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- EMDB-29790: 30S focus refined map of WT E.coli ribosome complexed with A-site... -

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Basic information

Entry
Database: EMDB / ID: EMD-29790
Title30S focus refined map of WT E.coli ribosome complexed with A-site ortho-aminobenzoic acid charged tRNA-Phe
Map data30S focus refined map
Sample
  • Complex: 70S E.coli ribosome
Keywordsribosome / non-natural monomers / aminobenzoic acids / unnatural monomers
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.14 Å
AuthorsMajumdar C / Cate JHD
Funding support United States, 1 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)CHE 2002182 United States
CitationJournal: ACS Cent Sci / Year: 2023
Title: Aminobenzoic Acid Derivatives Obstruct Induced Fit in the Catalytic Center of the Ribosome.
Authors: Chandrima Majumdar / Joshua A Walker / Matthew B Francis / Alanna Schepartz / Jamie H D Cate /
Abstract: The () ribosome can incorporate a variety of non-l-α-amino acid monomers into polypeptide chains but with poor efficiency. Although these monomers span a diverse set of compounds, there exists no ...The () ribosome can incorporate a variety of non-l-α-amino acid monomers into polypeptide chains but with poor efficiency. Although these monomers span a diverse set of compounds, there exists no high-resolution structural information regarding their positioning within the catalytic center of the ribosome, the peptidyl transferase center (PTC). Thus, details regarding the mechanism of amide bond formation and the structural basis for differences and defects in incorporation efficiency remain unknown. Within a set of three aminobenzoic acid derivatives-3-aminopyridine-4-carboxylic acid (Apy), aminobenzoic acid (ABZ), and aminobenzoic acid (ABZ)-the ribosome incorporates Apy into polypeptide chains with the highest efficiency, followed by ABZ and then ABZ, a trend that does not track with the nucleophilicity of the reactive amines. Here, we report high-resolution cryo-EM structures of the ribosome with each of these three aminobenzoic acid derivatives charged on tRNA bound in the aminoacyl-tRNA site (A-site). The structures reveal how the aromatic ring of each monomer sterically blocks the positioning of nucleotide U2506, thereby preventing rearrangement of nucleotide U2585 and the resulting induced fit in the PTC required for efficient amide bond formation. They also reveal disruptions to the bound water network that is believed to facilitate formation and breakdown of the tetrahedral intermediate. Together, the cryo-EM structures reported here provide a mechanistic rationale for differences in reactivity of aminobenzoic acid derivatives relative to l-α-amino acids and each other and identify stereochemical constraints on the size and geometry of non-monomers that can be accepted efficiently by wild-type ribosomes.
History
DepositionFeb 16, 2023-
Header (metadata) releaseApr 19, 2023-
Map releaseApr 19, 2023-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29790.png

Downloads & links

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Map

FileDownload / File: emd_29790.map.gz / Format: CCP4 / Size: 381.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation30S focus refined map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 464 pix.
= 381.965 Å		0.82 Å/pix.
x 464 pix.
= 381.965 Å		0.82 Å/pix.
x 464 pix.
= 381.965 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8232 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0167
Minimum - Maximum-0.084711894 - 0.21918154
Average (Standard dev.)0.00028465106 (±0.004125598)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions464464464
Spacing464464464
CellA=B=C: 381.96478 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: 30S focus refinement half map

Fileemd_29790_half_map_1.map
Annotation30S focus refinement half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 30S focus refinement half map

Fileemd_29790_half_map_2.map
Annotation30S focus refinement 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 : 70S E.coli ribosome

EntireName: 70S E.coli ribosome
Components
  • Complex: 70S E.coli ribosome

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Supramolecule #1: 70S E.coli ribosome

SupramoleculeName: 70S E.coli ribosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#55
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: 0.2 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN 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

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8ebb

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.14 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 339543
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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