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- EMDB-26744: Higher-order assembly of multiple MMTV strand transfer complex in... -

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Basic information

Entry
Database: EMDB / ID: EMD-26744
TitleHigher-order assembly of multiple MMTV strand transfer complex intasomes
Map datasharpened map
Sample
  • Complex: Higher-order assembly of multiple MMTV strand transfer complex intasomes
    • Protein or peptide: Integrase
    • DNA: vDNA strand (non-transferred)
    • DNA: vDNA-tDNA strand (transferred)
    • DNA: tDNA strand
  • Ligand: ZINC ION
KeywordsIntegrase-DNA complex / hydrolase / VIRAL PROTEIN / HYDROLASE-DNA complex
Function / homology
Function and homology information


dUTP diphosphatase activity / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA stem-loop binding / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / viral nucleocapsid / DNA recombination / structural constituent of virion ...dUTP diphosphatase activity / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA stem-loop binding / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / viral nucleocapsid / DNA recombination / structural constituent of virion / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / proteolysis / DNA binding / zinc ion binding
Similarity search - Function
GAG-polyprotein viral zinc-finger / Beta-retroviral matrix protein / Beta-retroviral matrix superfamily / Retroviral GAG p10 protein / dUTPase-like / dUTPase / dUTPase, trimeric / dUTPase-like superfamily / gag protein p24 N-terminal domain / Reverse transcriptase thumb ...GAG-polyprotein viral zinc-finger / Beta-retroviral matrix protein / Beta-retroviral matrix superfamily / Retroviral GAG p10 protein / 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. / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retropepsin-like catalytic domain / Ribonuclease H domain / RNase H type-1 domain profile. / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / 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. / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Gag-Pro-Pol polyprotein
Similarity search - Component
Biological speciesMouse mammary tumor virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsJozwik I / Lyumkis D
Funding support United States, 3 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)MCB-2048095 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U54 AI150472 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI136680 United States
CitationJournal: Nucleic Acids Res / Year: 2022
Title: B-to-A transition in target DNA during retroviral integration.
Authors: Ilona K Jóźwik / Wen Li / Da-Wei Zhang / Doris Wong / Julia Grawenhoff / Allison Ballandras-Colas / Sriram Aiyer / Peter Cherepanov / Alan N Engelman / Dmitry Lyumkis /
Abstract: Integration into host target DNA (tDNA), a hallmark of retroviral replication, is mediated by the intasome, a multimer of integrase (IN) assembled on viral DNA (vDNA) ends. To ascertain aspects of ...Integration into host target DNA (tDNA), a hallmark of retroviral replication, is mediated by the intasome, a multimer of integrase (IN) assembled on viral DNA (vDNA) ends. To ascertain aspects of tDNA recognition during integration, we have solved the 3.5 Å resolution cryo-EM structure of the mouse mammary tumor virus (MMTV) strand transfer complex (STC) intasome. The tDNA adopts an A-like conformation in the region encompassing the sites of vDNA joining, which exposes the sugar-phosphate backbone for IN-mediated strand transfer. Examination of existing retroviral STC structures revealed conservation of A-form tDNA in the analogous regions of these complexes. Furthermore, analyses of sequence preferences in genomic integration sites selectively targeted by six different retroviruses highlighted consistent propensity for A-philic sequences at the sites of vDNA joining. Our structure additionally revealed several novel MMTV IN-DNA interactions, as well as contacts seen in prior STC structures, including conserved Pro125 and Tyr149 residues interacting with tDNA. In infected cells, Pro125 substitutions impacted the global pattern of MMTV integration without significantly altering local base sequence preferences at vDNA insertion sites. Collectively, these data advance our understanding of retroviral intasome structure and function, as well as factors that influence patterns of vDNA integration in genomic DNA.
History
DepositionApr 26, 2022-
Header (metadata) releaseAug 24, 2022-
Map releaseAug 24, 2022-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26744.png

Downloads & links

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Map

FileDownload / File: emd_26744.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.36923516 - 0.8495139
Average (Standard dev.)0.005920584 (±0.03423979)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Mask #1

Fileemd_26744_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map #2

Fileemd_26744_half_map_1.map
Annotationhalf-map #2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map #1

Fileemd_26744_half_map_2.map
Annotationhalf-map #1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Higher-order assembly of multiple MMTV strand transfer complex in...

EntireName: Higher-order assembly of multiple MMTV strand transfer complex intasomes
Components
  • Complex: Higher-order assembly of multiple MMTV strand transfer complex intasomes
    • Protein or peptide: Integrase
    • DNA: vDNA strand (non-transferred)
    • DNA: vDNA-tDNA strand (transferred)
    • DNA: tDNA strand
  • Ligand: ZINC ION

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Supramolecule #1: Higher-order assembly of multiple MMTV strand transfer complex in...

SupramoleculeName: Higher-order assembly of multiple MMTV strand transfer complex intasomes
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Mouse mammary tumor virus
Molecular weightTheoretical: 0.6 kDa/nm

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Macromolecule #1: Integrase

MacromoleculeName: Integrase / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Mouse mammary tumor virus
Molecular weightTheoretical: 35.753332 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: ALESAQESHA LHHQNAAALR FQFHITREQA REIVKLCPNC PDWGHAPQLG VNPRGLKPRV LWQMDVTHVS EFGKLKYVHV TVDTYSHFT FATARTGEAT KDVLQHLAQS FAYMGIPQKI KTDNAPAYVS RSIQEFLARW KISHVTGIPY NPQGQAIVER T HQNIKAQL ...String:
ALESAQESHA LHHQNAAALR FQFHITREQA REIVKLCPNC PDWGHAPQLG VNPRGLKPRV LWQMDVTHVS EFGKLKYVHV TVDTYSHFT FATARTGEAT KDVLQHLAQS FAYMGIPQKI KTDNAPAYVS RSIQEFLARW KISHVTGIPY NPQGQAIVER T HQNIKAQL NKLQKAGKYY TPHHLLAHAL FVLNHVNMDN QGHTAAERHW GPISADPKPM VMWKDLLTGS WKGPDVLITA GR GYACVFP QDAESPIWVP DRFIRPFTER KEATPTPGTA EKTPPRDEKD QQESPKNESS PHQREDGLAT SAGVDLRSGG GP

UniProtKB: Gag-Pro-Pol polyprotein

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Macromolecule #2: vDNA strand (non-transferred)

MacromoleculeName: vDNA strand (non-transferred) / type: dna / ID: 2 / Number of copies: 4 / Classification: DNA
Source (natural)Organism: Mouse mammary tumor virus
Molecular weightTheoretical: 6.738343 KDa
SequenceString:
(DA)(DA)(DT)(DG)(DC)(DC)(DG)(DC)(DA)(DG) (DT)(DC)(DG)(DG)(DC)(DC)(DG)(DA)(DC)(DC) (DT)(DG)

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Macromolecule #3: vDNA-tDNA strand (transferred)

MacromoleculeName: vDNA-tDNA strand (transferred) / type: dna / ID: 3 / Number of copies: 4 / Classification: DNA
Source (natural)Organism: Mouse mammary tumor virus
Molecular weightTheoretical: 12.875227 KDa
SequenceString:
(DC)(DA)(DG)(DG)(DT)(DC)(DG)(DG)(DC)(DC) (DG)(DA)(DC)(DT)(DG)(DC)(DG)(DG)(DC)(DA) (DC)(DT)(DC)(DG)(DA)(DG)(DC)(DT)(DA) (DC)(DT)(DT)(DC)(DC)(DC)(DT)(DG)(DT)(DT) (DT) (DA)(DG)

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Macromolecule #4: tDNA strand

MacromoleculeName: tDNA strand / type: dna / ID: 4 / Number of copies: 4 / Classification: DNA
Source (natural)Organism: Mouse mammary tumor virus
Molecular weightTheoretical: 4.980269 KDa
SequenceString:
(DC)(DT)(DA)(DA)(DA)(DC)(DA)(DG)(DG)(DG) (DA)(DA)(DG)(DT)(DA)(DG)

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 12 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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: 6.5
Component:
ConcentrationNameFormula
25.0 mMTris-HCl
200.0 mMsodium chlorideNaCl
2.0 mMDTT
10.0 mMCalcium chlorideCaCl2
25.0 mMZinc chlorideZnCl2
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Cryo-EM grids were prepared by freezing using a manual plunger in cold room at 4C.
DetailsMMTV STC intasomes were applied onto R1.2/1.3 gold UltrAufoil grids, Au 300 mesh (Quantifoil). Cryo-EM grids were prepared by manually freezing using a manual plunger in cold room at 4C and stored in liquid nitrogen for future data acquisition.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-100 / Number grids imaged: 1 / Number real images: 1578 / Average exposure time: 10.0 sec. / Average electron dose: 67.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 38167 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1048508
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0) / Number images used: 86379
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 2.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 2.4)

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Atomic model buiding 1

DetailsMMTV STC intasomes were rigid body docked into the EM map in Chimera, and the complex was subjected to a refinement in Phenix.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: CC
Output model

PDB-7ut1:
Higher-order assembly of multiple MMTV strand transfer complex intasomes

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