[English] 日本語
Yorodumi
- EMDB-13592: Cryo-EM SPA reconstruction of an extended RNA origami 5 helix til... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-13592
TitleCryo-EM SPA reconstruction of an extended RNA origami 5 helix tile (5HT-B-3X)
Map dataUnsharpened map from cryoSPARC local refinement.
Sample
  • Complex: 5-helix tile B 3X extended version
  • RNA: 5HT-B-3X
KeywordsRNA / origami / nanostructure
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsMcRae EKS / Bogglid A / Boesen T / Andersen ES
Funding support Denmark, 1 items
OrganizationGrant numberCountry
Danish Council for Independent Research31789 Denmark
CitationJournal: Nat Nanotechnol / Year: 2023
Title: Structure, folding and flexibility of co-transcriptional RNA origami.
Authors: Ewan K S McRae / Helena Østergaard Rasmussen / Jianfang Liu / Andreas Bøggild / Michael T A Nguyen / Nestor Sampedro Vallina / Thomas Boesen / Jan Skov Pedersen / Gang Ren / Cody Geary / ...Authors: Ewan K S McRae / Helena Østergaard Rasmussen / Jianfang Liu / Andreas Bøggild / Michael T A Nguyen / Nestor Sampedro Vallina / Thomas Boesen / Jan Skov Pedersen / Gang Ren / Cody Geary / Ebbe Sloth Andersen /
Abstract: RNA origami is a method for designing RNA nanostructures that can self-assemble through co-transcriptional folding with applications in nanomedicine and synthetic biology. However, to advance the ...RNA origami is a method for designing RNA nanostructures that can self-assemble through co-transcriptional folding with applications in nanomedicine and synthetic biology. However, to advance the method further, an improved understanding of RNA structural properties and folding principles is required. Here we use cryogenic electron microscopy to study RNA origami sheets and bundles at sub-nanometre resolution revealing structural parameters of kissing-loop and crossover motifs, which are used to improve designs. In RNA bundle designs, we discover a kinetic folding trap that forms during folding and is only released after 10 h. Exploration of the conformational landscape of several RNA designs reveal the flexibility of helices and structural motifs. Finally, sheets and bundles are combined to construct a multidomain satellite shape, which is characterized by individual-particle cryo-electron tomography to reveal the domain flexibility. Together, the study provides a structural basis for future improvements to the design cycle of genetically encoded RNA nanodevices.
History
DepositionSep 17, 2021-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateAug 2, 2023-
Current statusAug 2, 2023Processing site: PDBe / Status: Released

-
Structure visualization

Supplemental images

emd_13592.png

Downloads & links

-
Map

FileDownload / File: emd_13592.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map from cryoSPARC local refinement.
Voxel sizeX=Y=Z: 2.64 Å
Density
Contour LevelBy AUTHOR: 2.25
Minimum - Maximum-0.77254045 - 7.5144925
Average (Standard dev.)-0.0042022993 (±0.18203257)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 675.84 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Mask #1

Fileemd_13592_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: Half-map A from cryoSPARC local refinement.

Fileemd_13592_half_map_1.map
AnnotationHalf-map A from cryoSPARC local refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: Half-map B from cryoSPARC local refinement.

Fileemd_13592_half_map_2.map
AnnotationHalf-map B from cryoSPARC local refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : 5-helix tile B 3X extended version

EntireName: 5-helix tile B 3X extended version
Components
  • Complex: 5-helix tile B 3X extended version
  • RNA: 5HT-B-3X

-
Supramolecule #1: 5-helix tile B 3X extended version

SupramoleculeName: 5-helix tile B 3X extended version / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 423 KDa

-
Macromolecule #1: 5HT-B-3X

MacromoleculeName: 5HT-B-3X / type: rna / ID: 1 / Details: In vitro transcribed RNA.
SequenceString: GGAACCUAAU CAUAAUUCCC UUUUCAUCCU CUUCUCAACU CUACUUUUCU UGAUCUUCCG CCUCUCAUCC UUUCAUUAUC UUCAUCCCUG CUUGCAUAUA GUGCCUUCGG GUACUAUGUG CAAGUAGGGG CGUCUGUGAU UCGUGGCUGU UCGCAGUCAC GAAUUACAGA ...String:
GGAACCUAAU CAUAAUUCCC UUUUCAUCCU CUUCUCAACU CUACUUUUCU UGAUCUUCCG CCUCUCAUCC UUUCAUUAUC UUCAUCCCUG CUUGCAUAUA GUGCCUUCGG GUACUAUGUG CAAGUAGGGG CGUCUGUGAU UCGUGGCUGU UCGCAGUCAC GAAUUACAGA CCAGGCUGUC GGAGAUGGGU UUUCGAAUCC AUCUGGCCAG GGGUUCGCCC UUGGGAACUU UUAUUCGUAG AAGUUCAAAA GUUUAGGAAU GGACAACCUA AGCUUUUCCA UCAGUCGUGC AAGAACGGAG CACGGCUGAU CCGACGGCCU GCGCUACAAA CUGACAGUAG CGGCUGGGAU GCGCUAAACG UUGAAGCGCG UCCCAAUGAA GGUAAUGGAA GGAUGAGGGG CGGAGGAUGG UACAGGUGAA GAACAACGUA CUUCAUCUGU AGGUUGGGUG UGACGUAACA AGUCAGUAGU UGCGUGGAGG AUUAUCCGAA CCGUUCACGG AUGAUCCUAC UUCGUGGUAG GAAUGUCCAA CCUACCGCGA AGUGGACUUC AAACAAUGGC UAAGUUUGGA GUCCCCGAGU GGGGACGAAU UUCCCACGUC CUCACUCCAC ACUCAACCAC GGUCAAGGUG UAAGACCGUC CUUAGAUCGG CUAACUAGUG AAGCCGGUCU AACAAGAAGA GUAGGGUUGA GAAGGGGAUG AGAAGCCCGA AAUGUGACAA CACUAGAGUC ACGUUUCGCA UACUCACGAC UAGAACAAUA CACCAGUUUU AGGGCUCCAU GCUACAAGGG AAAAGUAGCG UGGAGGGUCC CGAUACGCAA UAGCCAAGCG UAUUGGGACC UAGCAUACCC UAACUCCACA AGGGUGUGCU ACCGAGUGUA CGGGAACGGA UAACCCGUGC ACUCUCGUGG GUAUGACAAC AAAAGGUAGA UGUUGUGGUU CCUGACGGGA AUCGUCUACC CGUUAGGAAG GAAUUGUGAU UGGGUUCCAA AUUUCACCUA AUCCUGUCCU ACUCAAAAGA CGAAUGAGUG GGACACCGGG AGCGAACGCG GGGAACUACC UACCCUGCGG CCUCAGGAAG CGAAUAUCCG ACGCUUUCUG AGCUAGUCUU UCGGAAAGUG GAGAUCCGAA GGACUAGUAU GGCAGUCAUG AUGGUGGUUC GCCACCGUCA UGAUUGCCAU AGCGAUGCGC GGGUGAGCUG CUUCGGCGGC UCGCCCGUGC AUCUUCGCUU CCGGGGAGGG UUCUUCGGAA CCUUCUGGGA CGUCGUUCGC GGCGUCGGUU UAAGGUUCGC CUUAGACCAU UGGGUGAAGU UU

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

Concentration2.5 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
25.0 mMHEPES
5.0 mMMgCl2
100.0 mMKCl

Details: Freshly prepared and filtered through 0.22 micron filter prior to use.
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa / Details: 15mA current
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 294 K / Instrument: LEICA EM GP
Details: 3 microlitre droplet, 4 second delay before blotting, 6 second blot, 0 second delay before plunging..
DetailsSample was purified by size exclusion chromatography and concentrated in an Amicon spin concentrator prior to grid preparation.

-
Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 8620 / Average exposure time: 1.5 sec. / Average electron dose: 60.0 e/Å2 / Details: Images were collected as 56 frame movies.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

-
Image processing

Particle selectionNumber selected: 588769
Details: Template picker from cryoSPARC using templates created from ab initio model.
Startup modelType of model: NONE
Details: Initial volume was generated using a 3 class ab initio reconstruction given 30000 randomly selected particles.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Details: The entire set of 588769 particles were then 3D classified into one of the three initial ab initio volumes.
Final 3D classificationNumber classes: 5 / Avg.num./class: 70000
Details: The final classification was a heterogeneous refinement from 5 different ab initio volumes and 265000 particles. The three best classes were combined to make the final particle stack.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Details: The final angle assignment was determined using the local refinement beta job and a mask covering the entire volume.
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 173322
FSC plot (resolution estimation)

-
Atomic model buiding 1

RefinementOverall B value: 271.2

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more