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Entry
Database: PDB / ID: 8wif
TitleCryo- EM structure of Mycobacterium smegmatis 30S ribosomal subunit (body 2) of 70S ribosome and RafH.
Components
  • (30S ribosomal protein ...) x 20
  • 16S rRNA
  • 50S ribosomal protein L31
  • Ribosome hibernation promotion factor RafH
KeywordsRIBOSOME / protein synthesis / Mycobacterium smegmatis / hibernation promotion factor / HPF / RafH / hypoxia stress / Cryo- EM / Single particle reconstruction
Function / homology
Function and homology information


primary metabolic process / regulation of translation / small ribosomal subunit / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / ribonucleoprotein complex / translation / mRNA binding ...primary metabolic process / regulation of translation / small ribosomal subunit / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / ribonucleoprotein complex / translation / mRNA binding / metal ion binding / cytoplasm
Similarity search - Function
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 / Mitochondrial mRNA-processing protein COX24, C-terminal / Mitochondrial mRNA-processing protein COX24, C-terminal / Mitochondrial domain of unknown function (DUF1713) / Ribosomal protein S14, bacterial/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 / Mitochondrial mRNA-processing protein COX24, C-terminal / Mitochondrial mRNA-processing protein COX24, C-terminal / Mitochondrial domain of unknown function (DUF1713) / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L31 type A / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein L31 signature. / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein S3, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S19, bacterial-type / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S4, bacterial-type / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein S2, bacteria/mitochondria/plastid / Ribosomal protein S18, conserved site / Ribosomal protein S18 signature. / L28p-like / Ribosomal protein S16 / Ribosomal protein S16 / Ribosomal protein S16 domain superfamily / Ribosomal protein S15, bacterial-type / Ribosomal protein S6 / Ribosomal protein S6 / Ribosomal protein S6 superfamily / Ribosomal protein S12, bacterial-type / Translation elongation factor EF1B/ribosomal protein S6 / Ribosomal protein S18 / Ribosomal protein S18 / Ribosomal protein S18 superfamily / K Homology domain / K homology RNA-binding domain / Ribosomal protein S3, conserved site / Ribosomal protein S10, conserved site / : / K Homology domain, type 2 / Ribosomal protein S3, C-terminal / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein S15/S19, conserved site / KH domain / Ribosomal protein S19/S15 / Ribosomal protein S19/S15, superfamily / Ribosomal protein S10 / Ribosomal protein S3, C-terminal domain / Ribosomal protein S3 signature. / Ribosomal protein S10 signature. / Ribosomal protein S7, conserved site / Ribosomal protein S17, conserved site / K homology domain superfamily, prokaryotic type / Ribosomal protein S19 / Ribosomal protein S2 signature 1. / Ribosomal protein S13, conserved site / Ribosomal protein S2, conserved site / Ribosomal protein S13 / 30s ribosomal protein S13, C-terminal / Ribosomal protein S2 / Ribosomal protein S2, flavodoxin-like domain superfamily / Ribosomal protein S2 / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S14 / Ribosomal protein S4/S9, N-terminal / Ribosomal protein S4, conserved site / Type-2 KH domain profile. / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S13/S18 / Ribosomal protein S4/S9 / Ribosomal protein S19 signature. / K homology domain-like, alpha/beta / Ribosomal protein S14p/S29e / Ribosomal protein S8 / Ribosomal protein S8 superfamily / Ribosomal protein S5, N-terminal, conserved site / Ribosomal protein S5 signature. / Ribosomal protein S5 / Ribosomal protein S5, N-terminal / Ribosomal S11, conserved site / Ribosomal protein S7 signature. / Ribosomal protein S10p/S20e / Ribosomal protein S13-like, H2TH / S5 double stranded RNA-binding domain profile. / Ribosomal protein S5, C-terminal / Ribosomal protein S9, conserved site
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein bS6 / Conserved domain protein / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS7 ...: / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein bS6 / Conserved domain protein / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS7 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS17 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS15 / Sigma 54 modulation/S30EA ribosomal protein C-terminal domain-containing protein / Small ribosomal subunit protein bS20 / Large ribosomal subunit protein bL31 / Small ribosomal subunit protein uS14A / Small ribosomal subunit protein bS18B
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsKumar, N. / Sharma, S. / Kaushal, P.S.
Funding support India, 1items
OrganizationGrant numberCountry
Science and Engineering Research Board (SERB) India
CitationJournal: Nat Commun / Year: 2024
Title: Cryo- EM structure of the mycobacterial 70S ribosome in complex with ribosome hibernation promotion factor RafH.
Authors: Niraj Kumar / Shivani Sharma / Prem S Kaushal /
Abstract: Ribosome hibernation is a key survival strategy bacteria adopt under environmental stress, where a protein, hibernation promotion factor (HPF), transitorily inactivates the ribosome. Mycobacterium ...Ribosome hibernation is a key survival strategy bacteria adopt under environmental stress, where a protein, hibernation promotion factor (HPF), transitorily inactivates the ribosome. Mycobacterium tuberculosis encounters hypoxia (low oxygen) as a major stress in the host macrophages, and upregulates the expression of RafH protein, which is crucial for its survival. The RafH, a dual domain HPF, an orthologue of bacterial long HPF (HPF), hibernates ribosome in 70S monosome form, whereas in other bacteria, the HPF induces 70S ribosome dimerization and hibernates its ribosome in 100S disome form. Here, we report the cryo- EM structure of M. smegmatis, a close homolog of M. tuberculosis, 70S ribosome in complex with the RafH factor at an overall 2.8 Å resolution. The N- terminus domain (NTD) of RafH binds to the decoding center, similarly to HPF NTD. In contrast, the C- terminus domain (CTD) of RafH, which is larger than the HPF CTD, binds to a distinct site at the platform binding center of the ribosomal small subunit. The two domain-connecting linker regions, which remain mostly disordered in earlier reported HPF structures, interact mainly with the anti-Shine Dalgarno sequence of the 16S rRNA.
History
DepositionSep 24, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 28, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
a: 16S rRNA
v: 30S ribosomal protein S22
d: 30S ribosomal protein S3
e: 30S ribosomal protein S4
f: 30S ribosomal protein S5
g: 30S ribosomal protein S6
h: 30S ribosomal protein S7
i: 30S ribosomal protein S8
j: 30S ribosomal protein S9
k: 30S ribosomal protein S10
l: 30S ribosomal protein S11
m: 30S ribosomal protein S12
n: 30S ribosomal protein S13
o: 30S ribosomal protein S14A
p: 30S ribosomal protein S15
q: 30S ribosomal protein S16
r: 30S ribosomal protein S17
s: 30S ribosomal protein S18B
t: 30S ribosomal protein S19
u: 30S ribosomal protein S20
c: 30S ribosomal protein S2
x: 50S ribosomal protein L31
w: Ribosome hibernation promotion factor RafH


Theoretical massNumber of molelcules
Total (without water)837,62323
Polymers837,62323
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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RNA chain , 1 types, 1 molecules a

#1: RNA chain 16S rRNA /


Mass: 495373.656 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: MC2 155 / References: GenBank: 118168627

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30S ribosomal protein ... , 20 types, 20 molecules vdefghijklmnopqrstuc

#2: Protein/peptide 30S ribosomal protein S22 /


Mass: 4164.300 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0QR10
#3: Protein 30S ribosomal protein S3 /


Mass: 30191.227 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: MC2 155 / References: UniProt: A0QSD7
#4: Protein 30S ribosomal protein S4 /


Mass: 23415.787 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0A8B4QH64
#5: Protein 30S ribosomal protein S5 /


Mass: 21946.090 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0QSG6
#6: Protein 30S ribosomal protein S6 /


Mass: 10991.637 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0A8B4QKV6
#7: Protein 30S ribosomal protein S7 /


Mass: 17660.375 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0QS97
#8: Protein 30S ribosomal protein S8 /


Mass: 14492.638 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0QSG3
#9: Protein 30S ribosomal protein S9 /


Mass: 16794.365 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0QSP9
#10: Protein 30S ribosomal protein S10 /


Mass: 11454.313 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0QSD0
#11: Protein 30S ribosomal protein S11 /


Mass: 14671.762 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0QSL6
#12: Protein 30S ribosomal protein S12 /


Mass: 13896.366 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0QS96
#13: Protein 30S ribosomal protein S13 /


Mass: 14249.619 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0QSL5
#14: Protein 30S ribosomal protein S14A / Ribosome / 30S ribosomal protein S14


Mass: 11792.728 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0R550
#15: Protein 30S ribosomal protein S15 /


Mass: 10368.097 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0QVQ3
#16: Protein 30S ribosomal protein S16 /


Mass: 16795.207 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0QV37
#17: Protein 30S ribosomal protein S17 /


Mass: 11127.002 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0QSE0
#18: Protein 30S ribosomal protein S18B / Ribosome


Mass: 9524.188 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0R7F7
#19: Protein 30S ribosomal protein S19 /


Mass: 10800.602 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0QSD5
#20: Protein 30S ribosomal protein S20 /


Mass: 9556.104 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0R102
#21: Protein 30S ribosomal protein S2 /


Mass: 30145.230 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0QVB8

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Protein , 2 types, 2 molecules xw

#22: Protein 50S ribosomal protein L31 /


Mass: 8312.485 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: UniProt: A0R215
#23: Protein Ribosome hibernation promotion factor RafH


Mass: 29898.936 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycolicibacterium smegmatis MC2 155 (bacteria)
Gene: MSMEG_3935 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41 / References: UniProt: A0QZ86

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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

Component
IDNameTypeEntity IDParent-IDSource
170S ribosome + RafH proteinRIBOSOMEall0MULTIPLE SOURCES
270S ribsomeRIBOSOME#1-#221NATURAL
3RafHRIBOSOME#231RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Strain: mc(2)155
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C41(DE3) / Plasmid: pET28a(+)
Buffer solutionpH: 7.4 / Details: 20mM HEPES, 100mM NH4Cl, 20mM MgCl2, 3mM DTT,
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 70S ribosome + RafH protein
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 1.34 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 12343

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3.1.4particle selection
2EPUimage acquisitionThermo Fisher Scientific
4RELION3.1.4CTF correction
5CTFFINDctffind/4.1.14CTF correction
8Cootv.0.9.3model fitting
10RELION3.1.4initial Euler assignment
11RELION3.1.4final Euler assignment
12RELION3.1.4classification
13RELION3.1.43D reconstruction
14PHENIXmodel refinementphenix.real_space_refinement
CTF correctionDetails: CTF correction in Relion3.1.4 / Type: NONE
Particle selectionNum. of particles selected: 1202461
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110934 / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Details: phenix.real_space_refinement
Atomic model buildingPDB-ID: 8WHX

8whx


Accession code: 8WHX / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00656972
ELECTRON MICROSCOPYf_angle_d0.6284464
ELECTRON MICROSCOPYf_dihedral_angle_d14.45921097
ELECTRON MICROSCOPYf_chiral_restr0.03910660
ELECTRON MICROSCOPYf_plane_restr0.0055103

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