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- PDB-3jca: Core model of the Mouse Mammary Tumor Virus intasome -

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Basic information

Entry
Database: PDB / ID: 3jca
TitleCore model of the Mouse Mammary Tumor Virus intasome
Components
  • (Integrase) x 2
  • 5'-D(*AP*AP*TP*GP*CP*CP*GP*CP*AP*GP*TP*CP*GP*GP*CP*CP*GP*AP*CP*CP*TP*G)-3'
  • 5'-D(*CP*AP*GP*GP*TP*CP*GP*GP*CP*CP*GP*AP*CP*TP*GP*CP*GP*GP*CP*A)-3'
KeywordsVIRAL PROTEIN / integration / retrovirus / integrase / intasome
Function / homology
Function and homology information


dUTP diphosphatase / dUTP diphosphatase activity / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases ...dUTP diphosphatase / dUTP diphosphatase activity / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / RNA-DNA hybrid ribonuclease activity / viral nucleocapsid / DNA recombination / structural constituent of virion / Hydrolases; Acting on ester bonds / nucleic acid binding / DNA-directed DNA polymerase / 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. / Ribonuclease H superfamily / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / Integrase / Gag-Pro-Pol polyprotein
Similarity search - Component
Biological speciesMouse mammary tumor virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsLyumkis, D.L. / Ballandras-Colas, A. / Brown, M. / Cook, N.J. / Dewdney, T.G. / Demeler, B. / Cherepanov, P. / Engelman, A.N.
CitationJournal: Nature / Year: 2016
Title: Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function.
Authors: Allison Ballandras-Colas / Monica Brown / Nicola J Cook / Tamaria G Dewdney / Borries Demeler / Peter Cherepanov / Dmitry Lyumkis / Alan N Engelman /
Abstract: Retroviral integrase catalyses the integration of viral DNA into host target DNA, which is an essential step in the life cycle of all retroviruses. Previous structural characterization of integrase- ...Retroviral integrase catalyses the integration of viral DNA into host target DNA, which is an essential step in the life cycle of all retroviruses. Previous structural characterization of integrase-viral DNA complexes, or intasomes, from the spumavirus prototype foamy virus revealed a functional integrase tetramer, and it is generally believed that intasomes derived from other retroviral genera use tetrameric integrase. However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Here, using single-particle cryo-electron microscopy and X-ray crystallography, we determine an unexpected octameric integrase architecture for the intasome of the betaretrovirus mouse mammary tumour virus. The structure is composed of two core integrase dimers, which interact with the viral DNA ends and structurally mimic the integrase tetramer of prototype foamy virus, and two flanking integrase dimers that engage the core structure via their integrase carboxy-terminal domains. Contrary to the belief that tetrameric integrase components are sufficient to catalyse integration, the flanking integrase dimers were necessary for mouse mammary tumour virus integrase activity. The integrase octamer solves a conundrum for betaretroviruses as well as alpharetroviruses by providing critical carboxy-terminal domains to the intasome core that cannot be provided in cis because of evolutionarily restrictive catalytic core domain-carboxy-terminal domain linker regions. The octameric architecture of the intasome of mouse mammary tumour virus provides new insight into the structural basis of retroviral DNA integration.
History
DepositionNov 24, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 17, 2016Provider: repository / Type: Initial release
Revision 1.1Feb 24, 2016Group: Database references
Revision 1.2Apr 18, 2018Group: Data collection / Database references / Category: citation / em_software
Item: _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed ..._citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _em_software.name
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id
Revision 1.4Feb 21, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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 / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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Assembly

Deposited unit
A: Integrase
B: Integrase
C: Integrase
D: Integrase
E: Integrase
F: Integrase
G: Integrase
H: Integrase
I: 5'-D(*AP*AP*TP*GP*CP*CP*GP*CP*AP*GP*TP*CP*GP*GP*CP*CP*GP*AP*CP*CP*TP*G)-3'
J: 5'-D(*CP*AP*GP*GP*TP*CP*GP*GP*CP*CP*GP*AP*CP*TP*GP*CP*GP*GP*CP*A)-3'
K: 5'-D(*AP*AP*TP*GP*CP*CP*GP*CP*AP*GP*TP*CP*GP*GP*CP*CP*GP*AP*CP*CP*TP*G)-3'
L: 5'-D(*CP*AP*GP*GP*TP*CP*GP*GP*CP*CP*GP*AP*CP*TP*GP*CP*GP*GP*CP*A)-3'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)168,48316
Polymers168,22212
Non-polymers2624
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Integrase /


Mass: 30034.287 Da / Num. of mol.: 4 / Fragment: UNP residues 1437-1701
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mouse mammary tumor virus / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / Strain (production host): PC2 / References: UniProt: K9W608, UniProt: P03365*PLUS
#2: Protein/peptide
Integrase /


Mass: 5571.474 Da / Num. of mol.: 4 / Fragment: C-terminal domain (UNP residues 1653-1701)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mouse mammary tumor virus / Plasmid: pET15b / Production host: Escherichia coli (E. coli) / Strain (production host): PC2 / References: UniProt: K9W608, UniProt: P03365*PLUS
#3: DNA chain 5'-D(*AP*AP*TP*GP*CP*CP*GP*CP*AP*GP*TP*CP*GP*GP*CP*CP*GP*AP*CP*CP*TP*G)-3'


Mass: 6738.343 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: pre-cleaved viral DNA 3' strand / Source: (synth.) Mouse mammary tumor virus
#4: DNA chain 5'-D(*CP*AP*GP*GP*TP*CP*GP*GP*CP*CP*GP*AP*CP*TP*GP*CP*GP*GP*CP*A)-3'


Mass: 6160.968 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: pre-cleaved viral DNA 5' strand / Source: (synth.) Mouse mammary tumor virus
#5: Chemical
ChemComp-ZN / ZINC ION


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

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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
IDNameTypeDetailsParent-ID
1Mouse Mammary Tumor Virus intasome complexCOMPLEXIntegrase octamer bound to two vDNA strands0
2betaretroviral integrase1
3MMTV U5 DNA end1
Molecular weightValue: 2 MDa / Experimental value: NO
Buffer solutionName: size-exclusion buffer / pH: 7.4 / Details: size-exclusion buffer
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 400 mesh C-flat 1.2/1.3-4C, plasma-treated for 6 seconds
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temp: 77 K / Humidity: 90 %
Details: Sample containing MMTV intasomes in SEC buffer (to which the detergent NP-40 was added to a final concentration of 0.05%) was applied onto a freshly plasma-treated (6s, Gatan Solarus plasma ...Details: Sample containing MMTV intasomes in SEC buffer (to which the detergent NP-40 was added to a final concentration of 0.05%) was applied onto a freshly plasma-treated (6s, Gatan Solarus plasma cleaner) holey carbon C-flat grid (CF-1.2/1.3-4C, Protochips, Inc.), allowing the sample to adsorb for 30 seconds and then plunge-freezing in liquid ethane using a manual cryo-plunger in an ambient environment of 4 degrees C.
Method: 3 uL sample was applied to grid, adsorbed for 30 seconds, blotted, and plunge-frozen in liquid ethane.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Apr 15, 2015
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: 4000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Astigmatism: Objective lens astigmatism was corrected using Leginon, and coma-free alignment was established.
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)
Image scansNum. digital images: 2714

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Processing

EM software
IDNameCategory
1Rosettamodel fitting
2FREALIGN3D reconstruction
CTF correctionDetails: each particle
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionMethod: projection matching / Resolution: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30307 / Nominal pixel size: 1.31 Å / Actual pixel size: 1.31 Å
Details: Local FSC values range from 4-5 A. (Single particle details: IN-NTD and IN-CCD domains of flanking INs 5-8 were computationally removed using Relion after assignment of Euler angles to full ...Details: Local FSC values range from 4-5 A. (Single particle details: IN-NTD and IN-CCD domains of flanking INs 5-8 were computationally removed using Relion after assignment of Euler angles to full octameric particles and masking out of flanking regions.) (Single particle - Applied symmetry: C2)
Symmetry type: POINT
Atomic model buildingB value: 300 / Protocol: OTHER / Space: REAL / Target criteria: FSC, Rosetta energy
Details: REFINEMENT PROTOCOL--real-space refinement DETAILS--Initial components solved by X-ray were rigid-body docked. Linker regions were built de novo from the EM density. The full model was ...Details: REFINEMENT PROTOCOL--real-space refinement DETAILS--Initial components solved by X-ray were rigid-body docked. Linker regions were built de novo from the EM density. The full model was refined using real-space methods in Rosetta.
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
15CZ1

5cz1

15CZ11PDBexperimental model
25CZ2

5cz2

15CZ22PDBexperimental model
35D7U

5d7u

15D7U3PDBexperimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms9826 1636 4 0 11466

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