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- EMDB-20416: Cryo-EM structure of the full-length Bacillus subtilis glyQS T-bo... -

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Basic information

Entry
Database: EMDB / ID: EMD-20416
TitleCryo-EM structure of the full-length Bacillus subtilis glyQS T-box riboswitch in complex with tRNA-Gly
Map dataT leader complex
Sample
  • Complex: T-box-tRNAGly complex
    • RNA: T-box GlyQS leader (155-MER)
    • RNA: tRNAGly (75-MER)
KeywordsRNA complex / riboswitch / transcription attenuation / stacking. / RNA
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsLi S / Su Z
Funding support United States, 1 items
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, 2019-
Header (metadata) releaseAug 21, 2019-
Map releaseNov 20, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.046
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.046
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6pom
  • Surface level: 0.046
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20416.png

Downloads & links

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Map

FileDownload / File: emd_20416.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationT leader complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 224 pix.
= 183.68 Å		0.82 Å/pix.
x 224 pix.
= 183.68 Å		0.82 Å/pix.
x 224 pix.
= 183.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.046 / Movie #1: 0.046
Minimum - Maximum-0.31172895 - 0.323623
Average (Standard dev.)0.00039600974 (±0.007759916)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 183.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z183.680183.680183.680
α/β/γ90.00090.00090.000
start NX/NY/NZ929262
NX/NY/NZ290290360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.3120.3240.000

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Supplemental data

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Mask #1

Fileemd_20416_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : T-box-tRNAGly complex

EntireName: T-box-tRNAGly complex
Components
  • Complex: T-box-tRNAGly complex
    • RNA: T-box GlyQS leader (155-MER)
    • RNA: tRNAGly (75-MER)

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Supramolecule #1: T-box-tRNAGly complex

SupramoleculeName: T-box-tRNAGly complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 75 KDa

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

MacromoleculeName: T-box GlyQS leader (155-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 54.730551 KDa
SequenceString:
GUUGCAGUGA GAGAAAGAAG UACUUGCGUU ACCUCAUGAA AGCGACCUUA GGGCGGUGUA AGCUAAGGAU GAGCACGCAA CGAAAGGCA UUCUUGAGCA AUUUUAAAAA AGAGGCUGGG AUUUUGUUCU CAGCAACUAG GGUGGAACCG CGGGAGAACU C UCGUCCCU A

GENBANK: GENBANK: D84432.1

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Macromolecule #2: tRNAGly (75-MER)

MacromoleculeName: tRNAGly (75-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 24.156336 KDa
SequenceString:
GCGGAAGUAG UUCAGUGGUA GAACACCACC UUGCCAAGGU GGGGGUCGCG GGUUCGAAUC CCGUCUUCCG CUCCA

GENBANK: GENBANK: CP035163.1

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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

Concentration2.25 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
10.0 mMTris-HCl2-Amino-2-(hydroxymethyl)propane-1,3-diol hydrochloride
100.0 mMKClpotassium chloride
20.0 mMMgCl2magnesium chloride
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279 K / Instrument: FEI VITROBOT MARK IV / Details: 3 uL sample blot once for 3s.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 79.0 K / Max: 79.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-25 / Number grids imaged: 3 / Number real images: 5600 / Average exposure time: 5.0 sec. / Average electron dose: 38.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsMotion corrected by MotionCor2
Particle selectionNumber selected: 1962681 / Details: neural network particle picking module in EMAN2.2
Startup modelType of model: INSILICO MODEL / In silico model: EMAN2 generated initial model
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF
Software: (Name: RELION (ver. 3.0-beta), cryoSPARC (ver. 2.2))
Number images used: 189361
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0-beta)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0-beta)
Final 3D classificationNumber classes: 8 / Avg.num./class: 48663
Software: (Name: RELION (ver. 3.0-beta), cryoSPARC (ver. 2.2))
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

4lck

chain_id: E, residue_range: 5-79, source_name: PDB, initial_model_type: experimental model

4lck

chain_id: F, residue_range: 1-102, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: model map cross correlation
Output model

PDB-6pom:
Cryo-EM structure of the full-length Bacillus subtilis glyQS T-box riboswitch in complex with tRNA-Gly

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