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- PDB-6pom: Cryo-EM structure of the full-length Bacillus subtilis glyQS T-bo... -

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Basic information

Entry
Database: PDB / ID: 6pom
TitleCryo-EM structure of the full-length Bacillus subtilis glyQS T-box riboswitch in complex with tRNA-Gly
Components
  • T-box GlyQS leader (155-MER)
  • tRNAGly (75-MER)
KeywordsRNA / RNA complex / riboswitch / transcription attenuation / stacking.
Function / homology: / : / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsLi, S. / Su, Z. / Zhang, J. / Chiu, W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P50GM103297 United States
CitationJournal: Nat Struct Mol Biol / Year: 2019
Title: Structural basis of amino acid surveillance by higher-order tRNA-mRNA interactions.
Authors: Shuang Li / Zhaoming Su / Jean Lehmann / Vassiliki Stamatopoulou / Nikoleta Giarimoglou / Frances E Henderson / Lixin Fan / Grigore D Pintilie / Kaiming Zhang / Muyuan Chen / Steven J Ludtke ...Authors: Shuang Li / Zhaoming Su / Jean Lehmann / Vassiliki Stamatopoulou / Nikoleta Giarimoglou / Frances E Henderson / Lixin Fan / Grigore D Pintilie / Kaiming Zhang / Muyuan Chen / Steven J Ludtke / Yun-Xing Wang / Constantinos Stathopoulos / Wah Chiu / Jinwei Zhang /
Abstract: Amino acid availability in Gram-positive bacteria is monitored by T-box riboswitches. T-boxes directly bind tRNAs, assess their aminoacylation state, and regulate the transcription or translation of ...Amino acid availability in Gram-positive bacteria is monitored by T-box riboswitches. T-boxes directly bind tRNAs, assess their aminoacylation state, and regulate the transcription or translation of downstream genes to maintain nutritional homeostasis. Here, we report cocrystal and cryo-EM structures of Geobacillus kaustophilus and Bacillus subtilis T-box-tRNA complexes, detailing their multivalent, exquisitely selective interactions. The T-box forms a U-shaped molecular vise that clamps the tRNA, captures its 3' end using an elaborate 'discriminator' structure, and interrogates its aminoacylation state using a steric filter fashioned from a wobble base pair. In the absence of aminoacylation, T-boxes clutch tRNAs and form a continuously stacked central spine, permitting transcriptional readthrough or translation initiation. A modeled aminoacyl disrupts tRNA-T-box stacking, severing the central spine and blocking gene expression. Our data establish a universal mechanism of amino acid sensing on tRNAs and gene regulation by T-box riboswitches and exemplify how higher-order RNA-RNA interactions achieve multivalency and specificity.
History
DepositionJul 4, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 20, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2019Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Dec 18, 2019Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • EMDB-20416
  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: T-box GlyQS leader (155-MER)
B: tRNAGly (75-MER)


Theoretical massNumber of molelcules
Total (without water)78,8872
Polymers78,8872
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: isothermal titration calorimetry, native gel electrophoresis, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain T-box GlyQS leader (155-MER)


Mass: 54730.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: glyQS
Production host: in vitro transcription vector pT7-TP(deltai) (others)
References: GenBank: 2627063
#2: RNA chain tRNAGly (75-MER)


Mass: 24156.336 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria)
Production host: in vitro transcription vector pT7-TP(deltai) (others)
References: GenBank: 1560688081

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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

ComponentName: T-box-tRNAGly complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.075 MDa / Experimental value: NO
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: in vitro transcription vector pT7-TP(deltai) (others)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
110 mM2-Amino-2-(hydroxymethyl)propane-1,3-diol hydrochlorideTris-HClTris1
2100 mMpotassium chlorideKCl1
320 mMmagnesium chlorideMgCl21
SpecimenConc.: 2.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 279 K / Details: 3 uL sample blot once for 3s

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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 FIELDBright-field microscopy / Nominal magnification: 165000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 79 K / Temperature (min): 79 K
Image recordingAverage exposure time: 5 sec. / Electron dose: 38 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 5600
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 25 / Used frames/image: 1-25

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
2EPU1.1image acquisition
4CTFFIND4.1.18CTF correction
5RELION3.0-betaCTF correction
8UCSF Chimera1.13.1model fitting
10RELION3.0-betainitial Euler assignment
11RELION3.0-betafinal Euler assignment
12RELION3.0-betaclassification
13cryoSPARC2.2classification
14RELION3.0-beta3D reconstruction
15cryoSPARC2.23D reconstruction
16PHENIX1.15.2model refinement
Image processingDetails: Motion corrected by MotionCor2
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 1962681 / Details: neural network particle picking module in EMAN2.2
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 189361 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: model map cross correlation
Atomic model building

3D fitting-ID: 1 / Accession code: 4LCK / Initial refinement model-ID: 1 / PDB-ID: 4LCK

4lck

/ Source name: PDB / Type: experimental model

IDPdb chain-IDPdb chain residue range
1E5-79
2F1-102
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028010
ELECTRON MICROSCOPYf_angle_d0.80814309
ELECTRON MICROSCOPYf_dihedral_angle_d20.6973158
ELECTRON MICROSCOPYf_chiral_restr0.031150
ELECTRON MICROSCOPYf_plane_restr0.003414

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