at 4C |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 22500 X / Calibrated magnification: 38167 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 10 sec. / Electron dose: 67 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1578 |
Image scans | Width: 3838 / Height: 3710 / Movie frames/image: 100 / Used frames/image: 1-100 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 1048508 | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 86379 / Algorithm: FOURIER SPACE / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: CC Details: MMTV STC intasomes were rigid body docked into the EM map in Chimera, and the complex was subjected to a refinement in Phenix. |