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- EMDB-28256: 30S-focused map for: "Atomistic simulations of the E. coli riboso... -

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Basic information

Entry
Database: EMDB / ID: EMD-28256
Title30S-focused map for: "Atomistic simulations of the E. coli ribosome provide selection criteria for translationally active substrates"
Map dataPost-processed, masked 30S-focused map
Sample
  • Complex: 30S-focused map
Keywordsribosome / tRNA / e. coli
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsWatson ZL / Cate JHD
Funding support United States, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)CHE 2002182 United States
CitationJournal: Nat Chem / Year: 2023
Title: Atomistic simulations of the Escherichia coli ribosome provide selection criteria for translationally active substrates.
Authors: Zoe L Watson / Isaac J Knudson / Fred R Ward / Scott J Miller / Jamie H D Cate / Alanna Schepartz / Ara M Abramyan /
Abstract: As genetic code expansion advances beyond L-α-amino acids to backbone modifications and new polymerization chemistries, delineating what substrates the ribosome can accommodate remains a challenge. ...As genetic code expansion advances beyond L-α-amino acids to backbone modifications and new polymerization chemistries, delineating what substrates the ribosome can accommodate remains a challenge. The Escherichia coli ribosome tolerates non-L-α-amino acids in vitro, but few structural insights that explain how are available, and the boundary conditions for efficient bond formation are so far unknown. Here we determine a high-resolution cryogenic electron microscopy structure of the E. coli ribosome containing α-amino acid monomers and use metadynamics simulations to define energy surface minima and understand incorporation efficiencies. Reactive monomers across diverse structural classes favour a conformational space where the aminoacyl-tRNA nucleophile is <4 Å from the peptidyl-tRNA carbonyl with a Bürgi-Dunitz angle of 76-115°. Monomers with free energy minima that fall outside this conformational space do not react efficiently. This insight should accelerate the in vivo and in vitro ribosomal synthesis of sequence-defined, non-peptide heterooligomers.
History
DepositionSep 28, 2022-
Header (metadata) releaseMay 31, 2023-
Map releaseMay 31, 2023-
UpdateJul 19, 2023-
Current statusJul 19, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28256.png

Downloads & links

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Map

FileDownload / File: emd_28256.map.gz / Format: CCP4 / Size: 361.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed, masked 30S-focused map
Voxel sizeX=Y=Z: 0.8296 Å
Density
Contour LevelBy AUTHOR: 0.035
Minimum - Maximum-0.07642774 - 0.2257059
Average (Standard dev.)0.0002650672 (±0.0036743255)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions456456456
Spacing456456456
CellA=B=C: 378.29758 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28256_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

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Additional map: Map from 30S-focused 3D auto-refine, without post-processing

Fileemd_28256_additional_1.map
AnnotationMap from 30S-focused 3D auto-refine, without post-processing
Projections & Slices
AxesZYX

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Half map: Unfiltered half-map 1

Fileemd_28256_half_map_1.map
AnnotationUnfiltered half-map 1
Projections & Slices
AxesZYX

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Histogram

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Half map: Unfiltered half-map 2

Fileemd_28256_half_map_2.map
AnnotationUnfiltered half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : 30S-focused map

EntireName: 30S-focused map
Components
  • Complex: 30S-focused map

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Supramolecule #1: 30S-focused map

SupramoleculeName: 30S-focused map / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#54
Details: Overall complex includes 70S ribosome with mRNA, A- and P-site Met-NH-tRNAs
Source (natural)Organism: Escherichia coli (E. coli)

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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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 / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

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: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 129455
FSC plot (resolution estimation)

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