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Yorodumi- EMDB-26322: MVV cleaved synaptic complex (CSC) intasome at 3.4 A resolution -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-26322 | ||||||||||||||||||
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Title | MVV cleaved synaptic complex (CSC) intasome at 3.4 A resolution | ||||||||||||||||||
Map data | sharpened cryo-EM reconstruction of MVV CSC | ||||||||||||||||||
Sample |
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Keywords | Integrase-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 / 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 / RNA-directed DNA polymerase activity / viral capsid / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / aspartic-type endopeptidase activity / DNA recombination / DNA-directed DNA polymerase / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase activity / symbiont entry into host cell / proteolysis / DNA binding / zinc ion binding Similarity search - Function | ||||||||||||||||||
Biological species | Streptomyces griseus (bacteria) / Visna/maedi virus EV1 KV1772 / synthetic construct (others) | ||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.46 Å | ||||||||||||||||||
Authors | Shan Z / Pye VE / Cherepanov P / Lyumkis D | ||||||||||||||||||
Funding support | United States, United Kingdom, 5 items
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_26322.map.gz | 203.3 MB | EMDB map data format | |
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Header (meta data) | emd-26322-v30.xml emd-26322.xml | 28.3 KB 28.3 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_26322_fsc.xml | 13.6 KB | Display | FSC data file |
Images | emd_26322.png | 212.3 KB | ||
Masks | emd_26322_msk_1.map | 216 MB | Mask map | |
Filedesc metadata | emd-26322.cif.gz | 7.7 KB | ||
Others | emd_26322_additional_1.map.gz emd_26322_additional_2.map.gz emd_26322_half_map_1.map.gz emd_26322_half_map_2.map.gz | 102 MB 190.7 MB 197.7 MB 197.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-26322 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-26322 | HTTPS FTP |
-Validation report
Summary document | emd_26322_validation.pdf.gz | 826.7 KB | Display | EMDB validaton report |
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Full document | emd_26322_full_validation.pdf.gz | 826.3 KB | Display | |
Data in XML | emd_26322_validation.xml.gz | 21.3 KB | Display | |
Data in CIF | emd_26322_validation.cif.gz | 28 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26322 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26322 | HTTPS FTP |
-Related structure data
Related structure data | 7u32MC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_26322.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | sharpened cryo-EM reconstruction of MVV CSC | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.92 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_26322_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: unsharpened map
File | emd_26322_additional_1.map | ||||||||||||
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Annotation | unsharpened map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: Reconstructed map after DeepEMhancer
File | emd_26322_additional_2.map | ||||||||||||
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Annotation | Reconstructed map after DeepEMhancer | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map #2
File | emd_26322_half_map_1.map | ||||||||||||
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Annotation | half map #2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map #1
File | emd_26322_half_map_2.map | ||||||||||||
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Annotation | half map #1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : MVV cleaved synaptic complex intasome
Entire | Name: MVV cleaved synaptic complex intasome |
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Components |
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-Supramolecule #1: MVV cleaved synaptic complex intasome
Supramolecule | Name: MVV cleaved synaptic complex intasome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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Source (natural) | Organism: Streptomyces griseus (bacteria) |
Molecular weight | Theoretical: 0.48 kDa/nm |
-Macromolecule #1: Integrase
Macromolecule | Name: Integrase / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases |
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Source (natural) | Organism: Visna/maedi virus EV1 KV1772 / Strain: KV1772 |
Molecular weight | Theoretical: 32.368826 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #2: DNA EV273
Macromolecule | Name: DNA EV273 / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 8.943719 KDa |
Sequence | String: (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) |
-Macromolecule #3: DNA EV272
Macromolecule | Name: DNA EV272 / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 8.272326 KDa |
Sequence | String: (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) |
-Macromolecule #4: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 4 / Number of copies: 12 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Macromolecule #5: CALCIUM ION
Macromolecule | Name: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: CA |
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Molecular weight | Theoretical: 40.078 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 6.5 Component:
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Grid | Model: 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. | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Instrument: HOMEMADE PLUNGER Details: Cryo-EM grids were prepared by freezing using a manual plunger in cold room at 4C. | |||||||||||||||
Details | MVV 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. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Details | The stage was tilted to 40 degrees during data collection to account for the preferential orientation of the sample within the vitreous ice. |
Image recording | Film 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 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Calibrated magnification: 54347 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 45000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Details | The 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. |
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Refinement | Space: REAL / Protocol: FLEXIBLE FIT / Overall B value: 262 / Target criteria: CC |
Output model | PDB-7u32: |