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

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Basic information

Entry
Database: PDB / ID: 7usf
TitleMouse mammary tumor virus strand transfer complex intasome
Components
  • Integrase
  • tDNA strand
  • vDNA strand (non-transferred)
  • vDNA-tDNA strand (transferred)
KeywordsHYDROLASE/DNA / Integrase-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-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
DNA / DNA (> 10) / Gag-Pro-Pol polyprotein
Similarity search - Component
Biological speciesMouse mammary tumor virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsJozwik, I. / Lyumkis, D.
Funding support United States, 3items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 24, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 23, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Integrase
B: Integrase
C: Integrase
D: Integrase
I: vDNA strand (non-transferred)
J: vDNA-tDNA strand (transferred)
K: tDNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)170,40512
Polymers170,1047
Non-polymers3025
Water0
1
A: Integrase
B: Integrase
C: Integrase
D: Integrase
I: vDNA strand (non-transferred)
J: vDNA-tDNA strand (transferred)
K: tDNA strand
hetero molecules

A: Integrase
B: Integrase
C: Integrase
D: Integrase
I: vDNA strand (non-transferred)
J: vDNA-tDNA strand (transferred)
K: tDNA strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)340,81124
Polymers340,20814
Non-polymers60310
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation1
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C2 (2 fold cyclic))

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Components

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Protein , 1 types, 4 molecules ABCD

#1: Protein
Integrase /


Mass: 35753.332 Da / Num. of mol.: 4 / Mutation: T252S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mouse mammary tumor virus / Gene: gag-pro-pol / Production host: Escherichia coli (E. coli) / References: UniProt: O56220

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DNA chain , 3 types, 3 molecules IJK

#2: DNA chain vDNA strand (non-transferred)


Mass: 9234.931 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mouse mammary tumor virus
#3: DNA chain vDNA-tDNA strand (transferred)


Mass: 12875.227 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mouse mammary tumor virus
#4: DNA chain tDNA strand


Mass: 4980.269 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mouse mammary tumor virus

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Non-polymers , 2 types, 5 molecules

#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#6: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca

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Details

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

ComponentName: MMTV strand transfer complex intasome / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.3 kDa/nm / Experimental value: NO
Source (natural)Organism: Mouse mammary tumor virus
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HClTris1
2200 mMsodium chlorideNaClSodium chloride1
32 mMDTT1
410 mMCalcium chlorideCaCl21
525 mMZinc chlorideZnCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: MMTV 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 ...Details: MMTV 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.
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: Cryo-EM grids were prepared by freezing using a manual plunger in cold room at 4C

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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 FIELDBright-field microscopy / Nominal magnification: 22500 X / Calibrated magnification: 38167 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 67 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1578
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 100 / Used frames/image: 1-100

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
4cryoSPARC3CTF correction
7PHENIXdev-4213-000model fitting
9cryoSPARC2.4initial Euler assignment
10cryoSPARC2.4final Euler assignment
12cryoSPARC33D reconstruction
13Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1048508
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50196 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: CC
Details: Initial 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 ...Details: Initial 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.

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