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- PDB-7pel: CryoEM structure of simian T-cell lymphotropic virus intasome in ... -

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Entry
Database: PDB / ID: 7pel
TitleCryoEM structure of simian T-cell lymphotropic virus intasome in complex with PP2A regulatory subunit B56 gamma
Components
  • DNA (5'-D(*AP*CP*TP*GP*TP*GP*TP*TP*TP*GP*GP*CP*GP*CP*TP*TP*CP*TP*CP*TP*C)-3')
  • DNA (5'-D(*GP*AP*GP*AP*GP*AP*AP*GP*CP*GP*CP*CP*AP*AP*AP*CP*AP*CP*A)-3')
  • Isoform 3 of PC4 and SFRS1-interacting protein,Isoform Gamma-2 of Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit gamma isoform
  • Pol protein
KeywordsVIRAL PROTEIN / integrase / transferase / deltaretrovirus / PP2A / B56 / phosphatase / molecular mimicry / host factor / DNA-binding
Function / homology
Function and homology information


protein phosphatase type 2A complex / protein phosphatase regulator activity / APC truncation mutants have impaired AXIN binding / AXIN missense mutants destabilize the destruction complex / Truncations of AMER1 destabilize the destruction complex / Beta-catenin phosphorylation cascade / Signaling by GSK3beta mutants / CTNNB1 S33 mutants aren't phosphorylated / CTNNB1 S37 mutants aren't phosphorylated / CTNNB1 S45 mutants aren't phosphorylated ...protein phosphatase type 2A complex / protein phosphatase regulator activity / APC truncation mutants have impaired AXIN binding / AXIN missense mutants destabilize the destruction complex / Truncations of AMER1 destabilize the destruction complex / Beta-catenin phosphorylation cascade / Signaling by GSK3beta mutants / CTNNB1 S33 mutants aren't phosphorylated / CTNNB1 S37 mutants aren't phosphorylated / CTNNB1 S45 mutants aren't phosphorylated / CTNNB1 T41 mutants aren't phosphorylated / supercoiled DNA binding / Disassembly of the destruction complex and recruitment of AXIN to the membrane / Integration of viral DNA into host genomic DNA / Autointegration results in viral DNA circles / CTLA4 inhibitory signaling / Platelet sensitization by LDL / 2-LTR circle formation / Formation of WDR5-containing histone-modifying complexes / Vpr-mediated nuclear import of PICs / protein phosphatase activator activity / Integration of provirus / APOBEC3G mediated resistance to HIV-1 infection / mRNA 5'-splice site recognition / chromosome, centromeric region / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / heterochromatin / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Resolution of Sister Chromatid Cohesion / DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest / nuclear periphery / RHO GTPases Activate Formins / RAF activation / Degradation of beta-catenin by the destruction complex / euchromatin / DNA integration / RNA stem-loop binding / Negative regulation of MAPK pathway / Separation of Sister Chromatids / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / Regulation of TP53 Degradation / PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling / response to heat / proteasome-mediated ubiquitin-dependent protein catabolic process / DNA-binding transcription factor binding / DNA recombination / response to oxidative stress / transcription coactivator activity / chromatin remodeling / negative regulation of cell population proliferation / chromatin binding / Golgi apparatus / signal transduction / positive regulation of transcription by RNA polymerase II / RNA binding / zinc ion binding / nucleoplasm / nucleus / cytosol
Similarity search - Function
Protein phosphatase 2A, regulatory B subunit, B56 / Protein phosphatase 2A regulatory B subunit (B56 family) / Lens epithelium-derived growth factor, integrase-binding domain / HIV integrase-binding domain superfamily / Lens epithelium-derived growth factor (LEDGF) / TFIIS/LEDGF domain superfamily / domain with conserved PWWP motif / PWWP domain / PWWP domain profile. / PWWP domain ...Protein phosphatase 2A, regulatory B subunit, B56 / Protein phosphatase 2A regulatory B subunit (B56 family) / Lens epithelium-derived growth factor, integrase-binding domain / HIV integrase-binding domain superfamily / Lens epithelium-derived growth factor (LEDGF) / TFIIS/LEDGF domain superfamily / domain with conserved PWWP motif / PWWP domain / PWWP domain profile. / PWWP domain / Integrase Zinc binding domain / Integrase DNA binding domain / 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. / Ribonuclease H domain / RNase H type-1 domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Armadillo-like helical / Armadillo-type fold / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / PC4 and SFRS1-interacting protein / Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit gamma isoform / Pol protein
Similarity search - Component
Biological speciesSimian T-lymphotropic virus 1
Homo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.34 Å
AuthorsBarski, M. / Pye, V.E. / Nans, A. / Cherepanov, P. / Maertens, G.N.
Funding support United Kingdom, 5items
OrganizationGrant numberCountry
Wellcome Trust107005/Z/15Z United Kingdom
Royal SocietyRG120032 United Kingdom
Cancer Research UKFC001061 United Kingdom
Medical Research Council (MRC, United Kingdom)FC001061 United Kingdom
Wellcome TrustFC001061 United Kingdom
CitationJournal: Nat Commun / Year: 2020
Title: Cryo-EM structure of the deltaretroviral intasome in complex with the PP2A regulatory subunit B56γ.
Authors: Michał S Barski / Jordan J Minnell / Zuzana Hodakova / Valerie E Pye / Andrea Nans / Peter Cherepanov / Goedele N Maertens /
Abstract: Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor ...Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor disease, unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the retroviral integrase (IN) enzyme. Here, we use X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of the functional deltaretroviral IN assembled on viral DNA ends and bound to the B56γ subunit of its human host factor, protein phosphatase 2 A. The structure reveals a tetrameric IN assembly bound to two molecules of the phosphatase via a conserved short linear motif. Insight into the deltaretroviral intasome and its interaction with the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.
History
DepositionAug 10, 2021Deposition site: PDBE / Processing site: PDBE
SupersessionAug 25, 2021ID: 6Z2Y
Revision 1.0Aug 25, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_database_related / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Pol protein
B: Pol protein
C: Isoform 3 of PC4 and SFRS1-interacting protein,Isoform Gamma-2 of Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit gamma isoform
K: DNA (5'-D(*AP*CP*TP*GP*TP*GP*TP*TP*TP*GP*GP*CP*GP*CP*TP*TP*CP*TP*CP*TP*C)-3')
L: DNA (5'-D(*GP*AP*GP*AP*GP*AP*AP*GP*CP*GP*CP*CP*AP*AP*AP*CP*AP*CP*A)-3')
D: Pol protein
E: Pol protein
F: Isoform 3 of PC4 and SFRS1-interacting protein,Isoform Gamma-2 of Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit gamma isoform
M: DNA (5'-D(*AP*CP*TP*GP*TP*GP*TP*TP*TP*GP*GP*CP*GP*CP*TP*TP*CP*TP*CP*TP*C)-3')
N: DNA (5'-D(*GP*AP*GP*AP*GP*AP*AP*GP*CP*GP*CP*CP*AP*AP*AP*CP*AP*CP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)332,22414
Polymers331,96210
Non-polymers2624
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area29290 Å2
ΔGint-153 kcal/mol
Surface area89850 Å2

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Components

#1: Protein
Pol protein


Mass: 33885.504 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Simian T-lymphotropic virus 1 / Gene: pol / Production host: Escherichia coli (E. coli) / Strain (production host): DE3 pLacI (Rosetta-2) / References: UniProt: Q4QY51
#2: Protein Isoform 3 of PC4 and SFRS1-interacting protein,Isoform Gamma-2 of Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit gamma isoform / PP2A B subunit isoform B'-gamma / PP2A B subunit isoform B56-gamma / PP2A B subunit isoform PR61- ...PP2A B subunit isoform B'-gamma / PP2A B subunit isoform B56-gamma / PP2A B subunit isoform PR61-gamma / PP2A B subunit isoform R5-gamma / Renal carcinoma antigen NY-REN-29


Mass: 80394.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: fusion construct containing human LEDGF (residues 1-324 thus without the IBD domain) (gene PSIP1; O75475)) and human B56gammma (residues 11-380) regulatory subunit of PP2A (gene PPP2R5C) (no ...Details: fusion construct containing human LEDGF (residues 1-324 thus without the IBD domain) (gene PSIP1; O75475)) and human B56gammma (residues 11-380) regulatory subunit of PP2A (gene PPP2R5C) (no space for these details below),fusion construct containing human LEDGF (residues 1-324 thus without the IBD domain) (gene PSIP1; O75475)) and human B56gammma (residues 11-380) regulatory subunit of PP2A (gene PPP2R5C) (no space for these details below),fusion construct containing human LEDGF (residues 1-324 thus without the IBD domain) (gene PSIP1; O75475)) and human B56gammma (residues 11-380) regulatory subunit of PP2A (gene PPP2R5C) (no space for these details below),fusion construct containing human LEDGF (residues 1-324 thus without the IBD domain) (gene PSIP1; O75475)) and human B56gammma (residues 11-380) regulatory subunit of PP2A (gene PPP2R5C) (no space for these details below)
Source: (gene. exp.) Homo sapiens (human) / Gene: PPP2R5C, KIAA0044 / Production host: Escherichia coli (E. coli) / Strain (production host): LOBSTR(RIL) / References: UniProt: O75475, UniProt: Q13362
#3: DNA chain DNA (5'-D(*AP*CP*TP*GP*TP*GP*TP*TP*TP*GP*GP*CP*GP*CP*TP*TP*CP*TP*CP*TP*C)-3')


Mass: 9221.909 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (5'-D(*GP*AP*GP*AP*GP*AP*AP*GP*CP*GP*CP*CP*AP*AP*AP*CP*AP*CP*A)-3')


Mass: 8593.560 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Complex of STLV-1 MarB43 integrase with nascent viral DNA and the human PP2A B56 subunitCOMPLEXSample composition and source have been described in "macromolecules"#1-#40MULTIPLE SOURCES
2Pol proteinCOMPLEX#11RECOMBINANT
3Isoform 3 of PC4 and SFRS1-interacting protein,Isoform Gamma-2 of Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit gamma isoformCOMPLEX#21RECOMBINANT
4DNACOMPLEX#3-#41RECOMBINANT
Molecular weightValue: 0.3311 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Simian T-lymphotropic virus 133747
23Homo sapiens (human)9606
34synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
34synthetic construct (others)32630
Buffer solutionpH: 6
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMBis tris propane1
20.3 MSodium ChlorideNaCl1
SpecimenConc.: 0.76 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Complex has been purified by SEC chromatography
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 295.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 50.4 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18_3845: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatchparticle selection
4Gctf1.06CTF correction
7UCSF Chimera1.12model fitting
8NAMDNamdinatormodel fitting
9Coot0.8.9.2model fitting
11PHENIX1.18-3845model refinement
12Coot0.8.9.2model refinement
13cryoSPARC2initial Euler assignment
15RELION3.1classification
16RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4356108
Details: 2,198,454 (OH dataset) and 2,157,654 (GO dataset) 3D reconstructions using either separate subsets resulted in highly anisotropic maps due to severe preferential orientations. Because 3D-FCS ...Details: 2,198,454 (OH dataset) and 2,157,654 (GO dataset) 3D reconstructions using either separate subsets resulted in highly anisotropic maps due to severe preferential orientations. Because 3D-FCS analysis indicated favourable complementarity of the data, the datasets were merged as separate optic groups in Relion-3.1. 3D reconstruction, including Bayesian particle polishing, CTF and beam tilt refinement, as implemented in Relion-3.1, resulted in the final map with minimal anisotropy; 3D-FSC sphericity index of the final map was 0.967.
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 161921
Details: 3D reconstruction, including Bayesian particle polishing, CTF and beam tilt refinement, as implemented in Relion-3.1, resulted in the final map with minimal anisotropy; 3D-FSC sphericity ...Details: 3D reconstruction, including Bayesian particle polishing, CTF and beam tilt refinement, as implemented in Relion-3.1, resulted in the final map with minimal anisotropy; 3D-FSC sphericity index of the final map was 0.967.
Symmetry type: POINT
Atomic model buildingB value: 31.31 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-IDPdb chain residue range
16QBW

6qbw

A6QBW153-221
26TOQ

6toq

A6TOQ2212-218
36TJU

6tju

C6TJU3238-286
45JJA

5jja

A5JJA429-375
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00415588
ELECTRON MICROSCOPYf_angle_d0.5621558
ELECTRON MICROSCOPYf_dihedral_angle_d23.1485784
ELECTRON MICROSCOPYf_chiral_restr0.0412406
ELECTRON MICROSCOPYf_plane_restr0.0042452

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