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Entry
Database: PDB / ID: 8why
TitleCryo- EM structure of Mycobacterium smegmatis 50S ribosomal subunit (body 1) of 70S ribosome and RafH.
Components
  • (50S ribosomal protein ...) x 26
  • 23S rRNA23S ribosomal RNA
  • 5S rRNA5S ribosomal RNA
KeywordsRIBOSOME / protein synthesis / Mycobacterium smegmatis / hibernation promotion factor / RafH / hypoxia stress / Cryo- EM / Single particle reconstruction
Function / homology
Function and homology information


large ribosomal subunit / transferase activity / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / ribonucleoprotein complex / translation / metal ion binding / cytoplasm
Similarity search - Function
Ribosomal protein L31 type A / Ribosomal protein L31 signature. / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / : / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. ...Ribosomal protein L31 type A / Ribosomal protein L31 signature. / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / : / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L17 signature. / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L28/L24 superfamily / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / Ribosomal protein L28 / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L5, bacterial-type / Ribosomal protein L6, bacterial-type / Ribosomal protein L18, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein L20 signature. / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein L14P, bacterial-type / Ribosomal protein L34, conserved site / Ribosomal protein L34 signature. / Ribosomal protein L22, bacterial/chloroplast-type / Ribosomal protein L35 / Ribosomal protein L35 superfamily / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L35 / Ribosomal L28 family / Ribosomal protein L33 / Ribosomal protein L33 / Ribosomal protein L28/L24 / Ribosomal protein L33 superfamily / : / Ribosomal protein L30, bacterial-type / Ribosomal protein L18 / Ribosomal L18 of archaea, bacteria, mitoch. and chloroplast / L28p-like / Ribosomal protein L20 / Ribosomal protein L20 / Ribosomal protein L20, C-terminal / Ribosomal protein L21 / Ribosomal protein L27 / Ribosomal L27 protein / Ribosomal protein L19 / Ribosomal protein L19 superfamily / Ribosomal protein L19 / Ribosomal proteins 50S L24/mitochondrial 39S L24 / Ribosomal protein L17 / Ribosomal protein L17 superfamily / Ribosomal protein L17 / Ribosomal protein L21-like / L21-like superfamily / Ribosomal prokaryotic L21 protein / Ribosomal L32p protein family / Ribosomal protein L24 / Ribosomal protein L32p / Ribosomal protein L34 / Ribosomal protein L34 / Ribosomal protein L13, bacterial-type / Ribosomal protein L23/L25, conserved site / Ribosomal protein L3, bacterial/organelle-type / Ribosomal protein L15, bacterial-type / 50S ribosomal protein uL4 / Ribosomal protein L23 signature. / Ribosomal protein L30, conserved site / Ribosomal protein L29, conserved site / Ribosomal protein L30 signature. / Ribosomal protein L5, N-terminal / Ribosomal protein L15, conserved site / Ribosomal protein L5, C-terminal / Ribosomal protein L5 / Ribosomal protein L5 domain superfamily / Ribosomal protein L2, conserved site / Ribosomal protein L2 signature. / Ribosomal protein L29 signature. / Ribosomal protein L5 / Ribosomal protein L6, alpha-beta domain / Ribosomal protein L6 / ribosomal L5P family C-terminus / Ribosomal protein L6, alpha-beta domain superfamily / Ribosomal protein L6 / Ribosomal protein L2, domain 3 / Ribosomal protein L14P, conserved site
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Large ribosomal subunit protein bL33A / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein uL2 ...: / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Large ribosomal subunit protein bL33A / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL5 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein bL31 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL9 / Large ribosomal subunit protein bL34
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 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 23, 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
E: 50S ribosomal protein L2
F: 50S ribosomal protein L3
G: 50S ribosomal protein L4
H: 50S ribosomal protein L5
I: 50S ribosomal protein L6
J: 50S ribosomal protein L9
M: 50S ribosomal protein L13
N: 50S ribosomal protein L14
O: 50S ribosomal protein L15
Q: 50S ribosomal protein L17
R: 50S ribosomal protein L18
S: 50S ribosomal protein L19
T: 50S ribosomal protein L20
U: 50S ribosomal protein L21
V: 50S ribosomal protein L22
W: 50S ribosomal protein L23
X: 50S ribosomal protein L24
Z: 50S ribosomal protein L27
1: 50S ribosomal protein L28
2: 50S ribosomal protein L29
3: 50S ribosomal protein L30
5: 50S ribosomal protein L32
6: 50S ribosomal protein L33A
7: 50S ribosomal protein L34
8: 50S ribosomal protein L35
4: 50S ribosomal protein L31
A: 23S rRNA
B: 5S rRNA


Theoretical massNumber of molelcules
Total (without water)1,406,69128
Polymers1,406,69128
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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50S ribosomal protein ... , 26 types, 26 molecules EFGHIJMNOQRSTUVWXZ12356784

#1: Protein 50S ribosomal protein L2 /


Mass: 30425.945 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: A0QSD4
#2: Protein 50S ribosomal protein L3 /


Mass: 23011.393 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: A0QSD1
#3: Protein 50S ribosomal protein L4 /


Mass: 22920.949 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: A0QSD2
#4: Protein 50S ribosomal protein L5 /


Mass: 21152.328 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: A0QSG1
#5: Protein 50S ribosomal protein L6 /


Mass: 19483.361 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: A0QSG4
#6: Protein 50S ribosomal protein L9 /


Mass: 15949.253 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: A0R7F6
#7: Protein 50S ribosomal protein L13 /


Mass: 16146.689 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: A0QSP8
#8: Protein 50S ribosomal protein L14 /


Mass: 13400.625 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: A0QSF9
#9: Protein 50S ribosomal protein L15 /


Mass: 15602.244 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: A0QSG8
#10: Protein 50S ribosomal protein L17 /


Mass: 21892.246 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: A0QSL9
#11: Protein 50S ribosomal protein L18 /


Mass: 13711.728 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: A0QSG5
#12: Protein 50S ribosomal protein L19 /


Mass: 12892.806 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: A0QV42
#13: Protein 50S ribosomal protein L20 /


Mass: 14494.693 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: A0QYU6
#14: Protein 50S ribosomal protein L21 /


Mass: 11055.894 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: A0R151
#15: Protein 50S ribosomal protein L22 /


Mass: 16353.600 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: A0QSD6
#16: Protein 50S ribosomal protein L23 /


Mass: 11047.836 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: A0QSD3
#17: Protein 50S ribosomal protein L24 /


Mass: 11228.946 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: A0QSG0
#18: Protein 50S ribosomal protein L27 /


Mass: 9249.582 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: A0R150
#19: Protein 50S ribosomal protein L28 /


Mass: 6891.016 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: A0QV03
#20: Protein 50S ribosomal protein L29 /


Mass: 8790.895 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: A0QSD9
#21: Protein 50S ribosomal protein L30 /


Mass: 7022.147 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: A0QSG7
#22: Protein 50S ribosomal protein L32 /


Mass: 6467.719 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: A0R3I9
#23: Protein 50S ribosomal protein L33A / Ribosome


Mass: 6468.501 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: A0QS39
#24: Protein/peptide 50S ribosomal protein L34 /


Mass: 5522.580 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: A0R7K0
#25: Protein 50S ribosomal protein L35 /


Mass: 7298.505 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: A0QYU7
#26: 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

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RNA chain , 2 types, 2 molecules AB

#27: RNA chain 23S rRNA / 23S ribosomal RNA


Mass: 1011834.750 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
#28: RNA chain 5S rRNA / 5S ribosomal RNA


Mass: 38061.816 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: mc(2)155 / References: GenBank: 118168627

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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: 70S ribosome + RafH protein / Type: RIBOSOME / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Strain: mc(2)155
Buffer solutionpH: 7.4 / Details: 20mM HEPES, 20mM MgCl2, 100mM NH4Cl, 3mM DTT
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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
4CTFFINDctffind/4.1.14CTF correction
7Cootv.0.9.3model fitting
9RELION3.1.4initial Euler assignment
11RELION3.1.4classification
12RELION3.1.43D reconstruction
13PHENIXmodel refinementphenix.real_space_refinement
CTF correctionDetails: CTF correction in Relion 3.1.4 / Type: NONE
Particle selectionNum. of particles selected: 1202461
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110934 / 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_d0.00498052
ELECTRON MICROSCOPYf_angle_d0.579148081
ELECTRON MICROSCOPYf_dihedral_angle_d15.5840820
ELECTRON MICROSCOPYf_chiral_restr0.03619096
ELECTRON MICROSCOPYf_plane_restr0.0057120

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