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- PDB-6puz: Structure of HIV cleaved synaptic complex (CSC) intasome bound wi... -

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Basic information

Entry
Database: PDB / ID: 6puz
TitleStructure of HIV cleaved synaptic complex (CSC) intasome bound with magnesium and INSTI XZ446 (compound 4f)
Components
  • Chimeric Sso7d and HIV-1 integrase
  • viral DNA non-transferred strand
  • viral DNA transferred strand
KeywordsVIRAL PROTEIN/DNA / integrase / intasome / transposition / VIRAL PROTEIN-DNA complex
Function / homology
Function and homology information


endoribonuclease activity / DNA integration / RNA-directed DNA polymerase activity / endonuclease activity / DNA recombination / DNA binding / zinc ion binding / cytoplasm
Integrase Zinc binding domain / Ribonuclease H-like superfamily / Integrase core domain / Integrase DNA binding domain / 7kD DNA-binding domain / Integrase, C-terminal domain superfamily, retroviral / Ribonuclease H superfamily / Integrase-like, N-terminal / Chromo-like domain superfamily / Integrase, N-terminal zinc-binding domain ...Integrase Zinc binding domain / Ribonuclease H-like superfamily / Integrase core domain / Integrase DNA binding domain / 7kD DNA-binding domain / Integrase, C-terminal domain superfamily, retroviral / Ribonuclease H superfamily / Integrase-like, N-terminal / Chromo-like domain superfamily / Integrase, N-terminal zinc-binding domain / DNA-binding 7kDa protein / Integrase, catalytic core / Integrase, C-terminal, retroviral
DNA-binding protein 7d / Integrase
Biological speciesSaccharolobus solfataricus (archaea)
Human immunodeficiency virus 1
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsLyumkis, D. / Jozwik, I.K. / Passos, D.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI136680 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM069832 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI146017 United States
CitationJournal: Science / Year: 2020
Title: Structural basis for strand-transfer inhibitor binding to HIV intasomes.
Authors: Dario Oliveira Passos / Min Li / Ilona K Jóźwik / Xue Zhi Zhao / Diogo Santos-Martins / Renbin Yang / Steven J Smith / Youngmin Jeon / Stefano Forli / Stephen H Hughes / Terrence R Burke / ...Authors: Dario Oliveira Passos / Min Li / Ilona K Jóźwik / Xue Zhi Zhao / Diogo Santos-Martins / Renbin Yang / Steven J Smith / Youngmin Jeon / Stefano Forli / Stephen H Hughes / Terrence R Burke / Robert Craigie / Dmitry Lyumkis /
Abstract: The HIV intasome is a large nucleoprotein assembly that mediates the integration of a DNA copy of the viral genome into host chromatin. Intasomes are targeted by the latest generation of ...The HIV intasome is a large nucleoprotein assembly that mediates the integration of a DNA copy of the viral genome into host chromatin. Intasomes are targeted by the latest generation of antiretroviral drugs, integrase strand-transfer inhibitors (INSTIs). Challenges associated with lentiviral intasome biochemistry have hindered high-resolution structural studies of how INSTIs bind to their native drug target. Here, we present high-resolution cryo-electron microscopy structures of HIV intasomes bound to the latest generation of INSTIs. These structures highlight how small changes in the integrase active site can have notable implications for drug binding and design and provide mechanistic insights into why a leading INSTI retains efficacy against a broad spectrum of drug-resistant variants. The data have implications for expanding effective treatments available for HIV-infected individuals.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJul 18, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 12, 2020Provider: repository / Type: Initial release

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Structure visualization

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  • Biological unit as complete point assembly
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  • Deposited structure unit
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Chimeric Sso7d and HIV-1 integrase
B: Chimeric Sso7d and HIV-1 integrase
C: Chimeric Sso7d and HIV-1 integrase
D: Chimeric Sso7d and HIV-1 integrase
E: viral DNA non-transferred strand
F: viral DNA transferred strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)185,94011
Polymers185,2466
Non-polymers6945
Water3,063170
1
A: Chimeric Sso7d and HIV-1 integrase
B: Chimeric Sso7d and HIV-1 integrase
C: Chimeric Sso7d and HIV-1 integrase
D: Chimeric Sso7d and HIV-1 integrase
E: viral DNA non-transferred strand
F: viral DNA transferred strand
hetero molecules

A: Chimeric Sso7d and HIV-1 integrase
B: Chimeric Sso7d and HIV-1 integrase
C: Chimeric Sso7d and HIV-1 integrase
D: Chimeric Sso7d and HIV-1 integrase
E: viral DNA non-transferred strand
F: viral DNA transferred strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)371,88122
Polymers370,49312
Non-polymers1,38810
Water21612
TypeNameSymmetry operationNumber
point symmetry operation2
2


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C2 (2 fold cyclic))

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Components

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Protein , 1 types, 4 molecules ABCD

#1: Protein
Chimeric Sso7d and HIV-1 integrase / 7 kDa DNA-binding protein d / Sso7d


Mass: 42321.258 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) (archaea), (gene. exp.) Human immunodeficiency virus 1
Strain: ATCC 35092 / DSM 1617 / JCM 11322 / P2 / Gene: sso7d, sso7d-1, SSO10610 / Production host: Escherichia coli (E. coli) / References: UniProt: P39476, UniProt: Q76353

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DNA chain , 2 types, 2 molecules EF

#2: DNA chain viral DNA non-transferred strand


Mass: 8188.271 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Human immunodeficiency virus 1
#3: DNA chain viral DNA transferred strand


Mass: 7773.023 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Human immunodeficiency virus 1

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Non-polymers , 4 types, 175 molecules

#4: Chemical ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#6: Chemical ChemComp-XXJ / 4-azanyl-N-[[2,4-bis(fluoranyl)phenyl]methyl]-1-oxidanyl-2-oxidanylidene-6-[2-(phenylsulfonyl)ethyl]-1,8-naphthyridine-3-carboxamide


Mass: 514.501 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H20F2N4O5S / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 170 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Assembly of HIV integrase and viral DNA / Type: COMPLEX / Entity ID: 1,2,3 / Source: RECOMBINANT
Molecular weightValue: 0.4 MDa / Experimental value: NO
Source (natural)Organism: hiv (virus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.2
Buffer component

Buffer-ID: 1

IDConc.NameFormula
120 mMBis-TrisBis-tris methane
2550 mMsodium chlorideNaClSodium chloride
35 mMmagnesium chlorideMgCl
40.5 mMTCEP
55 % (w/v)glycerol
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 80 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 37000 X / Calibrated magnification: 63291 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 42 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2072
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 80 / Used frames/image: 4-80

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Processing

SoftwareName: PHENIX / Version: (1.16_3549: ???) / Classification: refinement
EM software
IDNameVersionCategory
1Warpparticle selection
2Leginonimage acquisition
4WarpCTF correction
7Cootmodel fitting
9PHENIX1.16model refinement
10cisTEM1initial Euler assignment
11cisTEM1.0 betafinal Euler assignment
12cisTEM1.0 betaclassification
13cisTEM1.0 beta3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 412354
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 143698 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model building
IDPDB-ID3D fitting-ID
13L3U1
21K6Y1
35U1C1
RefinementResolution: 2.8→303.36 Å / SU ML: 1.19 / Phase error: 61.61 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.5352 215480 9.96 %
Rwork0.5355 --
Obs0.5355 2164399 99.97 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.0075868
f_angle_d0.8098066
f_dihedral_angle_d22.4232236
f_chiral_restr0.054882
f_plane_restr0.007884
LS refinement shell

Refinement-ID: ELECTRON MICROSCOPY

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.8-2.871247933.945318080995
2.87-2.94771489066.14316384186
2.9477-3.03441.8807901.605314793078

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