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- PDB-8dut: Duplex-G-quadruplex-duplex (DGD) class_1 -

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Basic information

Entry
Database: PDB / ID: 8dut
TitleDuplex-G-quadruplex-duplex (DGD) class_1
Components(Duplex-G-quadruplex-duplex (46-MER)) x 2
KeywordsDNA / G-quadruplex / promoter / duplex
Function / homologyDNA / DNA (> 10)
Function and homology information
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.4 Å
AuthorsMonsen, R.C. / Chua, E.Y.D. / Hopkins, J.B. / Chaires, J.B. / Trent, J.O.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)GM077422 United States
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Structure of a 28.5 kDa duplex-embedded G-quadruplex system resolved to 7.4 Å resolution with cryo-EM.
Authors: Robert C Monsen / Eugene Y D Chua / Jesse B Hopkins / Jonathan B Chaires / John O Trent /
Abstract: Genomic regions with high guanine content can fold into non-B form DNA four-stranded structures known as G-quadruplexes (G4s). Extensive in vivo investigations have revealed that promoter G4s are ...Genomic regions with high guanine content can fold into non-B form DNA four-stranded structures known as G-quadruplexes (G4s). Extensive in vivo investigations have revealed that promoter G4s are transcriptional regulators. Little structural information exists for these G4s embedded within duplexes, their presumed genomic environment. Here, we report the 7.4 Å resolution structure and dynamics of a 28.5 kDa duplex-G4-duplex (DGD) model system using cryo-EM, molecular dynamics, and small-angle X-ray scattering (SAXS) studies. The DGD cryo-EM refined model features a 53° bend induced by a stacked duplex-G4 interaction at the 5' G-tetrad interface with a persistently unstacked 3' duplex. The surrogate complement poly dT loop preferably stacks onto the 3' G-tetrad interface resulting in occlusion of both 5' and 3' tetrad interfaces. Structural analysis shows that the DGD model is quantifiably more druggable than the monomeric G4 structure alone and represents a new structural drug target. Our results illustrate how the integration of cryo-EM, MD, and SAXS can reveal complementary detailed static and dynamic structural information on DNA G4 systems.
History
DepositionJul 27, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 8, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 15, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Duplex-G-quadruplex-duplex (46-MER)
B: Duplex-G-quadruplex-duplex (46-MER)


Theoretical massNumber of molelcules
Total (without water)28,4932
Polymers28,4932
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: SAXS
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: DNA chain Duplex-G-quadruplex-duplex (46-MER)


Mass: 14086.031 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: DNA chain Duplex-G-quadruplex-duplex (46-MER)


Mass: 14407.232 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: synthetic duplex-G-quadruplex-duplex construct mimicking a promoter G4
Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: synthetic construct (others)
Buffer solutionpH: 7.2
Buffer component
IDConc.NameFormulaBuffer-ID
18 mMsodium phosphateNa2HPO41
2185 mMpotassium chlorideKCl1
31 mMsodium EDTA1
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was monodisperse.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2700 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: HELIUM / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.4 sec. / Electron dose: 68.54 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 12175

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
2Leginonimage acquisition
4cryoSPARC3.3.1CTF correction
12cryoSPARC3.3.1classification
13cryoSPARC3.3.13D reconstruction
Image processingDetails: Selected images were dose-weighted and corrected for beam-induced motion.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 666328
3D reconstructionResolution: 7.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110105 / Symmetry type: POINT
Atomic model buildingB value: 557.2 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: map correlation

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