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- EMDB-23035: Cryo-EM structure of Rous sarcoma virus cleaved synaptic complex ... -

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Entry
Database: EMDB / ID: EMD-23035
TitleCryo-EM structure of Rous sarcoma virus cleaved synaptic complex (CSC) with HIV-1 integrase strand transfer inhibitor MK-2048. Cluster identified by 3-dimensional variability analysis in cryoSPARC.
Map dataMap
Sample
  • Complex: Cleaved synaptic complex (CSC) formed with Rous sarcoma virus integrase and viral DNA in presence of HIV-1 integrase strand inhibitor MK-2048
    • Protein or peptide: integrase
    • DNA: DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*T)-3')
    • DNA: DNA (5'-D(*AP*TP*TP*GP*CP*AP*TP*AP*AP*GP*AP*CP*AP*AP*CP*A)-3')
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: (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
Keywordsintasome / 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 / 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 (strain Schmidt-Ruppin A)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsPandey KK / Bera S
Funding support United States, 2 items
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
DepositionNov 24, 2020-
Header (metadata) releaseMar 17, 2021-
Map releaseMar 17, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ku7
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kui
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7kui
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23035.png

Downloads & links

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Map

FileDownload / File: emd_23035.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy EMDB: 0.3 / Movie #1: 0.3
Minimum - Maximum-1.1492264 - 2.5057988
Average (Standard dev.)-0.0006445808 (±0.0597384)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 422.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z422.400422.400422.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-1.1492.506-0.001

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Supplemental data

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Half map: Half map A

Fileemd_23035_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_23035_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 : Cleaved synaptic complex (CSC) formed with Rous sarcoma virus int...

EntireName: Cleaved synaptic complex (CSC) formed with Rous sarcoma virus integrase and viral DNA in presence of HIV-1 integrase strand inhibitor MK-2048
Components
  • Complex: Cleaved synaptic complex (CSC) formed with Rous sarcoma virus integrase and viral DNA in presence of HIV-1 integrase strand inhibitor MK-2048
    • Protein or peptide: integrase
    • DNA: DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*T)-3')
    • DNA: DNA (5'-D(*AP*TP*TP*GP*CP*AP*TP*AP*AP*GP*AP*CP*AP*AP*CP*A)-3')
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
  • Ligand: (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

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Supramolecule #1: Cleaved synaptic complex (CSC) formed with Rous sarcoma virus int...

SupramoleculeName: 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 / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Rous sarcoma virus (strain Schmidt-Ruppin A)
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: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Rous sarcoma virus (strain Schmidt-Ruppin A) / Strain: Schmidt-Ruppin A
Molecular weightTheoretical: 30.926582 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*T)-3')

MacromoleculeName: DNA (5'-D(*AP*AP*TP*GP*TP*TP*GP*TP*CP*TP*TP*AP*TP*GP*CP*AP*AP*T)-3')
type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Rous sarcoma virus (strain Schmidt-Ruppin A)
Molecular weightTheoretical: 5.5206 KDa
SequenceString:
(DA)(DA)(DT)(DG)(DT)(DT)(DG)(DT)(DC)(DT) (DT)(DA)(DT)(DG)(DC)(DA)(DA)(DT)

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

MacromoleculeName: DNA (5'-D(*AP*TP*TP*GP*CP*AP*TP*AP*AP*GP*AP*CP*AP*AP*CP*A)-3')
type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Rous sarcoma virus (strain Schmidt-Ruppin A)
Molecular weightTheoretical: 4.899232 KDa
SequenceString:
(DA)(DT)(DT)(DG)(DC)(DA)(DT)(DA)(DA)(DG) (DA)(DC)(DA)(DA)(DC)(DA)

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: (6S)-2-(3-chloro-4-fluorobenzyl)-8-ethyl-10-hydroxy-N,6-dimethyl-...

MacromoleculeName: (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
type: ligand / ID: 6 / Number of copies: 2 / Formula: ZZX
Molecular weightTheoretical: 461.874 Da
Chemical component information

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 / inhibitor*YM / MK-2048

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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 R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
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 magnification: 105000
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: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 5187 / Average electron dose: 66.0 e/Å2
Details: Images were collected in movie mode at 0.2 seconds per frame.
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: 362307 / Software - Name: cryoSPARC
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Refined map is for one cluster identified by 3-dimensional variability analysis done in cryoSPARC.
Number images used: 64449
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7jn3


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-7ku7:
Cryo-EM structure of Rous sarcoma virus cleaved synaptic complex (CSC) with HIV-1 integrase strand transfer inhibitor MK-2048. Cluster identified by 3-dimensional variability analysis in cryoSPARC.

PDB-7kui:
Cryo-EM structure of Rous sarcoma virus cleaved synaptic complex (CSC) with HIV-1 integrase strand transfer inhibitor MK-2048. CIC region of a cluster identified by 3-dimensional variability analysis in cryoSPARC.

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