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- EMDB-26322: MVV cleaved synaptic complex (CSC) intasome at 3.4 A resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-26322
TitleMVV cleaved synaptic complex (CSC) intasome at 3.4 A resolution
Map datasharpened cryo-EM reconstruction of MVV CSC
Sample
  • Complex: MVV cleaved synaptic complex intasome
    • Protein or peptide: Integrase
    • DNA: DNA EV273
    • DNA: DNA EV272
  • Ligand: ZINC ION
  • Ligand: CALCIUM IONCalcium
KeywordsIntegrase-DNA complex / hydrolase / VIRAL PROTEIN / VIRAL PROTEIN-DNA complex
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 speciesStreptomyces griseus (bacteria) / Visna/maedi virus EV1 KV1772 / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsShan Z / Pye VE / Cherepanov P / Lyumkis D
Funding support United States, United Kingdom, 5 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
The Francis Crick InstituteFC10061 United Kingdom
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P50AI150481 United States
CitationJournal: Nat Commun / Year: 2022
Title: Multivalent interactions essential for lentiviral integrase function.
Authors: Allison Ballandras-Colas / Vidya Chivukula / Dominika T Gruszka / Zelin Shan / Parmit K Singh / Valerie E Pye / Rebecca K McLean / Gregory J Bedwell / Wen Li / Andrea Nans / Nicola J Cook / ...Authors: Allison Ballandras-Colas / Vidya Chivukula / Dominika T Gruszka / Zelin Shan / Parmit K Singh / Valerie E Pye / Rebecca K McLean / Gregory J Bedwell / Wen Li / Andrea Nans / Nicola J Cook / Hind J Fadel / Eric M Poeschla / David J Griffiths / Javier Vargas / Ian A Taylor / Dmitry Lyumkis / Hasan Yardimci / Alan N Engelman / Peter Cherepanov /
Abstract: A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi- ...A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits. Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 Å resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the α-helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching reveals that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 results in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes.
History
DepositionFeb 25, 2022-
Header (metadata) releaseMay 11, 2022-
Map releaseMay 11, 2022-
UpdateFeb 21, 2024-
Current statusFeb 21, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26322.png

Downloads & links

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Map

FileDownload / File: emd_26322.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened cryo-EM reconstruction of MVV CSC
Voxel sizeX=Y=Z: 0.92 Å
Density
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-3.0471601 - 6.298784
Average (Standard dev.)0.020010429 (±0.09795843)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 353.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26322_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: unsharpened map

Fileemd_26322_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Reconstructed map after DeepEMhancer

Fileemd_26322_additional_2.map
AnnotationReconstructed map after DeepEMhancer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map #2

Fileemd_26322_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_26322_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 : MVV cleaved synaptic complex intasome

EntireName: MVV cleaved synaptic complex intasome
Components
  • Complex: MVV cleaved synaptic complex intasome
    • Protein or peptide: Integrase
    • DNA: DNA EV273
    • DNA: DNA EV272
  • Ligand: ZINC ION
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: MVV cleaved synaptic complex intasome

SupramoleculeName: MVV cleaved synaptic complex intasome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Streptomyces griseus (bacteria)
Molecular weightTheoretical: 0.48 kDa/nm

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Macromolecule #1: Integrase

MacromoleculeName: Integrase / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Visna/maedi virus EV1 KV1772 / 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

UniProtKB: Gag-Pol polyprotein

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Macromolecule #2: DNA EV273

MacromoleculeName: DNA EV273 / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.943719 KDa
SequenceString:
(DG)(DC)(DT)(DG)(DC)(DG)(DA)(DG)(DA)(DT) (DC)(DC)(DG)(DC)(DT)(DC)(DC)(DG)(DG)(DT) (DG)(DT)(DT)(DG)(DC)(DA)(DC)(DG)(DG)

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Macromolecule #3: DNA EV272

MacromoleculeName: DNA EV272 / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.272326 KDa
SequenceString:
(DC)(DC)(DG)(DT)(DG)(DC)(DA)(DA)(DC)(DA) (DC)(DC)(DG)(DG)(DA)(DG)(DC)(DG)(DG)(DA) (DT)(DC)(DT)(DC)(DG)(DC)(DA)

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 12 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #5: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 2 / 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
350.0 mMsodium chlorideNaClSodium chloride
1.0 mMTCEP
3.0 mMCalcium chlorideCaCl2
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec.
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Details: Cryo-EM grids were prepared by freezing using a manual plunger in cold room at 4C.
DetailsMVV CSC intasomes, assembled and purified as previously described, 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 TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 54347 / 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: 45000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsThe stage was tilted to 40 degrees during data collection to account for the preferential orientation of the sample within the vitreous ice.
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: 2295 / Average exposure time: 10.0 sec. / Average electron dose: 43.6 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 926176
Startup modelType of model: EMDB MAP
EMDB ID:

4138
EMDB Unreleased entry

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0) / Number images used: 147860
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsThe STC model refined in study, with the tDNA removed, was docked into the CSC cryoEM map using UCSF Chimera. It was observed that there were some slight differences in some domain positions, to address this, individual domains that were not well fitted to the map were docked as individual domains to achieve a best-fit starting model. Two (C2 related) NTDs (aa 1-35) were removed from the model due to lack of supporting map. Adjustments were made to the model interactively using Coot and the coordinates were subjected to real-space refinement in Phenix dev-4213-000 employing C2 NCS constraints.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 262 / Target criteria: CC
Output model

PDB-7u32:
MVV cleaved synaptic complex (CSC) intasome at 3.4 A resolution

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