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- EMDB-27823: Cryo-EM structure of Rous sarcoma virus strand transfer complex -

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Basic information

Entry
Database: EMDB / ID: EMD-27823
TitleCryo-EM structure of Rous sarcoma virus strand transfer complex
Map dataFlipped map
Sample
  • Complex: RSV strand transfer complex
    • Protein or peptide: integrase
    • DNA: DNA (42-MER)
    • DNA: DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*TP*AP*CP*TP*C)-3')
    • DNA: DNA (5'-D(*AP*GP*TP*GP*TP*CP*TP*TP*CP*TP*TP*CP*TP*TP*TP*C)-3')
  • Ligand: ZINC ION
Keywordsintasome / integrase-viral DNA complex / strand transfer complex / VIRAL PROTEIN-DNA 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 / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / RNA-DNA hybrid ribonuclease activity / 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 / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / RNA-DNA hybrid ribonuclease activity / 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
Biological speciesRous sarcoma virus - Prague C
Methodsingle particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsPandey KK / Bera S / Shi K / Aihara H / Grandgenett DP
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI165081 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118047 United States
CitationJournal: J Biol Chem / Year: 2023
Title: Molecular determinants for Rous sarcoma virus intasome assemblies involved in retroviral integration.
Authors: Sibes Bera / Ke Shi / Hideki Aihara / Duane P Grandgenett / Krishan K Pandey /
Abstract: Integration of retroviral DNA into the host genome involves the formation of integrase (IN)-DNA complexes termed intasomes. Further characterization of these complexes is needed to understand their ...Integration of retroviral DNA into the host genome involves the formation of integrase (IN)-DNA complexes termed intasomes. Further characterization of these complexes is needed to understand their assembly process. Here, we report the single-particle cryo-EM structure of the Rous sarcoma virus (RSV) strand transfer complex (STC) intasome produced with IN and a preassembled viral/target DNA substrate at 3.36 Å resolution. The conserved intasome core region consisting of IN subunits contributing active sites interacting with viral/target DNA has a resolution of 3 Å. Our structure demonstrated the flexibility of the distal IN subunits relative to the IN subunits in the conserved intasome core, similar to results previously shown with the RSV octameric cleaved synaptic complex intasome produced with IN and viral DNA only. An extensive analysis of higher resolution STC structure helped in the identification of nucleoprotein interactions important for intasome assembly. Using structure-function studies, we determined the mechanisms of several IN-DNA interactions critical for assembly of both RSV intasomes. We determined the role of IN residues R244, Y246, and S124 in cleaved synaptic complex and STC intasome assemblies and their catalytic activities, demonstrating differential effects. Taken together, these studies advance our understanding of different RSV intasome structures and molecular determinants involved in their assembly.
History
DepositionAug 9, 2022-
Header (metadata) releaseApr 26, 2023-
Map releaseApr 26, 2023-
UpdateOct 18, 2023-
Current statusOct 18, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27823.png

Downloads & links

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Map

FileDownload / File: emd_27823.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFlipped map
Voxel sizeX=Y=Z: 1.16 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-0.6671884 - 2.136148
Average (Standard dev.)0.00077787286 (±0.054304652)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 375.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_27823_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_27823_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RSV strand transfer complex

EntireName: RSV strand transfer complex
Components
  • Complex: RSV strand transfer complex
    • Protein or peptide: integrase
    • DNA: DNA (42-MER)
    • DNA: DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*TP*AP*CP*TP*C)-3')
    • DNA: DNA (5'-D(*AP*GP*TP*GP*TP*CP*TP*TP*CP*TP*TP*CP*TP*TP*TP*C)-3')
  • Ligand: ZINC ION

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Supramolecule #1: RSV strand transfer complex

SupramoleculeName: RSV strand transfer complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Rous sarcoma virus - Prague C
Molecular weightTheoretical: 257 KDa

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Macromolecule #1: integrase

MacromoleculeName: integrase / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Aspartic endopeptidases
Source (natural)Organism: Rous sarcoma virus - Prague C / Strain: Prague C
Molecular weightTheoretical: 30.926582 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: PLREAKDLHT ALHIGPRALS KACNISMQQA REVVQTCPHC NSAPALEAGV NPRGLGPLQI WQTDFTLEPR MAPRSWLAVT VDTASSAIV VTQHGRVTSV AVQHHWATAI AVLGRPKAIK TDNGSCFTSK STREWLARWG IAHTTGIPGN SQGQAMVERA N RLLKDKIR ...String:
PLREAKDLHT ALHIGPRALS KACNISMQQA REVVQTCPHC NSAPALEAGV NPRGLGPLQI WQTDFTLEPR MAPRSWLAVT VDTASSAIV VTQHGRVTSV AVQHHWATAI AVLGRPKAIK TDNGSCFTSK STREWLARWG IAHTTGIPGN SQGQAMVERA N RLLKDKIR VLAEGDGFMK RIPTSKQGEL LAKAMYALNH FERGENTKTP IQKHWRPTVL TEGPPVKIRI ETGEWEKGWN VL VWGRGYA AVKNRDTDKV IWVPSRKVKP DITQKDEVTK K

UniProtKB: Gag-Pol polyprotein

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Macromolecule #2: DNA (42-MER)

MacromoleculeName: DNA (42-MER) / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Rous sarcoma virus - Prague C
Molecular weightTheoretical: 13.047456 KDa
SequenceString:
(DG)(DA)(DG)(DT)(DA)(DT)(DT)(DG)(DC)(DA) (DT)(DA)(DA)(DG)(DA)(DC)(DA)(DA)(DC)(DA) (DG)(DT)(DG)(DC)(DA)(DC)(DG)(DA)(DA) (DA)(DG)(DA)(DA)(DG)(DA)(DA)(DG)(DA)(DC) (DA) (DC)(DT)

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Macromolecule #3: DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*TP*...

MacromoleculeName: DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*TP*AP*CP*TP*C)-3')
type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Rous sarcoma virus - Prague C
Molecular weightTheoretical: 6.716363 KDa
SequenceString:
(DA)(DA)(DT)(DG)(DT)(DT)(DG)(DT)(DC)(DT) (DT)(DA)(DT)(DG)(DC)(DA)(DA)(DT)(DA)(DC) (DT)(DC)

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Macromolecule #4: DNA (5'-D(*AP*GP*TP*GP*TP*CP*TP*TP*CP*TP*TP*CP*TP*TP*TP*C)-3')

MacromoleculeName: DNA (5'-D(*AP*GP*TP*GP*TP*CP*TP*TP*CP*TP*TP*CP*TP*TP*TP*C)-3')
type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Rous sarcoma virus - Prague C
Molecular weightTheoretical: 4.821123 KDa
SequenceString:
(DA)(DG)(DT)(DG)(DT)(DC)(DT)(DT)(DC)(DT) (DT)(DC)(DT)(DT)(DT)(DC)

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 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

Concentration0.5 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 59000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 3000 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1811357
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5ejk

Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 5 / Avg.num./class: 130000 / Software - Name: cryoSPARC
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1.1) / Number images used: 141000

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Atomic model buiding 1

Initial modelPDB ID:

5ejk


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 30 / Target criteria: correlation coefficient
Output model

PDB-8e14:
Cryo-EM structure of Rous sarcoma virus strand transfer complex

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