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- EMDB-10043: SIVrcm intasome in complex with dolutegravir -

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Basic information

Entry
Database: EMDB / ID: EMD-10043
TitleSIVrcm intasome in complex with dolutegravir
Map dataMap sharpened with B factor -110
Sample
  • Complex: SIVrcm intasome in complex with dolutegravir
    • Complex: Pol protein
      • Protein or peptide: Pol protein
    • Complex: DNA
      • DNA: DNA (5'-D(*AP*AP*CP*TP*GP*GP*TP*AP*GP*AP*GP*AP*TP*TP*TP*TP*TP*CP*TP*TP*AP*GP*C)-3')
      • DNA: DNA (5'-D(P*GP*CP*TP*AP*AP*GP*AP*AP*AP*AP*AP*TP*CP*TP*CP*TP*AP*CP*CP*A)-3')
  • Ligand: ZINC ION
  • Ligand: CHLORIDE IONChloride
  • 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: MAGNESIUM ION
  • Ligand: water
Function / homology
Function and homology information


exoribonuclease H activity / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / DNA recombination / aspartic-type endopeptidase activity / symbiont entry into host cell / proteolysis ...exoribonuclease H activity / DNA integration / viral genome integration into host DNA / establishment of integrated proviral latency / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / DNA recombination / aspartic-type endopeptidase activity / symbiont entry into host cell / proteolysis / DNA binding / zinc ion binding
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. / RNase H / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / 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 / 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
Biological speciesSimian immunodeficiency virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsCherepanov P / Nans A / Cook N
Funding support United States, United Kingdom, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM082251 United States
The Francis Crick InstituteFC001061 United Kingdom
CitationJournal: Science / Year: 2020
Title: Structural basis of second-generation HIV integrase inhibitor action and viral resistance.
Authors: Nicola J Cook / Wen Li / Dénes Berta / Magd Badaoui / Allison Ballandras-Colas / Andrea Nans / Abhay Kotecha / Edina Rosta / Alan N Engelman / Peter Cherepanov /
Abstract: Although second-generation HIV integrase strand-transfer inhibitors (INSTIs) are prescribed throughout the world, the mechanistic basis for the superiority of these drugs is poorly understood. We ...Although second-generation HIV integrase strand-transfer inhibitors (INSTIs) are prescribed throughout the world, the mechanistic basis for the superiority of these drugs is poorly understood. We used single-particle cryo-electron microscopy to visualize the mode of action of the advanced INSTIs dolutegravir and bictegravir at near-atomic resolution. Glutamine-148→histidine (Q148H) and glycine-140→serine (G140S) amino acid substitutions in integrase that result in clinical INSTI failure perturb optimal magnesium ion coordination in the enzyme active site. The expanded chemical scaffolds of second-generation compounds mediate interactions with the protein backbone that are critical for antagonizing viruses containing the Q148H and G140S mutations. Our results reveal that binding to magnesium ions underpins a fundamental weakness of the INSTI pharmacophore that is exploited by the virus to engender resistance and provide a structural framework for the development of this class of anti-HIV/AIDS therapeutics.
History
DepositionJun 5, 2019-
Header (metadata) releaseJun 12, 2019-
Map releaseFeb 5, 2020-
UpdateMar 30, 2022-
Current statusMar 30, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6rwn
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6rwn
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10043.png

Downloads & links

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Map

FileDownload / File: emd_10043.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap sharpened with B factor -110
Voxel sizeX=Y=Z: 1.38 Å
Density
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.9
Minimum - Maximum-3.603641 - 6.915492
Average (Standard dev.)0.0013935855 (±0.18609895)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 496.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.381.381.38
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z496.800496.800496.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-3.6046.9150.001

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Supplemental data

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Mask #1

Fileemd_10043_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Locally filtered autosharpened CryoSPARC map

Fileemd_10043_additional_1.map
AnnotationLocally filtered autosharpened CryoSPARC map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened CryoSPARC map

Fileemd_10043_additional_2.map
AnnotationUnsharpened CryoSPARC map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_10043_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_10043_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SIVrcm intasome in complex with dolutegravir

EntireName: SIVrcm intasome in complex with dolutegravir
Components
  • Complex: SIVrcm intasome in complex with dolutegravir
    • Complex: Pol protein
      • Protein or peptide: Pol protein
    • Complex: DNA
      • DNA: DNA (5'-D(*AP*AP*CP*TP*GP*GP*TP*AP*GP*AP*GP*AP*TP*TP*TP*TP*TP*CP*TP*TP*AP*GP*C)-3')
      • DNA: DNA (5'-D(P*GP*CP*TP*AP*AP*GP*AP*AP*AP*AP*AP*TP*CP*TP*CP*TP*AP*CP*CP*A)-3')
  • Ligand: ZINC ION
  • Ligand: CHLORIDE IONChloride
  • 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: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: SIVrcm intasome in complex with dolutegravir

SupramoleculeName: SIVrcm intasome in complex with dolutegravir / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: Pol protein

SupramoleculeName: Pol protein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Simian immunodeficiency virus
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Simian immunodeficiency virus
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: Pol protein

MacromoleculeName: Pol protein / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Simian immunodeficiency virus
Molecular weightTheoretical: 32.732182 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GFLDGIEKAQ EEHEKYHNNW RAMAEDFQIP QVVAKEIVAQ CPKCQVKGEA MHGQVDASPK TWQMDCTHLE GKVIIVAVHV ASGYIEAEV LPAETGKETA HFLLKLAARW PVKHLHTDNG DNFTSSAVQA VCWWAQIEHT FGVPYNPQSQ GVVESMNHQL K TIITQIRD ...String:
GFLDGIEKAQ EEHEKYHNNW RAMAEDFQIP QVVAKEIVAQ CPKCQVKGEA MHGQVDASPK TWQMDCTHLE GKVIIVAVHV ASGYIEAEV LPAETGKETA HFLLKLAARW PVKHLHTDNG DNFTSSAVQA VCWWAQIEHT FGVPYNPQSQ GVVESMNHQL K TIITQIRD QAEKIETAVQ MAVLIHNFKR KGGIGGYSAG ERIIDIIASD LQTTKLQNQI SKIQNFRVYF REGRDQQWKG PA TLIWKGE GAVVIQDGQD LKVVPRRKCK IIKDYGRKDV DSETSMEGRQ EKD

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Macromolecule #2: DNA (5'-D(*AP*AP*CP*TP*GP*GP*TP*AP*GP*AP*GP*AP*TP*TP*TP*TP*TP*CP*...

MacromoleculeName: DNA (5'-D(*AP*AP*CP*TP*GP*GP*TP*AP*GP*AP*GP*AP*TP*TP*TP*TP*TP*CP*TP*TP*AP*GP*C)-3')
type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Simian immunodeficiency virus
Molecular weightTheoretical: 10.134541 KDa
SequenceString:
(DA)(DA)(DC)(DT)(DG)(DG)(DT)(DA)(DG)(DA) (DG)(DA)(DT)(DT)(DT)(DT)(DT)(DC)(DT)(DT) (DA)(DG)(DC)(DC)(DT)(DT)(DC)(DT)(DA) (DG)(DA)(DA)(DC)

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

MacromoleculeName: DNA (5'-D(P*GP*CP*TP*AP*AP*GP*AP*AP*AP*AP*AP*TP*CP*TP*CP*TP*AP*CP*CP*A)-3')
type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Simian immunodeficiency virus
Molecular weightTheoretical: 9.223995 KDa
SequenceString:
(DG)(DT)(DT)(DC)(DT)(DA)(DG)(DA)(DA)(DG) (DG)(DC)(DT)(DA)(DA)(DG)(DA)(DA)(DA)(DA) (DA)(DT)(DC)(DT)(DC)(DT)(DA)(DC)(DC) (DA)

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 8 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #5: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #6: (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: 6 / 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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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #8: water

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

ChemComp-HOH:
WATER / 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

BufferpH: 7
Component:
ConcentrationFormulaName
0.35 MNaClSodium chloridesodium chloride
0.025 MHepes4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
0.005 MMgSO4magnesium sulfate
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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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 / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.6 µm
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: COUNTING / Digitization - Frames/image: 1-30 / Number grids imaged: 1 / Number real images: 6838 / Average electron dose: 50.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf (ver. 1.06) / Software - details: per-particle estimation
Startup modelType of model: OTHER / Details: ab initio, as implemented in CryoSPARC
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 12 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 18302
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

2b4j

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1ex4

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1k6y

)
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6rwn:
SIVrcm intasome in complex with dolutegravir

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