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- EMDB-20853: P7A7 ribosome large subunit -

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Basic information

Entry
Database: EMDB / ID: EMD-20853
TitleP7A7 ribosome large subunit
Map dataRELION post-processed map
Sample
  • Complex: P7A7 E. coli 50S mutant
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsWatson ZL / Ward FR / Cate JHD
Funding support United States, 3 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)NSF CHE-1740549 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM R01-114454 United States
National Science Foundation (NSF, United States)1106400 United States
CitationJournal: Biochemistry / Year: 2019
Title: Defects in the Assembly of Ribosomes Selected for β-Amino Acid Incorporation.
Authors: Fred R Ward / Zoe L Watson / Omer Ad / Alanna Schepartz / Jamie H D Cate /
Abstract: Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve ...Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant ribosomes have been developed that improve the incorporation of several nonstandard monomers including d-amino acids, dipeptides, and β-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these ribosomes catalyze incorporation of these new substrates. Here, we probed the properties of a mutant ribosome-P7A7-evolved for better β-amino acid incorporation through biochemistry and cryo-electron microscopy. Although P7A7 is a functional ribosome , it is inactive , and assembles poorly into 70S ribosome complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient ribosome assembly during ribosome engineering toward new catalytic abilities.
History
DepositionOct 21, 2019-
Header (metadata) releaseOct 30, 2019-
Map releaseOct 30, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0153
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0153
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20853.png

Downloads & links

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Map

FileDownload / File: emd_20853.map.gz / Format: CCP4 / Size: 98.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRELION post-processed map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.11 Å/pix.
x 296 pix.
= 329.626 Å		1.11 Å/pix.
x 296 pix.
= 329.626 Å		1.11 Å/pix.
x 296 pix.
= 329.626 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1136 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0153 / Movie #1: 0.0153
Minimum - Maximum-0.046426296 - 0.06829227
Average (Standard dev.)0.00006028577 (±0.004241044)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions296296296
Spacing296296296
CellA=B=C: 329.6256 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11360135135141.11360135135141.1136013513514
M x/y/z296296296
origin x/y/z0.0000.0000.000
length x/y/z329.626329.626329.626
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS296296296
D min/max/mean-0.0460.0680.000

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Supplemental data

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Additional map: RELION 3d-autorefine map

Fileemd_20853_additional.map
AnnotationRELION 3d-autorefine map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: RELION 3d-autorefine map

Fileemd_20853_additional_1.map
AnnotationRELION 3d-autorefine map
Projections & Slices
AxesZYX

Projections

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1

Fileemd_20853_half_map_1.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2

Fileemd_20853_half_map_2.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : P7A7 E. coli 50S mutant

EntireName: P7A7 E. coli 50S mutant
Components
  • Complex: P7A7 E. coli 50S mutant

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Supramolecule #1: P7A7 E. coli 50S mutant

SupramoleculeName: P7A7 E. coli 50S mutant / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: 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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 10.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 37609
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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