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- EMDB-25244: Tertiary structure of an individual particle of self-folding RNA ... -

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Basic information

Entry
Database: EMDB / ID: EMD-25244
TitleTertiary structure of an individual particle of self-folding RNA polymer (particle #150)
Map data
Sample
  • Complex: RNA Origami 6 Helix Bundle with Clasp (6HBC)
    • Protein or peptide: 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: 26.0 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: bioRxiv / Year: 2023
Title: Tertiary structure of single-instant RNA molecule reveals folding landscape.
Authors: Jianfang Liu / Ewan K S McRae / Meng Zhang / Cody Geary / Ebbe Sloth Andersen / Gang Ren
Abstract: The folding of RNA and protein molecules during their synthesis is a crucial self-assembly process that nature employs to convert genetic information into the complex molecular machinery that ...The folding of RNA and protein molecules during their synthesis is a crucial self-assembly process that nature employs to convert genetic information into the complex molecular machinery that supports life. Misfolding events are the cause of several diseases, and the folding pathway of central biomolecules, such as the ribosome, is strictly regulated by programmed maturation processes and folding chaperones. However, the dynamic folding processes are challenging to study because current structure determination methods heavily rely on averaging, and existing computational methods do not efficiently simulate non-equilibrium dynamics. Here we utilize individual-particle cryo-electron tomography (IPET) to investigate the folding landscape of a rationally designed RNA origami 6-helix bundle that undergoes slow maturation from a "young" to "mature" conformation. By optimizing the IPET imaging and electron dose conditions, we obtain 3D reconstructions of 120 individual particles at resolutions ranging from 23-35 Å, enabling us first-time to observe individual RNA helices and tertiary structures without averaging. Statistical analysis of 120 tertiary structures confirms the two main conformations and suggests a possible folding pathway driven by helix-helix compaction. Studies of the full conformational landscape reveal both trapped states, misfolded states, intermediate states, and fully compacted states. The study provides novel insight into RNA folding pathways and paves the way for future studies of the energy landscape of molecular machines and self-assembly processes.
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_25244.png

Downloads & links

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Map

FileDownload / File: emd_25244.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-2.2240622 - 5.636576
Average (Standard dev.)0.022667043 (±0.31261533)
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)
    • Protein or peptide: 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: protein_or_peptide / ID: 1 / Enantiomer: LEVO
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: 25 / 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 was initially aligned by IMOD. Additionally, to reduce the image noise, the tilt series was further processed using machine learning, a median-filter process, and a contrast enhancement method.
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 26.0 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: 25
FSC plot (resolution estimation)

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