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- PDB-5nko: Solution structure of the C-terminal domain of S. aureus Hibernat... -

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Basic information

Entry
Database: PDB / ID: 5nko
TitleSolution structure of the C-terminal domain of S. aureus Hibernating Promoting Factor (CTD-SaHPF)
ComponentsRibosome hibernation promotion factor
KeywordsRIBOSOMAL PROTEIN / Staphylococcus aureus / Hibernation / Pathogen / Ribosome / Hibernating Promoting Factor / HPF
Function / homology
Function and homology information


negative regulation of translational elongation / ribosomal small subunit binding / cytosolic small ribosomal subunit
Similarity search - Function
Sigma 54 modulation/S30EA ribosomal protein, C-terminal domain / Ribosome hibernation promoting factor, long/plastid / Sigma 54 modulation/S30EA ribosomal protein, C-terminal / Sigma 54 modulation/S30EA ribosomal protein, C-terminal domain superfamily / Sigma 54 modulation/S30EA ribosomal protein C terminus / Ribosome hibernation promoting factor/RaiA / Ribosome hibernation promotion factor-like / Sigma 54 modulation protein / S30EA ribosomal protein / SHC Adaptor Protein / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Ribosome hibernation promotion factor
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria)
MethodSOLUTION NMR / simulated annealing
AuthorsUsachev, K.S. / Khusainov, I.S. / Ayupov, R.K. / Validov, S.Z. / Kieffer, B. / Yusupov, M.M.
Funding support Russian Federation, 1items
OrganizationGrant numberCountry
Russian Science Foundation16-14-10014 Russian Federation
CitationJournal: EMBO J / Year: 2017
Title: Structures and dynamics of hibernating ribosomes from mediated by intermolecular interactions of HPF.
Authors: Iskander Khusainov / Quentin Vicens / Rustam Ayupov / Konstantin Usachev / Alexander Myasnikov / Angelita Simonetti / Shamil Validov / Bruno Kieffer / Gulnara Yusupova / Marat Yusupov / Yaser Hashem /
Abstract: In bacteria, ribosomal hibernation shuts down translation as a response to stress, through reversible binding of stress-induced proteins to ribosomes. This process typically involves the formation of ...In bacteria, ribosomal hibernation shuts down translation as a response to stress, through reversible binding of stress-induced proteins to ribosomes. This process typically involves the formation of 100S ribosome dimers. Here, we present the structures of hibernating ribosomes from human pathogen containing a long variant of the hibernation-promoting factor (SaHPF) that we solved using cryo-electron microscopy. Our reconstructions reveal that the N-terminal domain (NTD) of SaHPF binds to the 30S subunit as observed for shorter variants of HPF in other species. The C-terminal domain (CTD) of SaHPF protrudes out of each ribosome in order to mediate dimerization. Using NMR, we characterized the interactions at the CTD-dimer interface. Secondary interactions are provided by helix 26 of the 16S ribosomal RNA We also show that ribosomes in the 100S particle adopt both rotated and unrotated conformations. Overall, our work illustrates a specific mode of ribosome dimerization by long HPF, a finding that may help improve the selectivity of antimicrobials.
History
DepositionMar 31, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 5, 2017Provider: repository / Type: Initial release
Revision 1.1Jul 26, 2017Group: Database references / Category: citation
Item: _citation.country / _citation.journal_id_ASTM ..._citation.country / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2May 8, 2019Group: Data collection / Category: pdbx_nmr_software / Item: _pdbx_nmr_software.name
Revision 1.3Jun 19, 2024Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_nmr_spectrometer
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_nmr_spectrometer.model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Ribosome hibernation promotion factor
B: Ribosome hibernation promotion factor


Theoretical massNumber of molelcules
Total (without water)14,2482
Polymers14,2482
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3370 Å2
ΔGint-14 kcal/mol
Surface area6770 Å2
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 500structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Ribosome hibernation promotion factor / HPF / Hibernation promoting factor SaHPF


Mass: 7124.070 Da / Num. of mol.: 2 / Fragment: C-terminal domain, UNP residues 131-190
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Staphylococcus aureus (strain NCTC 8325) (bacteria)
Strain: NCTC 8325 / Gene: hpf, SAOUHSC_00767 / Variant: SaHPF / Plasmid: pGS21A / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): star / References: UniProt: Q2G055

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic13D 1H-13C NOESY aliphatic
121isotropic13D 1H-13C NOESY aromatic
131isotropic13D 1H-15N NOESY
141isotropic12D 1H-1H NOESY

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Sample preparation

DetailsType: solution
Contents: 2.0 mM [U-99% 13C; U-99% 15N] C-terminal domain of SaHPF, 50 mM potassium phosphate, 250 mM NH4Cl, 90% H2O/10% D2O
Label: 15N, 13C / Solvent system: 90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
2.0 mMC-terminal domain of SaHPF[U-99% 13C; U-99% 15N]1
50 mMpotassium phosphatenatural abundance1
250 mMNH4Clnatural abundance1
Sample conditionsIonic strength: 250 mM / Label: conditions_1 / pH: 7.6 / Pressure: 1 bar / Temperature: 308 K

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NMR measurement

NMR spectrometerType: Bruker AVANCE III / Manufacturer: Bruker / Model: AVANCE III / Field strength: 700 MHz

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Processing

NMR software
NameDeveloperClassification
ARIALinge, O'Donoghue and Nilgeschemical shift assignment
ARIALinge, O'Donoghue and Nilgespeak picking
TopSpinBruker Biospinprocessing
CNSBrunger, Adams, Clore, Gros, Nilges and Readstructure calculation
PROCHECK / PROCHECK-NMRLaskowski, MacArthur, Smith, Jones, Hutchinson, Morris, Moss and Thorntondata analysis
CNSBrunger, Adams, Clore, Gros, Nilges and Readrefinement
RefinementMethod: simulated annealing / Software ordinal: 1
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 500 / Conformers submitted total number: 10

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