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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-6440 | |||||||||
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Title | Three-dimensional structure of the full MMTV intasome | |||||||||
![]() | Reconstruction of the full octameric MMTV intasome | |||||||||
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![]() | integration / retrovirus / integrase | |||||||||
Function / homology | ![]() dUTP diphosphatase / dUTP diphosphatase activity / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / 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 ...dUTP diphosphatase / dUTP diphosphatase activity / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / 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 / nucleic acid binding / structural constituent of virion / 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 | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 6.0 Å | |||||||||
![]() | Ballandras-Colas A / Brown M / Cook N / Demeler B / Cherepanov P / Lyumkis D / Engelman AN | |||||||||
![]() | ![]() Title: Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function. Authors: Allison Ballandras-Colas / Monica Brown / Nicola J Cook / Tamaria G Dewdney / Borries Demeler / Peter Cherepanov / Dmitry Lyumkis / Alan N Engelman / ![]() ![]() Abstract: Retroviral integrase catalyses the integration of viral DNA into host target DNA, which is an essential step in the life cycle of all retroviruses. Previous structural characterization of integrase- ...Retroviral integrase catalyses the integration of viral DNA into host target DNA, which is an essential step in the life cycle of all retroviruses. Previous structural characterization of integrase-viral DNA complexes, or intasomes, from the spumavirus prototype foamy virus revealed a functional integrase tetramer, and it is generally believed that intasomes derived from other retroviral genera use tetrameric integrase. However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Here, using single-particle cryo-electron microscopy and X-ray crystallography, we determine an unexpected octameric integrase architecture for the intasome of the betaretrovirus mouse mammary tumour virus. The structure is composed of two core integrase dimers, which interact with the viral DNA ends and structurally mimic the integrase tetramer of prototype foamy virus, and two flanking integrase dimers that engage the core structure via their integrase carboxy-terminal domains. Contrary to the belief that tetrameric integrase components are sufficient to catalyse integration, the flanking integrase dimers were necessary for mouse mammary tumour virus integrase activity. The integrase octamer solves a conundrum for betaretroviruses as well as alpharetroviruses by providing critical carboxy-terminal domains to the intasome core that cannot be provided in cis because of evolutionarily restrictive catalytic core domain-carboxy-terminal domain linker regions. The octameric architecture of the intasome of mouse mammary tumour virus provides new insight into the structural basis of retroviral DNA integration. | |||||||||
History |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 52.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 10.7 KB 10.7 KB | Display Display | ![]() |
Images | ![]() ![]() | 49.2 KB 3.8 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Reconstruction of the full octameric MMTV intasome | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.31 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Mouse Mammary Tumor Virus intasome complex
Entire | Name: Mouse Mammary Tumor Virus intasome complex |
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Components |
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-Supramolecule #1000: Mouse Mammary Tumor Virus intasome complex
Supramolecule | Name: Mouse Mammary Tumor Virus intasome complex / type: sample / ID: 1000 Oligomeric state: Integrase octamer bound to two vDNA strands Number unique components: 2 |
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Molecular weight | Experimental: 318.9 MDa / Theoretical: 313.6 MDa / Method: sedimentation velocity centrifugation |
-Macromolecule #1: betaretroviral integrase
Macromolecule | Name: betaretroviral integrase / type: protein_or_peptide / ID: 1 / Name.synonym: MMTV IN / Number of copies: 8 / Oligomeric state: octamer / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 36 KDa |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #2: MMTV U5 DNA end
Macromolecule | Name: MMTV U5 DNA end / type: dna / ID: 2 / Name.synonym: viral DNA / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 13 KDa |
Sequence | String: CAGGTCGGCC GACTGCGGCA |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.4 Details: 25 mM Tris-HCl, 200 mM NaCl, 2 mM DTT, 25 uM ZnCl2, 10 mM CaCl2 |
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Grid | Details: 400 mesh C-flat, plasma-treated for 6 seconds |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER Method: 3 uL of sample was applied to the grid, adsorbed for 30 seconds, blotted, and plunge-frozen in liquid ethane. |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected using Leginon, and coma-free alignment was established. |
Date | Apr 15, 2016 |
Image recording | Category: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 2714 / Average electron dose: 40 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 38167 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
CTF correction | Details: each particle |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: OTHER / Software - Name: Frealign / Details: Local FSC values range from 5 to 6 Angstrom. / Number images used: 41475 |
Final angle assignment | Details: Frealign, phi, theta, psi |