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- EMDB-70056: 30S focus refined map of E.coli 70S ribosome complexed with P-sit... -

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Basic information

Entry
Database: EMDB / ID: EMD-70056
Title30S focus refined map of E.coli 70S ribosome complexed with P-site fMet-tRNAfMet and A-site R-beta(2)hydroxyBocK-tRNAPyl
Map data
Sample
  • Complex: 70S ribosome particle
Keywordsribosome / non-natural monomers / beta-2-hydroxy acids / unnatural monomers
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.45 Å
AuthorsMajumdar C / Cate J
Funding support United States, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)CHE 2002182 United States
CitationJournal: J Am Chem Soc / Year: 2026
Title: Co-Translational Incorporation of - and -β-Hydroxy Acids : A Structural and Biochemical Study on the Ribosome.
Authors: Chandrima Majumdar / Alexandra D Kent / Noah X Hamlish / Cathy Zhu / Katelyn A Fitzgerald / Jamie H D Cate / Alanna Schepartz /
Abstract: Engineering the translation apparatus to accept backbone-modified amino acid analogues would enable the programmed synthesis of sequence-defined biopolymers with tunable properties. β-Hydroxy acids ...Engineering the translation apparatus to accept backbone-modified amino acid analogues would enable the programmed synthesis of sequence-defined biopolymers with tunable properties. β-Hydroxy acids are of particular interest because they could support the programmed biosynthesis of both biocompatible polyester materials as well as natural product-like depsipeptides. Previous work has reported that both enantiomers of β-hydroxy-N-Boc-lysine (β-OH-BocK) are substrates for the orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNA pair, but only one enantiomer is introduced into protein . Here we make use of high-resolution cryogenic electron microscopy (cryo-EM) to determine the structural basis for this observation. These structures reveal both β-OH-BocK isomers equally well-positioned within the ribosomal A site regardless of stereochemistry. Consistent with this observation, translation reactions charged with tRNAs acylated with - or -β-OH-BocK produced roughly equal amounts of translated product when quantified on the basis of either mass spectrometry or luminescence. Together, these experiments imply that the substantial preferential incorporation of one enantiomer over the other observed previously results primarily from deficiencies in the steps that precede bond formation by the ribosome. Indeed, as predicted by this work and demonstrated in an accompanying paper (Soni, C. Co-Translational Incorporation of ()- and ()-β-Hydroxyacids : Directed Evolution of Efficient Aminoacyl-tRNA Synthetases. 2026, 148, 10.1021/jacs.5c18595), when cells are provided with an active and orthogonal aminoacyl-tRNA synthetase/tRNA pair that accepts both - and -β-OH-BocK as substrates, both monomers are introduced into protein in good yield and with high fidelity.
History
DepositionApr 4, 2025-
Header (metadata) releaseMar 11, 2026-
Map releaseMar 11, 2026-
UpdateMar 11, 2026-
Current statusMar 11, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70056.png

Downloads & links

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Map

FileDownload / File: emd_70056.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 512 pix.
= 421.632 Å		0.82 Å/pix.
x 512 pix.
= 421.632 Å		0.82 Å/pix.
x 512 pix.
= 421.632 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8235 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.08209938 - 0.1721959
Average (Standard dev.)0.00016856239 (±0.0031190778)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 421.632 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_70056_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_70056_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : 70S ribosome particle

EntireName: 70S ribosome particle
Components
  • Complex: 70S ribosome particle

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Supramolecule #1: 70S ribosome particle

SupramoleculeName: 70S ribosome particle / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600

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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
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 765402
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8ebb

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.45 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 86644
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8ebb


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT

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