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- EMDB-28229: E. coli 70S ribosome with A-loop mutations U2554C and U2555C (30S... -

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Basic information

Entry
Database: EMDB / ID: EMD-28229
TitleE. coli 70S ribosome with A-loop mutations U2554C and U2555C (30S Focus Refinement)
Map dataE. coli 70S ribosome with A-loop mutations U2554C and U2555C (30S Focus Refinement)
Sample
  • Complex: E. coli 70S ribosome with A-loop mutations U2554C and U2555C
    • Complex: 50S SubunitProkaryotic large ribosomal subunit
    • Complex: 30S SubunitProkaryotic small ribosomal subunit
KeywordsRNA / thermophile / A loop / RIBOSOME
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.39 Å
AuthorsNissley AJ / Penev PI / Watson ZL / Banfield JF / Cate JHD
Funding support United States, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)CHE-2002182 United States
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome.
Authors: Amos J Nissley / Petar I Penev / Zoe L Watson / Jillian F Banfield / Jamie H D Cate /
Abstract: The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are ...The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3'-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.
History
DepositionSep 24, 2022-
Header (metadata) releaseFeb 1, 2023-
Map releaseFeb 1, 2023-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28229.png

Downloads & links

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Map

FileDownload / File: emd_28229.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationE. coli 70S ribosome with A-loop mutations U2554C and U2555C (30S Focus Refinement)
Voxel sizeX=Y=Z: 0.8279 Å
Density
Contour LevelBy AUTHOR: 0.0577
Minimum - Maximum-0.15123937 - 0.32878605
Average (Standard dev.)0.0006557304 (±0.008435037)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 364.276 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: E. coli 70S ribosome with A-loop mutations U2554C...

Fileemd_28229_half_map_1.map
AnnotationE. coli 70S ribosome with A-loop mutations U2554C and U2555C (Half Map)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: E. coli 70S ribosome with A-loop mutations U2554C...

Fileemd_28229_half_map_2.map
AnnotationE. coli 70S ribosome with A-loop mutations U2554C and U2555C (Half 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 : E. coli 70S ribosome with A-loop mutations U2554C and U2555C

EntireName: E. coli 70S ribosome with A-loop mutations U2554C and U2555C
Components
  • Complex: E. coli 70S ribosome with A-loop mutations U2554C and U2555C
    • Complex: 50S SubunitProkaryotic large ribosomal subunit
    • Complex: 30S SubunitProkaryotic small ribosomal subunit

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Supramolecule #1: E. coli 70S ribosome with A-loop mutations U2554C and U2555C

SupramoleculeName: E. coli 70S ribosome with A-loop mutations U2554C and U2555C
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli) / Strain: BL21
Molecular weightTheoretical: 850 KDa

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Supramolecule #2: 50S Subunit

SupramoleculeName: 50S Subunit / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#5, #27-#54
Source (natural)Organism: Escherichia coli (E. coli) / Strain: BL21

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Supramolecule #3: 30S Subunit

SupramoleculeName: 30S Subunit / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #6-#26
Source (natural)Organism: Escherichia coli (E. coli) / Strain: BL21

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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

Concentration0.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
20.0 mMTris-HClTris
100.0 mMAmmonium Chloride
0.5 mMDisodium EDTA
2.0 mMDTT
15.0 mMMagnesium Chloride
2.0 mMSpermidine
0.05 mMSpermine
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 12 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1614362
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7k00

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.39 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 114493
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7k00


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 38.8

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