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- EMDB-4139: Cryo-EM reconstruction of the maedi-visna virus (MVV) strand tran... -

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Basic information

Entry
Database: EMDB / ID: EMD-4139
TitleCryo-EM reconstruction of the maedi-visna virus (MVV) strand transfer complex
Map dataMVV STC reconstruction at 8.6 %u212B resolution.
Sample
  • Complex: MVV strand transfer complex
    • Complex: intergrase
      • Protein or peptide: integrase
    • Complex: nucleic acid
      • DNA: vDNA, non-transfered strand
      • DNA: vDNA-tDNA, transferred strand, joined to a model tDNA
    • Complex: nucleic acid
      • DNA: tDNA
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 / RNA-directed DNA polymerase / viral genome integration into host DNA ...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 / RNA-directed DNA polymerase / viral genome integration into host DNA / 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
Biological speciesMaedi visna virus (strain KV1772) / Visna lentivirus (strain 1514) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsPye VE / Ballandras-Colas A / Maskell D / Locke J / Kotecha A / Costa A / Cherepanov P
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, 2016-
Header (metadata) releaseOct 26, 2016-
Map releaseJan 18, 2017-
UpdateNov 21, 2018-
Current statusNov 21, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5m0r
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4139.png

Downloads & links

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Map

FileDownload / File: emd_4139.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMVV STC reconstruction at 8.6 %u212B resolution.
Voxel sizeX=Y=Z: 2.7 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.20115614 - 0.3331452
Average (Standard dev.)0.0012151365 (±0.011176318)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 405.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.72.72.7
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z405.000405.000405.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.2010.3330.001

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Supplemental data

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Half map: None

Fileemd_4139_half_map_1.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: None

Fileemd_4139_half_map_2.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MVV strand transfer complex

EntireName: MVV strand transfer complex
Components
  • Complex: MVV strand transfer complex
    • Complex: intergrase
      • Protein or peptide: integrase
    • Complex: nucleic acid
      • DNA: vDNA, non-transfered strand
      • DNA: vDNA-tDNA, transferred strand, joined to a model tDNA
    • Complex: nucleic acid
      • DNA: tDNA

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Supramolecule #1: MVV strand transfer complex

SupramoleculeName: MVV strand transfer complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: intergrase

SupramoleculeName: intergrase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Maedi visna virus (strain KV1772)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: nucleic acid

SupramoleculeName: nucleic acid / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Visna lentivirus (strain 1514)
Recombinant expressionOrganism: synthetic construct (others)

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Supramolecule #4: nucleic acid

SupramoleculeName: nucleic acid / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: integrase

MacromoleculeName: integrase / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases
Source (natural)Organism: Maedi visna virus (strain KV1772)
Molecular weightTheoretical: 32.368826 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: WIENIPLAEE EHNKWHQDAV SLHLEFGIPR TAAEDIVQQC DVCQENKMPS TLRGSNKRGI DHWQVDYTHY EDKIILVWVE TNSGLIYAE RVKGETGQEF RVQTMKWYAM FAPKSLQSDN GPAFVAESTQ LLMKYLGIEH TTGIPWNPQS QALVERTHQT L KNTLEKLI ...String:
WIENIPLAEE EHNKWHQDAV SLHLEFGIPR TAAEDIVQQC DVCQENKMPS TLRGSNKRGI DHWQVDYTHY EDKIILVWVE TNSGLIYAE RVKGETGQEF RVQTMKWYAM FAPKSLQSDN GPAFVAESTQ LLMKYLGIEH TTGIPWNPQS QALVERTHQT L KNTLEKLI PMFNAFESAL AGTLITLNIK RKGGLGTSPM DIFIFNKEQQ RIQQQSKSKQ EKIRFCYYRT RKRGHPGEWQ GP TQVLWGG DGAIVVKDRG TDRYLVIANK DVKFIPPPKE IQKE

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Macromolecule #2: vDNA, non-transfered strand

MacromoleculeName: vDNA, non-transfered strand / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Visna lentivirus (strain 1514)
Molecular weightTheoretical: 6.456146 KDa
SequenceString:
(DG)(DC)(DT)(DG)(DC)(DG)(DA)(DG)(DA)(DT) (DC)(DC)(DG)(DC)(DT)(DC)(DC)(DG)(DG)(DT) (DG)

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Macromolecule #3: vDNA-tDNA, transferred strand, joined to a model tDNA

MacromoleculeName: vDNA-tDNA, transferred strand, joined to a model tDNA / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Visna lentivirus (strain 1514)
Molecular weightTheoretical: 15.387863 KDa
SequenceString:
(DA)(DA)(DC)(DA)(DC)(DC)(DG)(DG)(DA)(DG) (DC)(DG)(DG)(DA)(DT)(DC)(DT)(DC)(DG)(DC) (DA)(DG)(DT)(DC)(DG)(DA)(DC)(DC)(DA) (DC)(DC)(DC)(DT)(DA)(DA)(DT)(DC)(DA)(DA) (DG) (DT)(DT)(DT)(DT)(DT)(DT)(DG)(DG) (DG)(DG)

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Macromolecule #4: tDNA

MacromoleculeName: tDNA / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.0736 KDa
SequenceString:
(DC)(DC)(DC)(DC)(DA)(DA)(DA)(DA)(DA)(DA) (DC)(DT)(DT)(DG)(DA)(DT)(DT)(DA)(DG)(DG) (DG)(DT)(DG)

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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

Concentration0.3 mg/mL
BufferpH: 6.5
Component:
ConcentrationFormulaName
1.0 MNaClSodium chloridesodium chloride
3.0 mMCaCl2calcium chloride
25.0 mMBis-TrisBis-tris methane
GridModel: Ted Pella, lacey carbon grids coated with ultrathin carbon
Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2.0 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
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

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 722 / Average electron dose: 1.47 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 37021
CTF correctionSoftware: (Name: CTFFIND (ver. 4), RELION (ver. 1.4))
Startup modelType of model: OTHER
Details: Initial model was produced with e2initialmodel.py from EMAN2 package using 2D classes of negatively stained particles
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 37021
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

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

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