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- PDB-3j2b: Dissecting the in vivo assembly of the 30S ribosomal subunit reve... -

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Entry
Database: PDB / ID: 3j2b
TitleDissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM
Components16S rRNA
KeywordsRIBOSOME / Ribosome biogenesis / 30S subunit assembly / RimM
Function / homology: / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000)
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 13.6 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, 2012Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Jan 16, 2013Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2013Group: Database references
Revision 1.2Mar 13, 2013Group: Other
Revision 1.3Dec 18, 2019Group: Data collection / Database references / Category: database_2 / em_image_scans / em_software
Item: _em_software.fitting_id / _em_software.image_processing_id
Revision 1.4Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
N: 16S rRNA


Theoretical massNumber of molelcules
Total (without water)496,8921
Polymers496,8921
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: RNA chain 16S rRNA


Mass: 496892.375 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: K-12 / References: GenBank: AP012306.1

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: immature ribosomal small subunit from rimm gene deleted E.coli strain
Type: RIBOSOME
Molecular weightValue: 0.8 MDa / Experimental value: NO
Buffer solutionpH: 7.5 / Details: 150mM NH4Cl, 10mM Tris-HCL,10mM MgCl2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 150mM NH4Cl, 10mM Tris-HCL,10mM MgCl2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Method: Blot for 1 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Jan 1, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 80000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1300 nm / Camera length: 0 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 20 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1MDFFmodel fitting
2SPIDER3D reconstruction
CTF correctionDetails: Weiner filter
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: Reference Projections / Resolution: 13.6 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 30892
Details: This is a classification volume (No. 4) using ML3D methods. )
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation
Details: REFINEMENT PROTOCOL--Initial local fitting was done using Chimera and then MDFF was used for flexible fitting DETAILS--ref- Trabuco, L.G., Villa, E., Mitra, K., Frank, J. and Schulten, K. ...Details: REFINEMENT PROTOCOL--Initial local fitting was done using Chimera and then MDFF was used for flexible fitting DETAILS--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
Atomic model buildingPDB-ID: 3OFA

3ofa
PDB Unreleased entry


Pdb chain-ID: A / Accession code: 3OFA / Source name: PDB / Type: experimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms0 32892 0 0 32892

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