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

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Basic information

Entry
Database: PDB / ID: 7u32
TitleMVV cleaved synaptic complex (CSC) intasome at 3.4 A resolution
Components
  • DNA EV272
  • DNA EV273
  • Integrase
KeywordsVIRAL PROTEIN/DNA / 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 / 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 speciesVisna/maedi virus EV1 KV1772
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsShan, Z. / Pye, V.E. / Cherepanov, P. / Lyumkis, D.
Funding support United States, United Kingdom, 5items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 11, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Integrase
B: Integrase
C: Integrase
D: Integrase
E: Integrase
F: Integrase
G: Integrase
H: Integrase
X: DNA EV273
W: DNA EV272
I: Integrase
J: Integrase
K: Integrase
L: Integrase
M: Integrase
N: Integrase
O: Integrase
P: Integrase
Y: DNA EV273
Z: DNA EV272
hetero molecules


Theoretical massNumber of molelcules
Total (without water)553,19834
Polymers552,33320
Non-polymers86514
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Integrase / / IN


Mass: 32368.826 Da / Num. of mol.: 16
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Visna/maedi virus EV1 KV1772 / Strain: KV1772 / Gene: pol / Production host: Escherichia coli (E. coli)
References: UniProt: P35956, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, Hydrolases; Acting on ester bonds
#2: DNA chain DNA EV273


Mass: 8943.719 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA EV272


Mass: 8272.326 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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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

ComponentName: MVV cleaved synaptic complex intasome / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.48 kDa/nm / Experimental value: NO
Source (natural)Organism: Streptomyces griseus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HClTris1
2350 mMsodium chlorideNaClSodium chloride1
31 mMTCEP1
43 mMCalcium chlorideCaCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 ...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.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: Cryo-EM grids were prepared by freezing using a manual plunger in cold room at 4C

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Details: The stage was tilted to 40 degrees during data collection to account for the preferential orientation of the sample within the vitreous ice.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 45000 X / Calibrated magnification: 54347 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 43.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2295
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 100 / Used frames/image: 1-100

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Processing

SoftwareName: PHENIX / Version: dev_4213: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3particle selection
2Leginonimage acquisition
4cryoSPARC3CTF correction
7PHENIXdev-4213-000model fitting
9cryoSPARC3initial Euler assignment
10cryoSPARC3final Euler assignment
12cryoSPARC33D reconstruction
19Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 926176
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 147860 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 262 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: CC
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 ...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.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00233038
ELECTRON MICROSCOPYf_angle_d0.47744982
ELECTRON MICROSCOPYf_dihedral_angle_d12.1834698
ELECTRON MICROSCOPYf_chiral_restr0.0414830
ELECTRON MICROSCOPYf_plane_restr0.0035574

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