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- EMDB-25253: Tertiary structure of an individual particle of self-folding RNA ... -

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Basic information

Entry
Database: EMDB / ID: EMD-25253
TitleTertiary structure of an individual particle of self-folding RNA polymer (particle #135)
Map data
Sample
  • Complex: RNA Origami 6 Helix Bundle with Clasp (6HBC)
    • RNA: RNA Origami 6 Helix Bundle with Clasp (6HBC)
KeywordsSingle molecule structure / Individual Particle cryo-Electron Tomography / IPET / Cryo-ET / tertiary structure / Structural Flexibility / Self-folding mechanism / RNA origami / RNA
Biological speciessynthetic construct (others)
Methodelectron tomography / cryo EM / Resolution: 27.0 Å
AuthorsLiu J / Ren G
Funding support United States, 5 items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-AC02-05CH11231 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL115153 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM104427 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)R01MH077303 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01DK042667 United States
CitationJournal: Nat Commun / Year: 2024
Title: Non-averaged single-molecule tertiary structures reveal RNA self-folding through individual-particle cryo-electron tomography.
Authors: Jianfang Liu / Ewan K S McRae / Meng Zhang / Cody Geary / Ebbe Sloth Andersen / Gang Ren /
Abstract: Large-scale and continuous conformational changes in the RNA self-folding process present significant challenges for structural studies, often requiring trade-offs between resolution and ...Large-scale and continuous conformational changes in the RNA self-folding process present significant challenges for structural studies, often requiring trade-offs between resolution and observational scope. Here, we utilize individual-particle cryo-electron tomography (IPET) to examine the post-transcriptional self-folding process of designed RNA origami 6-helix bundle with a clasp helix (6HBC). By avoiding selection, classification, averaging, or chemical fixation and optimizing cryo-ET data acquisition parameters, we reconstruct 120 three-dimensional (3D) density maps from 120 individual particles at an electron dose of no more than 168 eÅ, achieving averaged resolutions ranging from 23 to 35 Å, as estimated by Fourier shell correlation (FSC) at 0.5. Each map allows us to identify distinct RNA helices and determine a unique tertiary structure. Statistical analysis of these 120 structures confirms two reported conformations and reveals a range of kinetically trapped, intermediate, and highly compacted states, demonstrating a maturation folding landscape likely driven by helix-helix compaction interactions.
History
DepositionOct 29, 2021-
Header (metadata) releaseMay 10, 2023-
Map releaseMay 10, 2023-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25253.png

Downloads & links

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Map

FileDownload / File: emd_25253.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.88 Å/pix.
x 128 pix.
= 240.64 Å		1.88 Å/pix.
x 128 pix.
= 240.64 Å		1.88 Å/pix.
x 128 pix.
= 240.64 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.88 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-2.6099048 - 5.1925826
Average (Standard dev.)0.026048018 (±0.32756633)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 240.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : RNA Origami 6 Helix Bundle with Clasp (6HBC)

EntireName: RNA Origami 6 Helix Bundle with Clasp (6HBC)
Components
  • Complex: RNA Origami 6 Helix Bundle with Clasp (6HBC)
    • RNA: RNA Origami 6 Helix Bundle with Clasp (6HBC)

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Supramolecule #1: RNA Origami 6 Helix Bundle with Clasp (6HBC)

SupramoleculeName: RNA Origami 6 Helix Bundle with Clasp (6HBC) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The RNA was transcribed from linearized DNA template using T7 RNA polymerase purified in house.
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 230 KDa

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Macromolecule #1: RNA Origami 6 Helix Bundle with Clasp (6HBC)

MacromoleculeName: RNA Origami 6 Helix Bundle with Clasp (6HBC) / type: rna / ID: 1
Source (natural)Organism: synthetic construct (others)
SequenceString: GGGAGAGUAC UAUUCAGAUG CAGACCGCAA GUUCAGAGCG GUUUGCAUCU AGGGUACGUU UUCGAACGUA UCCUCCGACU AAGUGUAUUC GUAUACUUAG UGCCUUGUGC CUGCUUCGGC AGGCAUGACC CAAAUGUGCC UUUCGGGGCA CAUUUCCGGU CAUCCAAGUU ...String:
GGGAGAGUAC UAUUCAGAUG CAGACCGCAA GUUCAGAGCG GUUUGCAUCU AGGGUACGUU UUCGAACGUA UCCUCCGACU AAGUGUAUUC GUAUACUUAG UGCCUUGUGC CUGCUUCGGC AGGCAUGACC CAAAUGUGCC UUUCGGGGCA CAUUUCCGGU CAUCCAAGUU CGCUUGGGUG AUGCGGGCGU AUAGGUUCGU CUAUACGUCC GCGUUUUCCG AGAAGAGGUA ACUCGGGAAA CCGGUCCACG UGACAAAGGU AGAGUUACGU GGAGGGAGCA GCUGCAAAGG GAUAAUGCAG UUGCUGGCUG GAUGCCAGAA CUCACGACUG GCAUCUACGG GGAUGGUGCU CUCCCAAUUC UCCAUUUACC GCCGAAUCGA CCCCAACGUG AGAGGGGUCG GUUCCCCGAG CAUAGACCAA UAUCCCAGGU UUAUGCUCCC CAACGCUGGA CGAACUACCU ACGUCUAGCG UUCCGGCAAA UGAGUCAAUA CCUCAGACUU AUUUGCGGUG CCUGAGCCUA AACUGAACAU GGGUUCAGGC AUCUUGGCUC CAGUUCGCUG GAGCCGACGG UAGCGCUGCG UUCGCGCAGU GCUAGGGAGC AUCCGUUUUC GAGCGGAUGC UGGGCGGUUG CCUGUUCGCA GGCAAUCGGG CCUACUCAUG AUUCGUCAUG AGUGGUGACA GCGUGAUGUU CGCAUUACGC UGUCGGGUAG AUGGAGAAUU

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
25.0 mMHEPES
5.0 mMmagnesium chlorideMgCl2
100.0 mMpotassium chlorideKCl
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: LEICA EM GP
DetailsIn vitro transcribed RNA was purified by size exclusion chromatography and spin concentrated in amicon spin columns.
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 17 / Average exposure time: 1.43 sec. / Average electron dose: 13.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsMotion correction of the multi-frame movie was conducted by MotionCor2. The tilt series of whole micrographs were initial aligned by IMOD. Additionally, to reduce the image noise, tilt series were further conducted by a machine learning, a median-filter process and a contrast enhancement method.
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 27.0 Å / Resolution method: FSC 0.5 CUT-OFF
Details: The targeted images of individual particle were reconstructed by Individual-Particle Electron Tomography (IPET). To reduce the missing-wedge artifact caused by the limited tilt angle range, ...Details: The targeted images of individual particle were reconstructed by Individual-Particle Electron Tomography (IPET). To reduce the missing-wedge artifact caused by the limited tilt angle range, the final 3D map was submitted to a low-tilt tomographic 3D reconstruction method (LoTToR). IPET 3D map was low-pass filtered to 0.8 nm following by Gaussian filtering (standard deviation is 3.0) and median filter (3x3x3) using EMAN and UCSF Chimera, and displayed by Chimera with applied the hidden dust function.
Number images used: 17
FSC plot (resolution estimation)

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