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- EMDB-42404: RORC mRNA 3'UTR riboswitch 117-AC mutant class B -

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Basic information

Entry
Database: EMDB / ID: EMD-42404
TitleRORC mRNA 3'UTR riboswitch 117-AC mutant class B
Map dataRORC mRNA 3'UTR riboswitch 77-GA mutant class C main refinement map
Sample
  • Complex: RORC mRNA 3'UTR riboswitch 117-AC mutant
    • RNA: RORC 3'UTR riboswitch 117-AC mutant
KeywordsRNA / riboswitch / RORC / mRNA
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.2 Å
AuthorsAsarnow D / Khoroshkin M / Goodarzi H / Cheng Y
Funding support United States, 7 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R35GM140847 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD020054 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021741 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD026881 United States
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA240984 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01CA244634 United States
CitationJournal: Nat Methods / Year: 2024
Title: A systematic search for RNA structural switches across the human transcriptome.
Authors: Matvei Khoroshkin / Daniel Asarnow / Shaopu Zhou / Albertas Navickas / Aidan Winters / Jackson Goudreau / Simon K Zhou / Johnny Yu / Christina Palka / Lisa Fish / Ashir Borah / Kian Yousefi ...Authors: Matvei Khoroshkin / Daniel Asarnow / Shaopu Zhou / Albertas Navickas / Aidan Winters / Jackson Goudreau / Simon K Zhou / Johnny Yu / Christina Palka / Lisa Fish / Ashir Borah / Kian Yousefi / Christopher Carpenter / K Mark Ansel / Yifan Cheng / Luke A Gilbert / Hani Goodarzi /
Abstract: RNA structural switches are key regulators of gene expression in bacteria, but their characterization in Metazoa remains limited. Here, we present SwitchSeeker, a comprehensive computational and ...RNA structural switches are key regulators of gene expression in bacteria, but their characterization in Metazoa remains limited. Here, we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3' untranslated region of the RORC (RAR-related orphan receptor C) transcript. In vivo dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identify trans factors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated messenger RNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape.
History
DepositionOct 19, 2023-
Header (metadata) releaseJul 31, 2024-
Map releaseJul 31, 2024-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42404.png

Downloads & links

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Map

FileDownload / File: emd_42404.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRORC mRNA 3'UTR riboswitch 77-GA mutant class C main refinement map
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

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel size
XYZ
EMDB info.2.172.172.17
CCP4 map header1.881.881.88
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-0.43192568 - 1.9285413
Average (Standard dev.)0.008825952 (±0.1024396)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 208.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_42404_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RORC mRNA 3'UTR riboswitch 77-GA mutant class C refinement half-map B

Fileemd_42404_half_map_1.map
AnnotationRORC mRNA 3'UTR riboswitch 77-GA mutant class C refinement half-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 : RORC mRNA 3'UTR riboswitch 117-AC mutant

EntireName: RORC mRNA 3'UTR riboswitch 117-AC mutant
Components
  • Complex: RORC mRNA 3'UTR riboswitch 117-AC mutant
    • RNA: RORC 3'UTR riboswitch 117-AC mutant

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Supramolecule #1: RORC mRNA 3'UTR riboswitch 117-AC mutant

SupramoleculeName: RORC mRNA 3'UTR riboswitch 117-AC mutant / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.27 KDa

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Macromolecule #1: RORC 3'UTR riboswitch 117-AC mutant

MacromoleculeName: RORC 3'UTR riboswitch 117-AC mutant / type: rna / ID: 1
Source (natural)Organism: Homo sapiens (human)
SequenceString:
UUCUAAUACG ACUCACUAUA GGUGCUCCAG UCCACUGAUC UUGGGUCUGG GGUGAUCCAA AUACCACCCC AGCUCCAGCU GUCUUCUACC ACUAGAAGAC CCAAGAGAAG CAGAAGUCGC UCGCACUGGU CAGUCGACAG GCAAGAUCAG AUCCUGGAGG ACU

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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

Concentration1.5 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 1502 / Average exposure time: 7.0 sec. / Average electron dose: 61.0 e/Å2 / Details: Super-res mode, 117 frames, 0.4745 A/px
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.62 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 43000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 645401
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 10.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 43446
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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