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- EMDB-24285: Fusobacterium nucleatum FMN riboswitch bound to FMN, synthetic tr... -

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Basic information

Entry
Database: EMDB / ID: EMD-24285
TitleFusobacterium nucleatum FMN riboswitch bound to FMN, synthetic trimeric construct
Map dataFusobacterium nucleatum FMN-riboswitch, synthetic trimeric construct
Sample
  • Complex: Fusobacterium nucleatum FMN riboswitch bound to FMN, synthetic trimeric construct
    • RNA: Engineered FMN riboswitch for trimeric assembly
Biological speciesFusobacterium nucleatum (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.9 Å
AuthorsThelot F / Liu D / Liao M / Yin P
Funding support United States, 5 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM122797 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5DP1GM133052 United States
National Science Foundation (NSF, United States)CMMI-1333215 United States
National Science Foundation (NSF, United States)CMMI-1344915 United States
National Science Foundation (NSF, United States)CBET-1729397 United States
CitationJournal: Nat Methods / Year: 2022
Title: Sub-3-Å cryo-EM structure of RNA enabled by engineered homomeric self-assembly.
Authors: Di Liu / François A Thélot / Joseph A Piccirilli / Maofu Liao / Peng Yin /
Abstract: High-resolution structural studies are essential for understanding the folding and function of diverse RNAs. Herein, we present a nanoarchitectural engineering strategy for efficient structural ...High-resolution structural studies are essential for understanding the folding and function of diverse RNAs. Herein, we present a nanoarchitectural engineering strategy for efficient structural determination of RNA-only structures using single-particle cryogenic electron microscopy (cryo-EM). This strategy-ROCK (RNA oligomerization-enabled cryo-EM via installing kissing loops)-involves installing kissing-loop sequences onto the functionally nonessential stems of RNAs for homomeric self-assembly into closed rings with multiplied molecular weights and mitigated structural flexibility. ROCK enables cryo-EM reconstruction of the Tetrahymena group I intron at 2.98-Å resolution overall (2.85 Å for the core), allowing de novo model building of the complete RNA, including the previously unknown peripheral domains. ROCK is further applied to two smaller RNAs-the Azoarcus group I intron and the FMN riboswitch, revealing the conformational change of the former and the bound ligand in the latter. ROCK holds promise to greatly facilitate the use of cryo-EM in RNA structural studies.
History
DepositionJun 22, 2021-
Header (metadata) releaseMay 4, 2022-
Map releaseMay 4, 2022-
UpdateJun 1, 2022-
Current statusJun 1, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24285.png

Downloads & links

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Map

FileDownload / File: emd_24285.map.gz / Format: CCP4 / Size: 2.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFusobacterium nucleatum FMN-riboswitch, synthetic trimeric construct
Voxel sizeX=Y=Z: 1.23 Å
Density
Contour LevelBy AUTHOR: 0.0942
Minimum - Maximum-1.8571008 - 1.7311313
Average (Standard dev.)4.6316054e-12 (±0.097204305)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-70-65-23
Dimensions12212848
Spacing12812248
CellA: 157.44 Å / B: 150.06 Å / C: 59.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Fusobacterium nucleatum FMN riboswitch bound to FMN, synthetic tr...

EntireName: Fusobacterium nucleatum FMN riboswitch bound to FMN, synthetic trimeric construct
Components
  • Complex: Fusobacterium nucleatum FMN riboswitch bound to FMN, synthetic trimeric construct
    • RNA: Engineered FMN riboswitch for trimeric assembly

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Supramolecule #1: Fusobacterium nucleatum FMN riboswitch bound to FMN, synthetic tr...

SupramoleculeName: Fusobacterium nucleatum FMN riboswitch bound to FMN, synthetic trimeric construct
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Fusobacterium nucleatum (bacteria)
Molecular weightTheoretical: 130 KDa

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Macromolecule #1: Engineered FMN riboswitch for trimeric assembly

MacromoleculeName: Engineered FMN riboswitch for trimeric assembly / type: rna / ID: 1
Source (natural)Organism: Fusobacterium nucleatum (bacteria)
SequenceString:
GGAUCGCUGA UUGAUCCGCU UCGGGGCAGG GUGAAAUUCC CGACCGGUGG UAUAGUCCAC GAAAGCGCGA AUCAGCCGCG CUUUGAUUUG GUGAAAUUCC AAAACCGACA GUAGAGUCUG GAUGAGAGAA GC

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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

Concentration1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTris-acetateTris-acetate
30.0 mMMgCl2Magnesium chloride
VitrificationCryogen name: ETHANE / Instrument: GATAN CRYOPLUNGE 3

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Nominal magnification: 31000
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 266623

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