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- PDB-6ues: Apo SAM-IV Riboswitch -

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Basic information

Entry
Database: PDB / ID: 6ues
TitleApo SAM-IV Riboswitch
ComponentsRNA (119-MER)
KeywordsRNA / SAM-IV riboswitch / Cryo-EM / Small RNA
Function / homology: / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesMycobacterium sp. MCS (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsZhang, K. / Li, S. / Kappel, K. / Pintilie, G. / Su, Z. / Mou, T. / Schmid, M. / Das, R. / Chiu, W.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)P41GM103832 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R01GM079429 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)S10OD021600 United States
CitationJournal: Nat Commun / Year: 2019
Title: Cryo-EM structure of a 40 kDa SAM-IV riboswitch RNA at 3.7 Å resolution.
Authors: Kaiming Zhang / Shanshan Li / Kalli Kappel / Grigore Pintilie / Zhaoming Su / Tung-Chung Mou / Michael F Schmid / Rhiju Das / Wah Chiu /
Abstract: Specimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The high flexibility of pure RNAs makes it difficult to obtain ...Specimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The high flexibility of pure RNAs makes it difficult to obtain high-resolution structures by cryo-EM. In bacteria, riboswitches regulate sulfur metabolism through binding to the S-adenosylmethionine (SAM) ligand and offer compelling targets for new antibiotics. SAM-I, SAM-I/IV, and SAM-IV are the three most commonly found SAM riboswitches, but the structure of SAM-IV is still unknown. Here, we report the structures of apo and SAM-bound SAM-IV riboswitches (119-nt, ~40 kDa) to 3.7 Å and 4.1 Å resolution, respectively, using cryo-EM. The structures illustrate homologies in the ligand-binding core but distinct peripheral tertiary contacts in SAM-IV compared to SAM-I and SAM-I/IV. Our results demonstrate the feasibility of resolving small RNAs with enough detail to enable detection of their ligand-binding pockets and suggest that cryo-EM could play a role in structure-assisted drug design for RNA.
History
DepositionSep 23, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 18, 2019Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: RNA (119-MER)


Theoretical massNumber of molelcules
Total (without water)38,5231
Polymers38,5231
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain RNA (119-MER)


Mass: 38522.906 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: RNA was prepared by in vitro transcription with T7 RNA polymerase
Source: (synth.) Mycobacterium sp. MCS (bacteria) / References: GenBank: 108767400

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Apo SAM-IV RiboswitchCOMPLEXall0MULTIPLE SOURCES
2Apo SAM-IV riboswitchCOMPLEXall1MULTIPLE SOURCES
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.040 MDaYES
210.040 MDaYES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
11Helicobacter pylori (bacteria)21060190
22Helicobacter pylori (bacteria)21060190
Buffer solutionpH: 7.2
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Apo SAM-IV riboswitch
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R3.5/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 7.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 7200
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 30 / Used frames/image: 1-30

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.13.1model fitting
9RELION2.1initial Euler assignment
10cryoSPARC2final Euler assignment
11cryoSPARC2classification
12cryoSPARC23D reconstruction
13PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2102569
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 796923 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL

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