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- EMDB-29329: Cryo-EM map of the unliganded Lettuce aptamer -

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Basic information

Entry
Database: EMDB / ID: EMD-29329
TitleCryo-EM map of the unliganded Lettuce aptamer
Map dataMap of file (mrc format) for apo Lettuce
Sample
  • Complex: Lettuce
KeywordsDNA / aptamer / fluorescence / turn-on / fluorogenic / fluorophore
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsBanco MT / Passalacqua LFM / Ferre-D'Amare AR
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI) United States
CitationJournal: Nature / Year: 2023
Title: Intricate 3D architecture of a DNA mimic of GFP.
Authors: Luiz F M Passalacqua / Michael T Banco / Jared D Moon / Xing Li / Samie R Jaffrey / Adrian R Ferré-D'Amaré /
Abstract: Numerous studies have shown how RNA molecules can adopt elaborate three-dimensional (3D) architectures. By contrast, whether DNA can self-assemble into complex 3D folds capable of sophisticated ...Numerous studies have shown how RNA molecules can adopt elaborate three-dimensional (3D) architectures. By contrast, whether DNA can self-assemble into complex 3D folds capable of sophisticated biochemistry, independent of protein or RNA partners, has remained mysterious. Lettuce is an in vitro-evolved DNA molecule that binds and activates conditional fluorophores derived from GFP. To extend previous structural studies of fluorogenic RNAs, GFP and other fluorescent proteins to DNA, we characterize Lettuce-fluorophore complexes by X-ray crystallography and cryogenic electron microscopy. The results reveal that the 53-nucleotide DNA adopts a four-way junction (4WJ) fold. Instead of the canonical L-shaped or H-shaped structures commonly seen in 4WJ RNAs, the four stems of Lettuce form two coaxial stacks that pack co-linearly to form a central G-quadruplex in which the fluorophore binds. This fold is stabilized by stacking, extensive nucleobase hydrogen bonding-including through unusual diagonally stacked bases that bridge successive tiers of the main coaxial stacks of the DNA-and coordination of monovalent and divalent cations. Overall, the structure is more compact than many RNAs of comparable size. Lettuce demonstrates how DNA can form elaborate 3D structures without using RNA-like tertiary interactions and suggests that new principles of nucleic acid organization will be forthcoming from the analysis of complex DNAs.
History
DepositionDec 28, 2022-
Header (metadata) releaseMay 10, 2023-
Map releaseMay 10, 2023-
UpdateJul 26, 2023-
Current statusJul 26, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29329.png

Downloads & links

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Map

FileDownload / File: emd_29329.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of file (mrc format) for apo Lettuce
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.9 Å/pix.
x 240 pix.
= 216. Å		0.9 Å/pix.
x 240 pix.
= 216. Å		0.9 Å/pix.
x 240 pix.
= 216. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.544
Minimum - Maximum-0.24120912 - 1.19973
Average (Standard dev.)0.0002462436 (±0.032214385)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 216.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_29329_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_29329_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Lettuce

EntireName: Lettuce
Components
  • Complex: Lettuce

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Supramolecule #1: Lettuce

SupramoleculeName: Lettuce / type: complex / ID: 1 / Parent: 0 / Details: Synthetic in vitro evolved DNA aptamer

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 3327 / Average electron dose: 52.5 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm

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Image processing

Particle selectionNumber selected: 4144838
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 55288
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.3.2)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 3.3.2) / Software - details: Heterogenous Refinement
FSC plot (resolution estimation)

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