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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 data
SamplePFV intasome in complex with D02 nucleosome:
Intasome / Nucleosome / nucleic-acidNucleic acid
Keywordsretroviral integration / integrase / nucleosome
Function / homology
Function and homology information


Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / virion component => GO:0044423 / viral genome integration into host DNA / viral penetration into host nucleus / RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / DNA integration / establishment of integrated proviral latency / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases ...Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / virion component => GO:0044423 / viral genome integration into host DNA / viral penetration into host nucleus / RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / DNA integration / establishment of integrated proviral latency / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / RNA-DNA hybrid ribonuclease activity / DNA recombination / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / aspartic-type endopeptidase activity / Hydrolases; Acting on ester bonds / viral entry into host cell / host cell cytoplasm / host cell nucleus / RNA binding / identical protein binding / metal ion binding
Similarity search - Function
Foamy virus protease (FV PR) domain profile. / Spumavirus aspartic protease A9 / Spumavirus aspartic protease (A9) / Retroviral integrase, C-terminal SH3 domain / Retroviral integrase C-terminal SH3 domain / 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 ...Foamy virus protease (FV PR) domain profile. / Spumavirus aspartic protease A9 / Spumavirus aspartic protease (A9) / Retroviral integrase, C-terminal SH3 domain / Retroviral integrase C-terminal SH3 domain / 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 domain profile. / Integrase, catalytic core / RNase H type-1 domain profile. / Ribonuclease H domain / Reverse transcriptase (RT) catalytic domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Aspartic peptidase domain 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
  • Download
  • 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

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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 196 pix.
= 258.72 Å
1.32 Å/pix.
x 196 pix.
= 258.72 Å
1.32 Å/pix.
x 196 pix.
= 258.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

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 / Number of Components: 3
MassTheoretical: 390 kDa / Experimental: 390 kDa

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Component #1: protein, Intasome

ProteinName: Intasome
Oligomeric Details: Heterodimer of 2 Nucleoprotein complexes (Intasome: 4 proteins + two 19bp DNA, Nucleosome: 8 proteins + 145bp DNA)
Number of Copies: 1 / Recombinant expression: Yes
MassExperimental: 399 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Vector: pET
External referencesUniProt: Pro-Pol polyprotein

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Component #2: protein, Nucleosome

ProteinName: Nucleosome
Oligomeric Details: Heterodimer of 2 Nucleoprotein complexes (Intasome: 4 proteins + two 19bp DNA, Nucleosome: 8 proteins + 145bp DNA)
Recombinant expression: Yes / Number of Copies: 1
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Vector: pet

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Component #3: nucleic-acid, DNA

nucleic acidName: DNA / Class: DNA / Structure: SINGLE STRANDED / Synthetic: Yes
SourceSpecies: synthetic construct (others)

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.1 mg/mL / Buffer solution: 320 mM NaCl, 25 mM BisTris propane-HCl / pH: 7.45
Support film400 mesh C-flat copper grids CF-1/1
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen Name: ETHANE / Temperature: 101 K / Humidity: 80 %
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 imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Oct 8, 2013
Electron gunElectron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 40 e/Å2 / Illumination Mode: FLOOD BEAM
LensMagnification: 59000 X (nominal), 104500 X (calibrated) / Cs: 2.7 mm / Imaging Mode: BRIGHT FIELD / Defocus: 1500 - 3000 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON I (4k x 4k)

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Image acquisition

Image acquisitionNumber of Digital Images: 932 / Sampling Size: 14 µm / Bit depth: 32

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Image processing

ProcessingMethod: single particle reconstruction / Applied Symmetry: C1 (asymmetric) / Number of Projections: 53887
Details: Particles were picked in Xmipp 3.0; Contrast Transfer Function was estimated using CTFFIND3. All further processing was performed within the RELION 1.2 environment.
3D reconstructionSoftware: RELION / CTF correction: each particle / Resolution: 7.8 Å / Resolution Method: FSC 0.143, gold-standard
FSC plot (resolution estimation)

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