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- PDB-6rwm: SIVrcm intasome in complex with bictegravir -

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Basic information

Entry
Database: PDB / ID: 6rwm
TitleSIVrcm intasome in complex with bictegravir
Components
  • 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')
  • Pol protein
KeywordsRECOMBINATION / retroviral integrase / lentivirus / strand transfer inhibior / protein-DNA complex
Function / homology
Function and homology information


exoribonuclease H activity / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase activity / establishment of integrated proviral latency / RNA-DNA hybrid ribonuclease activity / viral entry into host cell / DNA recombination / aspartic-type endopeptidase activity / DNA binding / zinc ion binding
Integrase Zinc binding domain / Ribonuclease H domain / Integrase core domain / Integrase DNA binding domain / Integrase, C-terminal domain superfamily, retroviral / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Retropepsins / Integrase-like, N-terminal / Ribonuclease H-like superfamily ...Integrase Zinc binding domain / Ribonuclease H domain / Integrase core domain / Integrase DNA binding domain / Integrase, C-terminal domain superfamily, retroviral / Ribonuclease H superfamily / Aspartic peptidase domain superfamily / Retropepsins / 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
Pol protein
Biological speciesSimian immunodeficiency virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.81 Å
AuthorsCherepanov, P. / 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: Despite worldwide prescription, the mechanistic basis for superiority of second-generation HIV integrase (IN) strand transfer inhibitors (INSTIs) is poorly understood. We use single-particle cryo- ...Despite worldwide prescription, the mechanistic basis for superiority of second-generation HIV integrase (IN) strand transfer inhibitors (INSTIs) is poorly understood. We use single-particle cryo-electron microscopy to visualize the mode of action of the advanced INSTIs dolutegravir and bictegravir at near atomic resolution. Q148H/G140S amino acid substitutions in IN that pervade 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, which are critical for antagonizing Q148H/G140S mutant virus. 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 important 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

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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  • Superimposition on EM map
  • EMDB-10042
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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)433,09332
Polymers431,50316
Non-polymers1,59016
Water1,946108
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 area48810 Å2
ΔGint-298 kcal/mol
Surface area110730 Å2
MethodPISA

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Components

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Protein , 1 types, 12 molecules ABCDFMIJKLNE

#1: Protein
Pol protein


Mass: 32732.182 Da / Num. of mol.: 12 / Details: SIVrcm integrase, S119D / Mutation: S119D
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

#2: 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 end / Variant: SIVrcmNg409 / Production host: synthetic construct (others)
#3: 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 end / Variant: SIVrcmNg409 / Production host: synthetic construct (others)

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Non-polymers , 5 types, 124 molecules

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


Mass: 65.409 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Formula: Zn
#5: Chemical
ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-KLQ / Bictegravir / Bictegravir


Mass: 449.380 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H18F3N3O5 / Feature type: SUBJECT OF INVESTIGATION / Comment: inhibitor*YM
#7: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 108 / Source method: isolated from a natural source / Formula: H2O

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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

Type: COMPLEX

IDNameEntity IDParent-IDSource
1SIVrcm intasome in complex with bictegravir1,2,30MULTIPLE SOURCES
2Pol Protein11RECOMBINANT
3DNA2,31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)

Ncbi tax-ID: 11723 / Organism: Simian immunodeficiency virus

IDEntity assembly-ID
12
23
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23synthetic construct (others)32630
Buffer solutionpH: 7
Buffer component

Buffer-ID: 1

IDConc.NameFormula
10.35 Msodium chlorideNaClSodium chloride
20.025 M4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHepes
30.005 Mmagnesium sulfateMgSO4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: no pretreatment / Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 4C
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: 55.7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 11769
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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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: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 149778 / Algorithm: FOURIER SPACE / Details: Non-uniform refinement as implemented in CryoSPARC / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID3D fitting-ID
12B4J

2b4j

1
21EX4

1ex4

1
31K6Y

1k6y

1

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