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- EMDB-21151: CryoEM structure of HIV-1 conserved Intasome Core -

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Basic information

Entry
Database: EMDB / ID: EMD-21151
TitleCryoEM structure of HIV-1 conserved Intasome Core
Map dataHIV-1 conserved Intasome Core
Sample
  • Complex: HIV-1 conserved intasome core
    • Protein or peptide: Integrase
    • DNA: DNA (27-MER)
    • DNA: DNA (25-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: (4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazine-9-carboxamide
Keywordssite-specific recombination / retroviruses / integrase / integration / nucleoprotein complex / DNA complex / integrase strand transfer inhibitor / TRANSFERASE-DNA complex
Function / homology
Function and homology information


nucleotidyltransferase activity / HIV-1 retropepsin / : / retroviral ribonuclease H / exoribonuclease H / : / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA ...nucleotidyltransferase activity / HIV-1 retropepsin / : / retroviral ribonuclease H / exoribonuclease H / : / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / viral penetration into host nucleus / RNA-directed DNA polymerase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / RNA-DNA hybrid ribonuclease activity / endonuclease activity / viral nucleocapsid / DNA recombination / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase / nucleic acid binding / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / symbiont-mediated suppression of host gene expression / lipid binding / host cell nucleus / structural molecule activity / host cell plasma membrane / virion membrane / proteolysis / DNA binding / RNA binding / zinc ion binding / membrane
Similarity search - Function
Reverse transcriptase connection / Reverse transcriptase connection 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 ...Reverse transcriptase connection / Reverse transcriptase connection 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. / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / 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 / 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. / 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
Integrase / Gag-Pol polyprotein
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1 / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsLi M / Chen X
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)A1070042 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK036169 United States
CitationJournal: J Mol Biol / Year: 2020
Title: A Peptide Derived from Lens Epithelium-Derived Growth Factor Stimulates HIV-1 DNA Integration and Facilitates Intasome Structural Studies.
Authors: Min Li / Xuemin Chen / Huaibin Wang / Kellie A Jurado / Alan N Engelman / Robert Craigie /
Abstract: The low solubility and aggregation properties of HIV-1 integrase (IN) are major obstacles for biochemical and structural studies. The lens epithelium-derived growth factor (LEDGF) is a cellular ...The low solubility and aggregation properties of HIV-1 integrase (IN) are major obstacles for biochemical and structural studies. The lens epithelium-derived growth factor (LEDGF) is a cellular factor that binds IN and tethers preintegration complexes to chromatin before integration. The LEDGF also stimulates HIV-1 IN DNA strand transfer activity and improves its solubility in vitro. We show that these properties are conferred by a short peptide spanning residues 178 to 197 of the LEDGF that encompasses its AT-hook DNA-binding elements. The peptide stimulates HIV-1 IN activity both in trans and in cis. Fusion of the peptide to either the N- or C-terminus of IN results in maximal stimulation of concerted integration activity and greatly improves the solubility of the protein and nucleoprotein complexes of IN with viral DNA ends (intasomes). High-resolution structures of HIV-1 intasomes are required to understand the mechanism of IN strand transfer inhibitors (INSTIs), which are front-line drugs for the treatment of HIV-1, and how the virus can develop resistance to INSTIs. We have previously determined the structure of the HIV-1 strand transfer complex intasome. The improved biophysical properties of intasomes assembled with LEDGF peptide fusion IN have enabled us to determine the structure of the cleaved synaptic complex intasome, which is the direct target of INSTIs.
History
DepositionDec 27, 2019-
Header (metadata) releaseJan 29, 2020-
Map releaseFeb 5, 2020-
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: 0.009
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.009
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vdk
  • Surface level: 0.009
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21151.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHIV-1 conserved Intasome Core
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.009 / Movie #1: 0.009
Minimum - Maximum-0.01674593 - 0.045504976
Average (Standard dev.)0.0000704237 (±0.00095504685)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 381.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z381.600381.600381.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ512512512
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0170.0460.000

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Supplemental data

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Sample components

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Entire : HIV-1 conserved intasome core

EntireName: HIV-1 conserved intasome core
Components
  • Complex: HIV-1 conserved intasome core
    • Protein or peptide: Integrase
    • DNA: DNA (27-MER)
    • DNA: DNA (25-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: (4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazine-9-carboxamide

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Supramolecule #1: HIV-1 conserved intasome core

SupramoleculeName: HIV-1 conserved intasome core / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Human immunodeficiency virus 1

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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: Human immunodeficiency virus 1
Molecular weightTheoretical: 39.898355 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSHMPKRGRP AATEVKIPKP RGRPPLPAGT NSKGPPDFSS DEEREPTPVL GSGAAAAGQS RAAVGRKATK KTDGGGFLDG IDKAQEEHE KYHSNWRAMA SDFNLPPVVA KEIVASCDKC QLKGEAMHGQ VDCSPGIWQL DCTHLEGKVI LVAVHVASGY I EAEVIPAE ...String:
GSHMPKRGRP AATEVKIPKP RGRPPLPAGT NSKGPPDFSS DEEREPTPVL GSGAAAAGQS RAAVGRKATK KTDGGGFLDG IDKAQEEHE KYHSNWRAMA SDFNLPPVVA KEIVASCDKC QLKGEAMHGQ VDCSPGIWQL DCTHLEGKVI LVAVHVASGY I EAEVIPAE TGQETAYFLL KLAGRWPVKT VHTDNGSNFT STTVKAACWW AGIKQEFGIP YNPQSQGVIE SMNKELKKII GQ VRDQAEH LKTAVQMAVF IHNFKRKGGI GGYSAGERIV DIIATDIQTK ELQKQITKIQ NFRVYYRDSR DPVWKGPAKL LWK GEGAVV IQDNSDIKVV PRRKAKIIRD YGKQMAGDDC VASRQDED

UniProtKB: Integrase

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Macromolecule #2: DNA (27-MER)

MacromoleculeName: DNA (27-MER) / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 8.188271 KDa
SequenceString:
(DA)(DC)(DT)(DG)(DC)(DT)(DA)(DG)(DA)(DG) (DA)(DT)(DT)(DT)(DT)(DC)(DC)(DC)(DG)(DC) (DC)(DC)(DA)(DC)(DG)(DC)(DT)

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Macromolecule #3: DNA (25-MER)

MacromoleculeName: DNA (25-MER) / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 7.773023 KDa
SequenceString:
(DA)(DG)(DC)(DG)(DT)(DG)(DG)(DG)(DC)(DG) (DG)(DG)(DA)(DA)(DA)(DA)(DT)(DC)(DT)(DC) (DT)(DA)(DG)(DC)(DA)

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Macromolecule #4: MAGNESIUM ION

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

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Macromolecule #5: (4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4...

MacromoleculeName: (4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazine-9-carboxamide
type: ligand / ID: 5 / Number of copies: 2 / Formula: DLU
Molecular weightTheoretical: 419.379 Da
Chemical component information

ChemComp-DLU:
(4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido[1',2':4,5]pyrazino[2,1-b][1,3]oxazine-9-carboxamide / medication, antiretroviral*YM / Dolutegravir

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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

BufferpH: 6.2
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 75.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: COMMON LINE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 134763

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