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- EMDB-27726: Duplex-G-quadruplex-duplex (DGD) class_1 -

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Basic information

Entry
Database: EMDB / ID: EMD-27726
TitleDuplex-G-quadruplex-duplex (DGD) class_1
Map data
Sample
  • Complex: synthetic duplex-G-quadruplex-duplex construct mimicking a promoter G4
    • DNA: Duplex-G-quadruplex-duplex (46-MER)
    • DNA: Duplex-G-quadruplex-duplex (46-MER)
KeywordsG-quadruplex / promoter / duplex / DNA
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.4 Å
AuthorsMonsen RC / Chua EYD / Hopkins JB / Chaires JB / Trent JO
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseFeb 8, 2023-
Map releaseFeb 8, 2023-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27726.png

Downloads & links

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Map

FileDownload / File: emd_27726.map.gz / Format: CCP4 / Size: 34.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.0252 Å
Density
Contour LevelBy AUTHOR: 0.403
Minimum - Maximum-0.53539526 - 2.4556203
Average (Standard dev.)0.002265104 (±0.071268685)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions208208208
Spacing208208208
CellA=B=C: 213.24161 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_27726_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_27726_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : synthetic duplex-G-quadruplex-duplex construct mimicking a promoter G4

EntireName: synthetic duplex-G-quadruplex-duplex construct mimicking a promoter G4
Components
  • Complex: synthetic duplex-G-quadruplex-duplex construct mimicking a promoter G4
    • DNA: Duplex-G-quadruplex-duplex (46-MER)
    • DNA: Duplex-G-quadruplex-duplex (46-MER)

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Supramolecule #1: synthetic duplex-G-quadruplex-duplex construct mimicking a promoter G4

SupramoleculeName: synthetic duplex-G-quadruplex-duplex construct mimicking a promoter G4
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)

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

MacromoleculeName: Duplex-G-quadruplex-duplex (46-MER) / type: dna / ID: 1 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.086031 KDa
SequenceString:
(DG)(DC)(DG)(DT)(DG)(DC)(DC)(DG)(DC)(DA) (DT)(DT)(DA)(DA)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DG)(DT)(DA) (DT) (DA)(DC)(DA)(DT)(DA)(DG)

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Macromolecule #2: Duplex-G-quadruplex-duplex (46-MER)

MacromoleculeName: Duplex-G-quadruplex-duplex (46-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 14.407232 KDa
SequenceString:
(DC)(DT)(DA)(DT)(DG)(DT)(DA)(DT)(DA)(DC) (DA)(DA)(DA)(DG)(DA)(DG)(DG)(DG)(DT)(DG) (DG)(DG)(DT)(DA)(DG)(DG)(DG)(DT)(DG) (DG)(DG)(DT)(DT)(DT)(DA)(DA)(DT)(DG)(DC) (DG) (DG)(DC)(DA)(DC)(DG)(DC)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state2D array

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Sample preparation

Concentration2.5 mg/mL
BufferpH: 7.2
Component:
ConcentrationFormulaName
8.0 mMNa2HPO4sodium phosphate
185.0 mMKClpotassium chloride
1.0 mMsodium EDTA
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 7 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was monodisperse.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 12175 / Average exposure time: 0.4 sec. / Average electron dose: 68.54 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: HELIUM
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsSelected images were dose-weighted and corrected for beam-induced motion.
Particle selectionNumber selected: 666328
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 110105
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 3 / Avg.num./class: 101000 / Software - Name: cryoSPARC (ver. 3.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 557.2 / Target criteria: map correlation
Output model

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

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