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- EMDB-6440: Three-dimensional structure of the full MMTV intasome -

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Basic information

Entry
Database: EMDB / ID: EMD-6440
TitleThree-dimensional structure of the full MMTV intasome
Map dataReconstruction of the full octameric MMTV intasome
Sample
  • Sample: Mouse Mammary Tumor Virus intasome complex
  • Protein or peptide: betaretroviral integrase
  • DNA: MMTV U5 DNA end
Keywordsintegration / retrovirus / integrase
Function / homology
Function and homology information


dUTP diphosphatase / dUTP diphosphatase activity / ribonuclease H / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / RNA-directed DNA polymerase activity / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / telomerase activity ...dUTP diphosphatase / dUTP diphosphatase activity / ribonuclease H / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / RNA-directed DNA polymerase activity / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / telomerase activity / RNA stem-loop binding / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / viral nucleocapsid / DNA recombination / DNA-directed DNA polymerase / structural constituent of virion / aspartic-type endopeptidase activity / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase activity / symbiont entry into host cell / viral translational frameshifting / proteolysis / DNA binding / zinc ion binding
Similarity search - Function
Beta-retroviral matrix protein / Beta-retroviral matrix superfamily / Retroviral GAG p10 protein / GAG-polyprotein viral zinc-finger / dUTPase-like / dUTPase / dUTPase, trimeric / dUTPase-like superfamily / gag protein p24 N-terminal domain / Reverse transcriptase thumb ...Beta-retroviral matrix protein / Beta-retroviral matrix superfamily / Retroviral GAG p10 protein / GAG-polyprotein viral zinc-finger / dUTPase-like / dUTPase / dUTPase, trimeric / dUTPase-like superfamily / gag protein p24 N-terminal domain / Reverse transcriptase thumb / Reverse transcriptase thumb domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / Integrase DNA binding domain / Integrase, C-terminal domain superfamily, retroviral / Integrase, N-terminal zinc-binding domain / Integrase, C-terminal, retroviral / Integrase DNA binding domain profile. / RNase H / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Retropepsin-like catalytic domain / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / RNase H type-1 domain profile. / Ribonuclease H domain / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / Retrovirus capsid, C-terminal / Retroviral matrix protein / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Pol / Gag-Pro-Pol polyprotein
Similarity search - Component
Biological speciesMouse mammary tumor virus
Methodsingle particle reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsBallandras-Colas A / Brown M / Cook N / Demeler B / Cherepanov P / Lyumkis D / Engelman AN
CitationJournal: Nature / Year: 2016
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
DepositionAug 26, 2015-
Header (metadata) releaseSep 23, 2015-
Map releaseFeb 17, 2016-
UpdateApr 13, 2016-
Current statusApr 13, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6440.gif

Downloads & links

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Map

FileDownload / File: emd_6440.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the full octameric MMTV intasome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 256 pix.
= 335.36 Å		1.31 Å/pix.
x 256 pix.
= 335.36 Å		1.31 Å/pix.
x 256 pix.
= 335.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.31 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.0317705 - 0.11796439
Average (Standard dev.)-0.00048652 (±0.00419351)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 335.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z335.360335.360335.360
α/β/γ90.00090.00090.000
start NX/NY/NZ-8211244
NX/NY/NZ115138149
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0320.118-0.000

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Supplemental data

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Sample components

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Entire : Mouse Mammary Tumor Virus intasome complex

EntireName: Mouse Mammary Tumor Virus intasome complex
Components
  • Sample: Mouse Mammary Tumor Virus intasome complex
  • Protein or peptide: betaretroviral integrase
  • DNA: MMTV U5 DNA end

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Supramolecule #1000: Mouse Mammary Tumor Virus intasome complex

SupramoleculeName: Mouse Mammary Tumor Virus intasome complex / type: sample / ID: 1000
Oligomeric state: Integrase octamer bound to two vDNA strands
Number unique components: 2
Molecular weightExperimental: 318.9 MDa / Theoretical: 313.6 MDa / Method: sedimentation velocity centrifugation

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Macromolecule #1: betaretroviral integrase

MacromoleculeName: betaretroviral integrase / type: protein_or_peptide / ID: 1 / Name.synonym: MMTV IN / Number of copies: 8 / Oligomeric state: octamer / Recombinant expression: Yes
Source (natural)Organism: Mouse mammary tumor virus / synonym: MMTV
Molecular weightTheoretical: 36 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: PC2 / Recombinant plasmid: pET-15b

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Macromolecule #2: MMTV U5 DNA end

MacromoleculeName: MMTV U5 DNA end / type: dna / ID: 2 / Name.synonym: viral DNA / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: Mouse mammary tumor virus / synonym: MMTV
Molecular weightTheoretical: 13 KDa
SequenceString:
CAGGTCGGCC GACTGCGGCA

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Details: 25 mM Tris-HCl, 200 mM NaCl, 2 mM DTT, 25 uM ZnCl2, 10 mM CaCl2
GridDetails: 400 mesh C-flat, plasma-treated for 6 seconds
VitrificationCryogen 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

MicroscopeFEI TITAN KRIOS
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected using Leginon, and coma-free alignment was established.
DateApr 15, 2016
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 2714 / Average electron dose: 40 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionAlgorithm: 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 assignmentDetails: Frealign, phi, theta, psi

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