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- PDB-8b0j: CryoEM structure of bacterial RNaseE.RapZ.GlmZ complex central to... -

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Basic information

Entry
Database: PDB / ID: 8b0j
TitleCryoEM structure of bacterial RNaseE.RapZ.GlmZ complex central to the control of cell envelope biogenesis
Components
  • GlmZ small RNA
  • RNase adapter protein RapZ
  • Ribonuclease E
KeywordsRNA BINDING PROTEIN / endonuclease RNase E / adaptor protein RapZ / small regulatory RNA GlmZ
Function / homology
Function and homology information


regulation of RNA helicase activity / rRNA 5'-end processing / ribonuclease E / ribonuclease E activity / bacterial degradosome / RNA destabilization / endoribonuclease complex / DEAD/H-box RNA helicase binding / carbohydrate derivative binding / 7S RNA binding ...regulation of RNA helicase activity / rRNA 5'-end processing / ribonuclease E / ribonuclease E activity / bacterial degradosome / RNA destabilization / endoribonuclease complex / DEAD/H-box RNA helicase binding / carbohydrate derivative binding / 7S RNA binding / RNA catabolic process / tRNA processing / mRNA catabolic process / RNA nuclease activity / RNA processing / RNA endonuclease activity / cytoplasmic side of plasma membrane / rRNA processing / protein complex oligomerization / protein homotetramerization / tRNA binding / rRNA binding / molecular adaptor activity / GTP binding / magnesium ion binding / protein-containing complex / RNA binding / zinc ion binding / ATP binding / membrane / identical protein binding / cytoplasm
Similarity search - Function
RapZ-like family / P-loop ATPase protein family / Polyribonucleotide phosphorylase C-terminal / Polyribonucleotide phosphorylase C terminal / : / RNase E/G, Thioredoxin-like domain / Ribonuclease E/G / RNA-binding protein AU-1/Ribonuclease E/G / Ribonuclease E / Ribonuclease E/G family ...RapZ-like family / P-loop ATPase protein family / Polyribonucleotide phosphorylase C-terminal / Polyribonucleotide phosphorylase C terminal / : / RNase E/G, Thioredoxin-like domain / Ribonuclease E/G / RNA-binding protein AU-1/Ribonuclease E/G / Ribonuclease E / Ribonuclease E/G family / S1 domain profile. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / RNase adapter protein RapZ / Ribonuclease E
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.99 Å
AuthorsIslam, M.S. / Hardwick, H.W. / Chirgadze, D.Y. / Luisi, B.F.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust200873/Z/16/Z United Kingdom
Citation
Journal: EMBO J / Year: 2023
Title: Structure of a bacterial ribonucleoprotein complex central to the control of cell envelope biogenesis.
Authors: Md Saiful Islam / Steven W Hardwick / Laura Quell / Svetlana Durica-Mitic / Dimitri Y Chirgadze / Boris Görke / Ben F Luisi /
Abstract: Biogenesis of the essential precursor of the bacterial cell envelope, glucosamine-6-phosphate (GlcN6P), is controlled by intricate post-transcriptional networks mediated by GlmZ, a small regulatory ...Biogenesis of the essential precursor of the bacterial cell envelope, glucosamine-6-phosphate (GlcN6P), is controlled by intricate post-transcriptional networks mediated by GlmZ, a small regulatory RNA (sRNA). GlmZ stimulates translation of the mRNA encoding GlcN6P synthtase in Escherichia coli, but when bound by RapZ protein, the sRNA becomes inactivated through cleavage by the endoribonuclease RNase E. Here, we report the cryoEM structure of the RapZ:GlmZ complex, revealing a complementary match of the RapZ tetrameric quaternary structure to structural repeats in the sRNA. The nucleic acid is contacted by RapZ mostly through a highly conserved domain that shares an evolutionary relationship with phosphofructokinase and suggests links between metabolism and riboregulation. We also present the structure of a precleavage intermediate formed between the binary RapZ:GlmZ complex and RNase E that reveals how GlmZ is presented and recognised by the enzyme. The structures provide a framework for understanding how other encounter complexes might guide recognition and action of endoribonucleases on target transcripts, and how structured substrates in polycistronic precursors may be recognised for processing by RNase E.
#1: Journal: Biorxiv / Year: 2022
Title: Structure of a bacterial ribonucleoprotein complex central to the control of cell envelope biogenesis
Authors: Islam, M.S. / Hardwick, S.W. / Quell, L. / Chirgadze, D.Y. / Gorke, B. / Luisi, B.F.
History
DepositionSep 7, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 5, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 7, 2022Group: Database references / Category: citation / citation_author
Revision 1.2Dec 21, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Jan 25, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNase adapter protein RapZ
B: RNase adapter protein RapZ
D: RNase adapter protein RapZ
C: RNase adapter protein RapZ
L: Ribonuclease E
N: Ribonuclease E
K: GlmZ small RNA


Theoretical massNumber of molelcules
Total (without water)325,2527
Polymers325,2527
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
RNase adapter protein RapZ


Mass: 32538.320 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: rapZ, yhbJ, b3205, JW3172 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A894
#2: Protein Ribonuclease E / / RNase E


Mass: 64518.363 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Strain: K12 / Gene: rne, ams, hmp1, b1084, JW1071 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P21513, ribonuclease E
#3: RNA chain GlmZ small RNA


Mass: 66061.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Production host: Escherichia coli K-12 (bacteria)

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: RapZ tetramer, GlmZ stem loops I & II / Type: COMPLEX / Details: In vitro reconstituted RapZ and GlmZ complex / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: 25 mM HEPES pH 7.5, 300 KCl, and 1 mM MgCl2
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: DIFFRACTION / Nominal defocus max: 300 nm / Nominal defocus min: 100 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 47.3 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameCategory
4WarpCTF correction
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 286172
3D reconstructionResolution: 3.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33595 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00819050
ELECTRON MICROSCOPYf_angle_d0.80226723
ELECTRON MICROSCOPYf_dihedral_angle_d20.0257384
ELECTRON MICROSCOPYf_chiral_restr0.0453291
ELECTRON MICROSCOPYf_plane_restr0.0062854

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