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- EMDB-26737: Mouse mammary tumor virus strand transfer complex intasome -

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Basic information

Entry
Database: EMDB / ID: EMD-26737
TitleMouse mammary tumor virus strand transfer complex intasome
Map datasharpened cryo-EM map from cryoSPARC used for refinement
Sample
  • Complex: MMTV strand transfer complex intasome
    • Protein or peptide: Integrase
    • DNA: vDNA strand (non-transferred)
    • DNA: vDNA-tDNA strand (transferred)
    • DNA: tDNA strand
  • Ligand: ZINC ION
  • Ligand: CALCIUM IONCalcium
Function / homology
Function and homology information


dUTP diphosphatase activity / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / viral nucleocapsid / DNA recombination / structural constituent of virion / aspartic-type endopeptidase activity ...dUTP diphosphatase activity / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / 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 / RNA 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: 3.5 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 25, 2022-
Header (metadata) releaseAug 24, 2022-
Map releaseAug 24, 2022-
UpdateNov 23, 2022-
Current statusNov 23, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26737.png

Downloads & links

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Map

FileDownload / File: emd_26737.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened cryo-EM map from cryoSPARC used for refinement
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.53075886 - 1.1645713
Average (Standard dev.)0.002247109 (±0.029403819)
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_26737_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: deepEMhanced map for visualization and building

Fileemd_26737_additional_1.map
AnnotationdeepEMhanced map for visualization and building
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map #2

Fileemd_26737_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_26737_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 : MMTV strand transfer complex intasome

EntireName: MMTV strand transfer complex intasome
Components
  • Complex: MMTV strand transfer complex intasome
    • Protein or peptide: Integrase
    • DNA: vDNA strand (non-transferred)
    • DNA: vDNA-tDNA strand (transferred)
    • DNA: tDNA strand
  • Ligand: ZINC ION
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: MMTV strand transfer complex intasome

SupramoleculeName: MMTV strand transfer complex intasome / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Mouse mammary tumor virus
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 0.3 kDa/nm

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Macromolecule #1: Integrase

MacromoleculeName: Integrase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / 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

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Macromolecule #2: vDNA strand (non-transferred)

MacromoleculeName: vDNA strand (non-transferred) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mouse mammary tumor virus
Molecular weightTheoretical: 9.234931 KDa
SequenceString:
(DA)(DA)(DT)(DG)(DC)(DC)(DG)(DC)(DA)(DG) (DT)(DC)(DG)(DG)(DC)(DC)(DG)(DA)(DC)(DC) (DT)(DG)(DA)(DG)(DG)(DG)(DT)(DC)(DA) (DC)

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Macromolecule #3: vDNA-tDNA strand (transferred)

MacromoleculeName: vDNA-tDNA strand (transferred) / type: dna / ID: 3 / Number of copies: 1 / 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: 1 / 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: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 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-HClTris
200.0 mMsodium chlorideNaClSodium chloride
2.0 mMDTT
10.0 mMCalcium chlorideCaCl2
25.0 mMZinc chlorideZnCl2
GridModel: UltrAuFoil / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
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
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 FIELDBright-field microscopy / 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
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1048508
CTF correctionSoftware - Name: cryoSPARC (ver. 3.0)
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 2.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 2.4)
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0) / Number images used: 50196

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Atomic model buiding 1

DetailsInitial model building was accomplished by rigid-body fitting of the MMTV CSC structure downloaded from the Protein Data Bank (PDB ID: 3JCA) into the EM map in Chimera 1.14 by Fit in Map tool. Unmodeled protein and DNA residues were manually built in Coot 0.9.4.1 and the structure underwent a few iterative cycles of manual model re-building and real-space refinement in Phenix. Ramachandran and secondary structure restraints were applied. To model the full octameric intasome, we additionally rigid-body docked the flanking IN dimers (PDB ID: 5CZ2) into the map. The density connecting the flanking dimers and the core was evident, but broken, and therefore a model was not derived for the linker regions. The final model accounts for the complete octameric MMTV STC intasome with connections for the linker regions.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: CC
Output model

PDB-7usf:
Mouse mammary tumor virus strand transfer complex intasome

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