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- EMDB-5508: Dissecting the in vivo assembly of the 30S ribosomal subunit reve... -

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Entry
Database: EMDB / ID: EMD-5508
TitleDissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM
Map dataThe map has been normalized to N(0,1)
Sample
  • Sample: immature ribosomal small subunit from rimm gene deleted E.coli strain treated with RimM in vitro
  • Complex: small subunit from rimm gene deleted E.coli strain
  • Protein or peptide: RimM
KeywordsRibosome biogenesis / 30S subunit assembly / RimM
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 17.6 Å
AuthorsGuo Q / Goto S / Chen Y / Muto A / Himeno H / Deng H / Lei J / Gao N
CitationJournal: Nucleic Acids Res / Year: 2013
Title: Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM and general features of the assembly process.
Authors: Qiang Guo / Simon Goto / Yuling Chen / Boya Feng / Yanji Xu / Akira Muto / Hyouta Himeno / Haiteng Deng / Jianlin Lei / Ning Gao /
Abstract: Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo ...Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo in bacteria. Although the assembly process has been extensively studied in vitro for over 40 years, very limited information is known for the in vivo process and specific roles of assembly factors. Such an example is ribosome maturation factor M (RimM), a factor involved in the late-stage assembly of the 30S subunit. Here, we combined quantitative mass spectrometry and cryo-electron microscopy to characterize the in vivo 30S assembly intermediates isolated from mutant Escherichia coli strains with genes for assembly factors deleted. Our compositional and structural data show that the assembly of the 3'-domain of the 30S subunit is severely delayed in these intermediates, featured with highly underrepresented 3'-domain proteins and large conformational difference compared with the mature 30S subunit. Further analysis indicates that RimM functions not only to promote the assembly of a few 3'-domain proteins but also to stabilize the rRNA tertiary structure. More importantly, this study reveals intriguing similarities and dissimilarities between the in vitro and the in vivo assembly pathways, suggesting that they are in general similar but with subtle differences.
History
DepositionSep 28, 2012-
Header (metadata) releaseDec 12, 2012-
Map releaseJan 16, 2013-
UpdateMar 6, 2013-
Current statusMar 6, 2013Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: -2.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: -2.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j2f
  • Surface level: -2.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5508.gif

Downloads & links

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Map

FileDownload / File: emd_5508.map.gz / Format: CCP4 / Size: 7.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe map has been normalized to N(0,1)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3 Å/pix.
x 125 pix.
= 375. Å		3 Å/pix.
x 125 pix.
= 375. Å		3 Å/pix.
x 125 pix.
= 375. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: -2.8 / Movie #1: -2.8
Minimum - Maximum-8.45064449 - 6.06328487
Average (Standard dev.)-4.95746183 (±0.71110576)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-62-62-62
Dimensions125125125
Spacing125125125
CellA=B=C: 375.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z333
M x/y/z125125125
origin x/y/z0.0000.0000.000
length x/y/z375.000375.000375.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-5029166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS-62-62-62
NC/NR/NS125125125
D min/max/mean-8.4516.063-4.957

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Supplemental data

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Sample components

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Entire : immature ribosomal small subunit from rimm gene deleted E.coli st...

EntireName: immature ribosomal small subunit from rimm gene deleted E.coli strain treated with RimM in vitro
Components
  • Sample: immature ribosomal small subunit from rimm gene deleted E.coli strain treated with RimM in vitro
  • Complex: small subunit from rimm gene deleted E.coli strain
  • Protein or peptide: RimM

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Supramolecule #1000: immature ribosomal small subunit from rimm gene deleted E.coli st...

SupramoleculeName: immature ribosomal small subunit from rimm gene deleted E.coli strain treated with RimM in vitro
type: sample / ID: 1000 / Number unique components: 1
Molecular weightExperimental: 800 KDa / Theoretical: 800 KDa

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Supramolecule #1: small subunit from rimm gene deleted E.coli strain

SupramoleculeName: small subunit from rimm gene deleted E.coli strain / type: complex / ID: 1 / Name.synonym: immature 30S / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: SSU 30S
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 800 KDa / Theoretical: 800 KDa

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Macromolecule #1: RimM

MacromoleculeName: RimM / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant plasmid: pET28b

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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 / Details: 150 mM NH4Cl,10mM Tris-HCL,10mM MgCl2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 1 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateJan 1, 2012
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.8 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThis is a classification volume (No. 3) using ML3D methods.
CTF correctionDetails: Weiner filter
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 9609

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Atomic model buiding 1

Initial modelPDB ID:

3ofa
PDB Unreleased entry


Chain - Chain ID: A
SoftwareName: MDFF
DetailsProtocol: Initial local fitting was done using Chimera and then MDFF was used for flexible fitting. ref: Trabuco, L.G., Villa, E., Mitra, K., Frank, J. and Schulten, K. (2008) Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure, 16, 673-683
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation
Output model

PDB-3j2f:
Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM

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