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- PDB-5m0r: Cryo-EM reconstruction of the maedi-visna virus (MVV) strand tran... -

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Basic information

Entry
Database: PDB / ID: 5m0r
TitleCryo-EM reconstruction of the maedi-visna virus (MVV) strand transfer complex
Components
  • integrase
  • tDNA
  • vDNA, non-transfered strand
  • vDNA-tDNA, transferred strand, joined to a model tDNA
KeywordsHYDROLASE / retrovirus / lentivirus / integrase / DNA-binding / Zn-binding / RNAseH fold
Function / homology
Function and homology information


dUTP diphosphatase / dUTP diphosphatase activity / nucleotide metabolic process / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / exoribonuclease H / exoribonuclease H activity / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase ...dUTP diphosphatase / dUTP diphosphatase activity / nucleotide metabolic process / Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / exoribonuclease H / exoribonuclease H activity / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / viral capsid / RNA-directed DNA polymerase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / RNA-DNA hybrid ribonuclease activity / DNA recombination / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / proteolysis / DNA binding / zinc ion binding
Similarity search - Function
dUTPase-like / dUTPase / dUTPase, trimeric / dUTPase-like superfamily / gag protein p24 N-terminal domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / Integrase, C-terminal domain superfamily, retroviral / Integrase, N-terminal zinc-binding domain ...dUTPase-like / dUTPase / dUTPase, trimeric / dUTPase-like superfamily / gag protein p24 N-terminal domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / 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 / 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 / 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) / Gag-Pol polyprotein
Similarity search - Component
Biological speciesMaedi visna virus
Visna lentivirus
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsPye, V.E. / Ballandras-Colas, A. / Maskell, D. / Locke, J. / Kotecha, A. / Costa, A. / Cherepanov, P.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of HealthGM082251 United States
CitationJournal: Science / Year: 2017
Title: A supramolecular assembly mediates lentiviral DNA integration.
Authors: Allison Ballandras-Colas / Daniel P Maskell / Erik Serrao / Julia Locke / Paolo Swuec / Stefán R Jónsson / Abhay Kotecha / Nicola J Cook / Valerie E Pye / Ian A Taylor / Valgerdur ...Authors: Allison Ballandras-Colas / Daniel P Maskell / Erik Serrao / Julia Locke / Paolo Swuec / Stefán R Jónsson / Abhay Kotecha / Nicola J Cook / Valerie E Pye / Ian A Taylor / Valgerdur Andrésdóttir / Alan N Engelman / Alessandro Costa / Peter Cherepanov /
Abstract: Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo-electron microscopy, we now visualize the ...Retroviral integrase (IN) functions within the intasome nucleoprotein complex to catalyze insertion of viral DNA into cellular chromatin. Using cryo-electron microscopy, we now visualize the functional maedi-visna lentivirus intasome at 4.9 angstrom resolution. The intasome comprises a homo-hexadecamer of IN with a tetramer-of-tetramers architecture featuring eight structurally distinct types of IN protomers supporting two catalytically competent subunits. The conserved intasomal core, previously observed in simpler retroviral systems, is formed between two IN tetramers, with a pair of C-terminal domains from flanking tetramers completing the synaptic interface. Our results explain how HIV-1 IN, which self-associates into higher-order multimers, can form a functional intasome, reconcile the bulk of early HIV-1 IN biochemical and structural data, and provide a lentiviral platform for design of HIV-1 IN inhibitors.
History
DepositionOct 5, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 18, 2017Provider: repository / Type: Initial release
Revision 1.1Jan 25, 2017Group: Source and taxonomy
Revision 1.2Aug 2, 2017Group: Data collection / Derived calculations / Category: em_software / struct_conn / Item: _em_software.name
Revision 1.3Oct 24, 2018Group: Advisory / Data collection / Derived calculations / Category: pdbx_validate_close_contact / struct_conn
Revision 1.4Nov 21, 2018Group: Advisory / Data collection / Derived calculations
Category: pdbx_validate_close_contact / struct_conn / struct_conn_type

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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: integrase
J: integrase
K: integrase
L: integrase
M: integrase
N: integrase
O: integrase
P: integrase
Q: vDNA, non-transfered strand
R: vDNA-tDNA, transferred strand, joined to a model tDNA
U: tDNA
S: vDNA, non-transfered strand
T: vDNA-tDNA, transferred strand, joined to a model tDNA
V: tDNA


Theoretical massNumber of molelcules
Total (without water)575,73622
Polymers575,73622
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area81700 Å2
ΔGint-504 kcal/mol
Surface area192480 Å2
MethodPISA

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Components

#1: Protein
integrase /


Mass: 32368.826 Da / Num. of mol.: 16 / Fragment: UNP residues 821-1101
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Maedi visna virus (strain KV1772) / Gene: pol / Production host: Escherichia coli (E. coli) / Strain (production host): PC2
References: UniProt: P35956, Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases, RNA-directed DNA polymerase, retroviral ribonuclease H, exoribonuclease H, dUTP diphosphatase, ...References: UniProt: P35956, Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases, RNA-directed DNA polymerase, retroviral ribonuclease H, exoribonuclease H, dUTP diphosphatase, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, Hydrolases; Acting on ester bonds
#2: DNA chain vDNA, non-transfered strand


Mass: 6456.146 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Visna lentivirus (strain 1514)
#3: DNA chain vDNA-tDNA, transferred strand, joined to a model tDNA


Mass: 15387.863 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Visna lentivirus (strain 1514)
#4: DNA chain tDNA


Mass: 7073.600 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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
IDNameTypeEntity IDParent-IDSource
1MVV strand transfer complexCOMPLEXall0MULTIPLE SOURCES
2intergraseCOMPLEX#11RECOMBINANT
3nucleic acidCOMPLEX#2-#31RECOMBINANT
4nucleic acidCOMPLEX#41RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Maedi visna virus (strain KV1772)36374
23Visna lentivirus (strain 1514)11742
34synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23synthetic construct (others)32630
34synthetic construct (others)32630
Buffer solutionpH: 6.5
Buffer component
IDConc.NameFormulaBuffer-ID
11 Msodium chlorideNaClSodium chloride1
23 mMcalcium chlorideCaCl21
325 mMBis-TrisBis-tris methane1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in.
Grid type: Ted Pella, lacey carbon grids coated with ultrathin carbon
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K
Details: To lower salt concentration before plunge-freezing, the grids were blotted for 0.5 s, immediately hydrated with a 4-ul drop of 200 mM NaCl, 3 mM CaCl2 and 25 mM BisTris-HCl pH 6.5 and ...Details: To lower salt concentration before plunge-freezing, the grids were blotted for 0.5 s, immediately hydrated with a 4-ul drop of 200 mM NaCl, 3 mM CaCl2 and 25 mM BisTris-HCl pH 6.5 and blotted again for 2.5 s followed by plunging into liquid ethane.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 1.47 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 722
Image scansMovie frames/image: 30

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Processing

EM software
IDNameVersionCategory
1RELION1.4particle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
5RELION1.4CTF correction
8UCSF Chimeramodel fitting
10RELION1.4initial Euler assignment
11RELION1.4final Euler assignment
13RELION1.43D reconstruction
14Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 37021
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37021 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL

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