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- EMDB-2992: Structure of a pre-catalytic retroviral Intasome bound to a human... -

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Basic information

Entry
Database: EMDB / ID: EMD-2992
TitleStructure of a pre-catalytic retroviral Intasome bound to a human nucleosome
Map dataReconstruction of a pre-catalytic Intasome/Nucleosome complex
Sample
  • Sample: PFV intasome in complex with D02 nucleosome
  • Protein or peptide: Intasome
  • Protein or peptide: Nucleosome
  • DNA: DNA
Keywordsretroviral integration / integrase / nucleosome
Function / homology
Function and homology information


Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / virion component / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA / viral penetration into host nucleus / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases ...Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / virion component / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA / viral penetration into host nucleus / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / RNA-DNA hybrid ribonuclease activity / DNA recombination / host cell cytoplasm / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / host cell nucleus / proteolysis / RNA binding / metal ion binding
Similarity search - Function
Spumavirus aspartic protease A9 / Retroviral integrase, C-terminal SH3 domain / Spumavirus aspartic protease (A9) / Retroviral integrase C-terminal SH3 domain / Foamy virus protease (FV PR) domain profile. / Integrase zinc-binding domain / Integrase zinc binding domain / Reverse transcriptase/retrotransposon-derived protein, RNase H-like domain / RNase H-like domain found in reverse transcriptase / RNase H ...Spumavirus aspartic protease A9 / Retroviral integrase, C-terminal SH3 domain / Spumavirus aspartic protease (A9) / Retroviral integrase C-terminal SH3 domain / Foamy virus protease (FV PR) domain profile. / Integrase zinc-binding domain / Integrase zinc binding domain / Reverse transcriptase/retrotransposon-derived protein, RNase H-like domain / RNase H-like domain found in reverse transcriptase / RNase H / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Ribonuclease H domain / RNase H type-1 domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 Å
AuthorsRenault L / Maskell D / Cherepanov P / Costa A
CitationJournal: Nature / Year: 2015
Title: Structural basis for retroviral integration into nucleosomes.
Authors: Daniel P Maskell / Ludovic Renault / Erik Serrao / Paul Lesbats / Rishi Matadeen / Stephen Hare / Dirk Lindemann / Alan N Engelman / Alessandro Costa / Peter Cherepanov /
Abstract: Retroviral integration is catalysed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which ...Retroviral integration is catalysed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration.
History
DepositionMay 1, 2015-
Header (metadata) releaseMay 27, 2015-
Map releaseJun 17, 2015-
UpdateJul 15, 2015-
Current statusJul 15, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2992.png

Downloads & links

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Map

FileDownload / File: emd_2992.map.gz / Format: CCP4 / Size: 28.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of a pre-catalytic Intasome/Nucleosome complex
Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.45667186 - 0.62969732
Average (Standard dev.)0.00332227 (±0.02546307)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions196196196
Spacing196196196
CellA=B=C: 258.72 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z196196196
origin x/y/z0.0000.0000.000
length x/y/z258.720258.720258.720
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS196196196
D min/max/mean-0.4570.6300.003

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Supplemental data

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Sample components

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Entire : PFV intasome in complex with D02 nucleosome

EntireName: PFV intasome in complex with D02 nucleosome
Components
  • Sample: PFV intasome in complex with D02 nucleosome
  • Protein or peptide: Intasome
  • Protein or peptide: Nucleosome
  • DNA: DNA

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Supramolecule #1000: PFV intasome in complex with D02 nucleosome

SupramoleculeName: PFV intasome in complex with D02 nucleosome / type: sample / ID: 1000 / Number unique components: 3
Molecular weightExperimental: 390 KDa / Theoretical: 390 KDa

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Macromolecule #1: Intasome

MacromoleculeName: Intasome / type: protein_or_peptide / ID: 1 / Number of copies: 1
Oligomeric state: Heterodimer of 2 Nucleoprotein complexes (Intasome: 4 proteins + two 19bp DNA, Nucleosome: 8 proteins + 145bp DNA)
Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 399 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET
SequenceUniProtKB: Pro-Pol polyprotein

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Macromolecule #2: Nucleosome

MacromoleculeName: Nucleosome / type: protein_or_peptide / ID: 2 / Number of copies: 1
Oligomeric state: Heterodimer of 2 Nucleoprotein complexes (Intasome: 4 proteins + two 19bp DNA, Nucleosome: 8 proteins + 145bp DNA)
Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pet

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Macromolecule #3: DNA

MacromoleculeName: DNA / type: dna / ID: 3 / Classification: DNA / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)

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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

Concentration0.1 mg/mL
BufferpH: 7.45 / Details: 320 mM NaCl, 25 mM BisTris propane-HCl
GridDetails: 400 mesh C-flat copper grids CF-1/1
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 101 K / Instrument: GATAN CRYOPLUNGE 3
Method: The sample was incubated for 1 minute on the grid and blotted for 3.8 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 104500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateOct 8, 2013
Image recordingCategory: CCD / Film or detector model: FEI FALCON I (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 932 / Average electron dose: 40 e/Å2 / Bits/pixel: 32
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 53887
DetailsParticles were picked in Xmipp 3.0; Contrast Transfer Function was estimated using CTFFIND3. All further processing was performed within the RELION 1.2 environment.
FSC plot (resolution estimation)

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