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- PDB-6rwl: SIVrcm intasome -

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Basic information

Entry
Database: PDB / ID: 6rwl
TitleSIVrcm intasome
Components
  • (Pol protein) x 3
  • 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 (5'-D(P*GP*CP*TP*AP*AP*GP*AP*AP*AP*AP*AP*TP*CP*TP*CP*TP*AP*CP*CP*A)-3')
KeywordsRECOMBINATION / retroviral integrase / lentivirus / strand transfer inhibior / protein-DNA complex
Function / homology
Function and homology information


exoribonuclease H / exoribonuclease H activity / retroviral ribonuclease H / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / establishment of integrated proviral latency / DNA recombination ...exoribonuclease H / exoribonuclease H activity / retroviral ribonuclease H / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / establishment of integrated proviral latency / DNA recombination / viral entry into host cell / aspartic-type endopeptidase activity / DNA binding / zinc ion binding
Ribonuclease H domain / Aspartic peptidase domain superfamily / Integrase-like, N-terminal / Ribonuclease H-like superfamily / Reverse transcriptase thumb / Reverse transcriptase connection / Integrase, N-terminal zinc-binding domain / Peptidase A2A, retrovirus, catalytic / Aspartic peptidase, active site / Integrase, catalytic core ...Ribonuclease H domain / Aspartic peptidase domain superfamily / Integrase-like, N-terminal / Ribonuclease H-like superfamily / Reverse transcriptase thumb / Reverse transcriptase connection / Integrase, N-terminal zinc-binding domain / Peptidase A2A, retrovirus, catalytic / Aspartic peptidase, active site / Integrase, catalytic core / Integrase, C-terminal, retroviral / Reverse transcriptase domain / Ribonuclease H superfamily / Integrase, C-terminal domain superfamily, retroviral / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily / Integrase DNA binding domain / Integrase core domain / Integrase Zinc binding domain / Retropepsins / Integrase, N-terminal zinc-binding domain / Arc Repressor Mutant, subunit A / Orthogonal Bundle / Mainly Alpha
Exoribonuclease H
Biological speciesSimian immunodeficiency virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsCherepanov, P. / Nans, A. / Cook, N.
Funding support United States, United Kingdom, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesGM082251 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 5, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 5, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 19, 2020Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Feb 26, 2020Group: Database references / Structure summary / Category: audit_author / citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Pol protein
B: Pol protein
C: Pol protein
D: Pol protein
F: Pol protein
M: Pol protein
T: 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')
S: 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')
I: Pol protein
J: Pol protein
K: Pol protein
L: Pol protein
N: Pol protein
E: Pol protein
W: 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')
Q: 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')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)431,82424
Polymers431,30116
Non-polymers5238
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, microscopy, negative-stain EM
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area47840 Å2
ΔGint-257 kcal/mol
Surface area112230 Å2
MethodPISA

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Components

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Protein , 3 types, 12 molecules ABCMIJKEDLFN

#1: Protein
Pol protein


Mass: 32732.182 Da / Num. of mol.: 8 / Mutation: A119D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Simian immunodeficiency virus / Gene: pol / Variant: red capped mangabey isolate from Cameroon / Production host: Escherichia coli (E. coli) / Variant (production host): PC2 / References: UniProt: E1ANT8
#2: Protein Pol protein


Mass: 32675.131 Da / Num. of mol.: 2 / Mutation: A119D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Simian immunodeficiency virus / Gene: pol / Variant: red capped mangabey isolate from Cameroon / Production host: Escherichia coli (E. coli) / Variant (production host): PC2 / References: UniProt: E1ANT8
#3: Protein Pol protein


Mass: 32688.170 Da / Num. of mol.: 2 / Mutation: A119D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Simian immunodeficiency virus / Gene: pol / Variant: red capped mangabey isolate from Cameroon / Production host: Escherichia coli (E. coli) / Variant (production host): PC2 / References: UniProt: E1ANT8

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DNA chain , 2 types, 4 molecules TWSQ

#4: DNA chain 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')


Mass: 9223.995 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Simian immunodeficiency virus / Gene: U5 vDNA LTR end / Variant: SIVrcmNg409 / Production host: synthetic construct (others)
#5: DNA chain 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')


Mass: 10134.541 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Simian immunodeficiency virus / Gene: U5 vDNA LTR end / Variant: SIVrcmNg409 / Production host: synthetic construct (others)

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Non-polymers , 1 types, 8 molecules

#6: Chemical
ChemComp-ZN / ZINC ION / Zinc


Mass: 65.409 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Zn

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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

Component
IDNameTypeEntity IDParent-IDSource
1SIVrcm intasome in complex with bictegravirCOMPLEX#1-#50MULTIPLE SOURCES
2Pol ProteinCOMPLEX#1-#31RECOMBINANT
3DNACOMPLEX#4-#51RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Simian immunodeficiency virus11723
23Simian immunodeficiency virus11723
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23synthetic construct (others)32630
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
10.35 Msodium chlorideNaClSodium chloride1
20.025 M4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHepes1
30.005 Mmagnesium sulfateMgSO41
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 295 K

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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 defocus max: 3500 nm / Nominal defocus min: 1600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 8027
Image scansMovie frames/image: 30 / Used frames/image: 1-30

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2RELION2.1particle selectionparticle picking
3cryoSPARC2particle selection2D classification
4EPUimage acquisition
6Gctf1.06CTF correctionper-particle estimation
9UCSF Chimeramodel fitting
11Cootmodel refinement
12PHENIX1.15.2-3472model refinementphenix.real_space_refine
13cryoSPARCinitial Euler assignment
14cryoSPARCfinal Euler assignment
15RELION2.1classification
16cryoSPARC23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 171893 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID3D fitting-ID
12B4J1
21EX41
31K6Y1

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