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- PDB-7jn3: Cryo-EM structure of Rous sarcoma virus cleaved synaptic complex ... -

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Basic information

Entry
Database: PDB / ID: 7jn3
TitleCryo-EM structure of Rous sarcoma virus cleaved synaptic complex (CSC) with HIV-1 integrase strand transfer inhibitor MK-2048
Components
  • DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*T)-3')
  • DNA (5'-D(*AP*TP*TP*GP*CP*AP*TP*AP*AP*GP*AP*CP*AP*AP*CP*A)-3')
  • integrase
KeywordsHYDROLASE/DNA/INHIBITOR / intasome / integrase-viral DNA complex / HYDROLASE-DNA-INHIBITOR complex
Function / homology
Function and homology information


Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / viral nucleocapsid ...Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases / ribonuclease H / DNA integration / RNA-directed DNA polymerase / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / viral nucleocapsid / 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 / RNA binding / zinc ion binding
Similarity search - Function
Retroviral Gag polyprotein, M / Retroviral M domain / gag protein p24 N-terminal domain / Reverse transcriptase thumb / Reverse transcriptase thumb domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / Integrase DNA binding domain / Integrase, C-terminal domain superfamily, retroviral ...Retroviral Gag polyprotein, M / Retroviral M domain / gag protein p24 N-terminal domain / Reverse transcriptase thumb / Reverse transcriptase thumb domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / Integrase DNA binding domain / Integrase, C-terminal domain superfamily, retroviral / Integrase, N-terminal zinc-binding domain / Integrase, C-terminal, retroviral / Integrase DNA binding domain profile. / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / 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 / Retroviral matrix protein / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Chem-ZZX / DNA / DNA (> 10) / Gag-Pol polyprotein
Similarity search - Component
Biological speciesRous sarcoma virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsPandey, K.K. / Bera, S. / Shi, K. / Aihara, H. / Grandgenett, D.P.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI127196 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118047 United States
CitationJournal: Commun Biol / Year: 2021
Title: Cryo-EM structure of the Rous sarcoma virus octameric cleaved synaptic complex intasome.
Authors: Krishan K Pandey / Sibes Bera / Ke Shi / Michael J Rau / Amarachi V Oleru / James A J Fitzpatrick / Alan N Engelman / Hideki Aihara / Duane P Grandgenett /
Abstract: Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate ...Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous sarcoma virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes.
History
DepositionAug 3, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 17, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 6, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2May 15, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: integrase
B: integrase
C: integrase
D: integrase
E: integrase
F: integrase
G: integrase
H: integrase
I: DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*T)-3')
J: DNA (5'-D(*AP*TP*TP*GP*CP*AP*TP*AP*AP*GP*AP*CP*AP*AP*CP*A)-3')
K: DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*T)-3')
L: DNA (5'-D(*AP*TP*TP*GP*CP*AP*TP*AP*AP*GP*AP*CP*AP*AP*CP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)269,40420
Polymers268,25212
Non-polymers1,1528
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 1 types, 8 molecules ABCDEFGH

#1: Protein
integrase /


Mass: 30926.582 Da / Num. of mol.: 8 / Fragment: UNP residues 1281-1558
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rous sarcoma virus (strain Schmidt-Ruppin A)
Strain: Schmidt-Ruppin A / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLysS
References: UniProt: P03354, Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases, RNA-directed DNA polymerase, DNA-directed DNA polymerase, ribonuclease H, Transferases; ...References: UniProt: P03354, Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases, RNA-directed DNA polymerase, DNA-directed DNA polymerase, ribonuclease H, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, Hydrolases; Acting on ester bonds

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DNA chain , 2 types, 4 molecules IKJL

#2: DNA chain DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*T)-3')


Mass: 5520.600 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Rous sarcoma virus (strain Schmidt-Ruppin A)
#3: DNA chain DNA (5'-D(*AP*TP*TP*GP*CP*AP*TP*AP*AP*GP*AP*CP*AP*AP*CP*A)-3')


Mass: 4899.232 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Rous sarcoma virus (strain Schmidt-Ruppin A)

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Non-polymers , 3 types, 8 molecules

#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-ZZX / (6S)-2-(3-chloro-4-fluorobenzyl)-8-ethyl-10-hydroxy-N,6-dimethyl-1,9-dioxo-1,2,6,7,8,9-hexahydropyrazino[1',2':1,5]pyrrolo[2,3-d]pyridazine-4-carboxamide / MK-2048


Mass: 461.874 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H21ClFN5O4 / Comment: inhibitor*YM
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestY

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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: Cleaved synaptic complex (CSC) formed with Rous sarcoma virus integrase and viral DNA in presence of HIV-1 integrase strand inhibitor MK-2048
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.257 MDa / Experimental value: NO
Source (natural)Organism: Rous sarcoma virus (strain Schmidt-Ruppin A)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Cs: 0.01 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 66 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 5187
Details: Images were collected in movie mode at 0.2 seconds per frame.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
7PHENIXmodel fitting
11cryoSPARCclassification
13PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1811357
3D reconstructionResolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 456948 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 30 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: correlation coefficient
Atomic model buildingPDB-ID: 5EJK

5ejk


Accession code: 5EJK / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00816245
ELECTRON MICROSCOPYf_angle_d1.25722340
ELECTRON MICROSCOPYf_dihedral_angle_d29.0012639
ELECTRON MICROSCOPYf_chiral_restr0.1122454
ELECTRON MICROSCOPYf_plane_restr0.0082584

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