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- EMDB-45364: HIV-1 intasome core bound with DTG -

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Basic information

Entry
Database: EMDB / ID: EMD-45364
TitleHIV-1 intasome core bound with DTG
Map data
Sample
  • Complex: HIV-1 intasomes Core
    • Protein or peptide: Integrase
    • Protein or peptide: Integrase
    • DNA: viral DNA
    • DNA: vDNA
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC 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
  • Ligand: water
KeywordsHIV-1 / integrase / nucleoprotein complexes / INSTI / intasome / CryoEM / VIRAL PROTEIN
Function / homology
Function and homology information


HIV-1 retropepsin / symbiont-mediated activation of host apoptosis / 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 ...HIV-1 retropepsin / symbiont-mediated activation of host apoptosis / 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 / symbiont-mediated suppression of host gene expression / RNA stem-loop binding / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / host cell / viral nucleocapsid / DNA recombination / DNA-directed DNA polymerase / Hydrolases; Acting on ester bonds / aspartic-type endopeptidase activity / DNA-directed DNA polymerase activity / symbiont entry into host cell / viral translational frameshifting / lipid binding / host cell nucleus / host cell plasma membrane / structural molecule activity / virion membrane / proteolysis / DNA 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. / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / RNase H type-1 domain profile. / Ribonuclease H domain / Reverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / 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. / Aspartic peptidase domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Biological speciesHuman immunodeficiency virus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 2.01 Å
AuthorsLi M / Craigie R
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK) United States
CitationJournal: Viruses / Year: 2024
Title: HIV-1 Intasomes Assembled with Excess Integrase C-Terminal Domain Protein Facilitate Structural Studies by Cryo-EM and Reveal the Role of the Integrase C-Terminal Tail in HIV-1 Integration.
Authors: Min Li / Zhen Li / Xuemin Chen / Yanxiang Cui / Alan N Engelman / Robert Craigie /
Abstract: Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 ...Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 intasomes provide detailed insights into the mechanism of integration and inhibition by clinical IN inhibitors. However, previously described HIV-1 intasomes are highly heterogeneous and have the tendency to form stacks, which is a limiting factor in determining high-resolution cryo-EM maps. We have assembled HIV-1 intasomes in the presence of excess IN C-terminal domain protein, which was readily incorporated into the intasomes. The purified intasomes were largely homogeneous and exhibited minimal stacking tendencies. The cryo-EM map resolution was further improved to 2.01 Å, which will greatly facilitate structural studies of IN inhibitor action and drug resistance mechanisms. The C-terminal 18 residues of HIV-1 IN, which are critical for virus replication and integration in vitro, have not been well resolved in previous intasome structures, and its function remains unclear. We show that the C-terminal tail participates in intasome assembly, resides within the intasome core, and forms a small alpha helix (residues 271-276). Mutations that disrupt alpha helix integrity impede IN activity in vitro and disrupt HIV-1 infection at the step of viral DNA integration.
History
DepositionJun 14, 2024-
Header (metadata) releaseJul 31, 2024-
Map releaseJul 31, 2024-
UpdateAug 7, 2024-
Current statusAug 7, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45364.png

Downloads & links

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Map

FileDownload / File: emd_45364.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 298.8 Å		0.83 Å/pix.
x 360 pix.
= 298.8 Å		0.83 Å/pix.
x 360 pix.
= 298.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.004
Minimum - Maximum-0.016545307 - 0.061857697
Average (Standard dev.)0.00003185576 (±0.0009256395)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 298.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: sharpened map

Fileemd_45364_additional_1.map
Annotationsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-2

Fileemd_45364_half_map_1.map
AnnotationHalf-2
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-1

Fileemd_45364_half_map_2.map
AnnotationHalf-1
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : HIV-1 intasomes Core

EntireName: HIV-1 intasomes Core
Components
  • Complex: HIV-1 intasomes Core
    • Protein or peptide: Integrase
    • Protein or peptide: Integrase
    • DNA: viral DNA
    • DNA: vDNA
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC 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
  • Ligand: water

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Supramolecule #1: HIV-1 intasomes Core

SupramoleculeName: HIV-1 intasomes Core / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: HIV-1 intasomes assembled with full length integrase and integrase c-terminal domain
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: 6 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 39.864641 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MATVKFKYKG EEKEVDISKI KKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKKGGGGGG FLDGIDKAQE EHEKYHSNW RAMASDFNLP PVVAKEIVAS CDKCQLKGEA MHGQVDCSPG IWQLDCTHLE GKVILVAVHV ASGYIEAEVI P AETGQETA ...String:
MATVKFKYKG EEKEVDISKI KKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKKGGGGGG FLDGIDKAQE EHEKYHSNW RAMASDFNLP PVVAKEIVAS CDKCQLKGEA MHGQVDCSPG IWQLDCTHLE GKVILVAVHV ASGYIEAEVI P AETGQETA YFLLKLAGRW PVKTVHTDNG SNFTSTTVKA ACWWAGIKQE FGIPYNPQSQ GVIESMNKEL KKIIGQVRDQ AE HLKTAVQ MAVFIHNFKR KGGIGGYSAG ERIVDIIATD IQTKELQKQI TKIQNFRVYY RDSRDPVWKG PAKLLWKGEG AVV IQDNSD IKVVPRRKAK IIRDYGKQMA GDDCVASRQD ED

UniProtKB: Gag-Pol polyprotein

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Macromolecule #2: Integrase

MacromoleculeName: Integrase / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 12.101872 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
HMFKRKGGIG GYSAGERIVD IIATDIQTKE LQKQITKIQN FRVYYRDSRD PVWKGPAKLL WKGEGAVVIQ DNSDIKVVPR RKAKIIRDY GKQMAGDDCV ASRQDED

UniProtKB: Gag-Pol polyprotein

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Macromolecule #3: viral DNA

MacromoleculeName: viral DNA / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Human immunodeficiency virus 1
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 #4: vDNA

MacromoleculeName: vDNA / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Human immunodeficiency virus 1
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 #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: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #7: (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: 7 / 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

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 81 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration0.4 mg/mL
BufferpH: 6.2
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 940736
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8w34

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.01 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 428779
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5u1c


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9c9m:
HIV-1 intasome core bound with DTG

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