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- EMDB-31278: Annealing synchronizes 70S ribosome into minimum-energy conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-31278
TitleAnnealing synchronizes 70S ribosome into minimum-energy conformation
Map data
Sample
  • Complex: 70S ribosome
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsSu X / Chu X / Shen Q
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Annealing synchronizes the 70 ribosome into a minimum-energy conformation.
Authors: Xiaofeng Chu / Xin Su / Mingdong Liu / Li Li / Tianhao Li / Yicheng Qin / Guoliang Lu / Lei Qi / Yunhui Liu / Jinzhong Lin / Qing-Tao Shen /
Abstract: Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate that simulated annealing on biological ...Researchers commonly anneal metals, alloys, and semiconductors to repair defects and improve microstructures via recrystallization. Theoretical studies indicate that simulated annealing on biological macromolecules helps predict the final structures with minimum free energy. Experimental validation of this homogenizing effect and further exploration of its applications are fascinating scientific questions that remain elusive. Here, we chose the apo-state 70 ribosome from as a model, wherein the 30 subunit undergoes a thermally driven intersubunit rotation and exhibits substantial structural flexibility as well as distinct free energy. We experimentally demonstrate that annealing at a fast cooling rate enhances the 70 ribosome homogeneity and improves local resolution on the 30 subunit. After annealing, the 70 ribosome is in a nonrotated state with respect to corresponding intermediate structures in unannealed or heated ribosomes. Manifold-based analysis further indicates that the annealed 70 ribosome takes a narrow conformational distribution and exhibits a minimum-energy state in the free-energy landscape. Our experimental results offer a facile yet robust approach to enhance protein stability, which is ideal for high-resolution cryogenic electron microscopy. Beyond structure determination, annealing shows great potential for synchronizing proteins on a single-molecule level and can be extended to study protein folding and explore conformational and energy landscapes.
History
DepositionMay 8, 2021-
Header (metadata) releaseFeb 23, 2022-
Map releaseFeb 23, 2022-
UpdateMar 16, 2022-
Current statusMar 16, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.19
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.19
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31278.png

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Map

FileDownload / File: emd_31278.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 400 pix.
= 424. Å		1.06 Å/pix.
x 400 pix.
= 424. Å		1.06 Å/pix.
x 400 pix.
= 424. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.19 / Movie #1: 0.19
Minimum - Maximum-0.2284516 - 1.4321967
Average (Standard dev.)-0.012380715 (±0.050503876)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 423.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z424.000424.000424.000
α/β/γ90.00090.00090.000
start NX/NY/NZ139118109
NX/NY/NZ123164187
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.2281.432-0.012

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Supplemental data

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Sample components

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Entire : 70S ribosome

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

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / 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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / 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: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / 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: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 200000
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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