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- PDB-7v2m: T.thermophilus 30S ribosome with KsgA, class K1k4 -

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Basic information

Entry
Database: PDB / ID: 7v2m
TitleT.thermophilus 30S ribosome with KsgA, class K1k4
Components
  • (30S ribosomal protein ...) x 20
  • 16s ribosomal RNA
  • Ribosomal RNA small subunit methyltransferase A
KeywordsRIBOSOME / 30S subunit / KsgA / rRNA methyltransferase
Function / homology
Function and homology information


16S rRNA (adenine1518-N6/adenine1519-N6)-dimethyltransferase / 16S rRNA (adenine(1518)-N(6)/adenine(1519)-N(6))-dimethyltransferase activity / rRNA (adenine-N6,N6-)-dimethyltransferase activity / rRNA methylation / ribosomal small subunit assembly / small ribosomal subunit / cytosolic small ribosomal subunit / tRNA binding / rRNA binding / ribosome ...16S rRNA (adenine1518-N6/adenine1519-N6)-dimethyltransferase / 16S rRNA (adenine(1518)-N(6)/adenine(1519)-N(6))-dimethyltransferase activity / rRNA (adenine-N6,N6-)-dimethyltransferase activity / rRNA methylation / ribosomal small subunit assembly / small ribosomal subunit / cytosolic small ribosomal subunit / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / translation / ribonucleoprotein complex / mRNA binding / RNA binding / zinc ion binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
rRNA adenine dimethylase-like, C-terminal / Ribosomal RNA adenine dimethylase / Ribosomal RNA adenine methylase transferase, conserved site / Ribosomal RNA adenine methylase transferase, N-terminal / Ribosomal RNA adenine dimethylases signature. / Ribosomal RNA adenine dimethylases / Ribosomal RNA adenine methyltransferase KsgA/Erm / Ribosomal RNA adenine dimethylase / rRNA adenine N(6)-methyltransferase family profile. / 30S ribosomal protein Thx ...rRNA adenine dimethylase-like, C-terminal / Ribosomal RNA adenine dimethylase / Ribosomal RNA adenine methylase transferase, conserved site / Ribosomal RNA adenine methylase transferase, N-terminal / Ribosomal RNA adenine dimethylases signature. / Ribosomal RNA adenine dimethylases / Ribosomal RNA adenine methyltransferase KsgA/Erm / Ribosomal RNA adenine dimethylase / rRNA adenine N(6)-methyltransferase family profile. / 30S ribosomal protein Thx / 30S ribosomal protein Thx / 30S ribosomal protein / Ribosomal protein S14, type Z / Ribosomal protein S14/S29 / Ribosomal protein S3, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S19, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, 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. / Ribosomal protein S16 / Ribosomal protein S16 domain superfamily / Ribosomal protein S16 / 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 S10, conserved site / Ribosomal protein S10 signature. / Ribosomal protein S3, conserved site / Ribosomal protein S3 signature. / Ribosomal protein S14, conserved site / Ribosomal protein S14 signature. / Ribosomal protein S2 signature 1. / KH domain / Type-2 KH domain profile. / K Homology domain, type 2 / Ribosomal protein S3, C-terminal / Ribosomal protein S3, C-terminal domain / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein S15/S19, conserved site / Ribosomal protein S19 signature. / Ribosomal protein S10 / : / Ribosomal protein S19/S15 / Ribosomal protein S19/S15, superfamily / Ribosomal protein S19 / Ribosomal protein S2, conserved site / Ribosomal protein S5, N-terminal, conserved site / Ribosomal protein S5 signature. / Ribosomal protein S7, conserved site / Ribosomal protein S7 signature. / Ribosomal protein S2 / Ribosomal protein S2, flavodoxin-like domain superfamily / Ribosomal protein S2 / Ribosomal protein S5 / Ribosomal protein S17, conserved site / Ribosomal protein S17 signature. / S5 double stranded RNA-binding domain profile. / K homology domain superfamily, prokaryotic type / Ribosomal protein S5, N-terminal / Ribosomal protein S5, N-terminal domain / Ribosomal protein S5, C-terminal / Ribosomal protein S5, C-terminal domain / Ribosomal protein S13, conserved site / Ribosomal protein S13 signature. / Ribosomal protein S13 / 30s ribosomal protein S13, C-terminal / Ribosomal protein S13/S18 / Ribosomal protein S13 family profile. / Ribosomal protein S8 signature. / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S4, conserved site / Ribosomal protein S4 signature. / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S4/S9, N-terminal / Ribosomal protein S15 signature. / Ribosomal protein S14 / Ribosomal protein S14p/S29e / Ribosomal protein S4/S9 / K homology domain-like, alpha/beta
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS7 / Ribosomal RNA small subunit methyltransferase A ...: / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS7 / Ribosomal RNA small subunit methyltransferase A / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein bTHX / Small ribosomal subunit protein uS15 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein bS18
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
Thermus thermophilus HB8 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsRaina, R. / Singh, J. / Anand, R. / Vinothkumar, K.R.
Funding support India, 3items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014 India
Science and Engineering Research Board (SERB)RJN-094/2017 India
Department of Biotechnology (DBT, India)IA/S/19/1/504293 India
CitationJournal: ACS Chem Biol / Year: 2022
Title: Decoding the Mechanism of Specific RNA Targeting by Ribosomal Methyltransferases.
Authors: Juhi Singh / Rahul Raina / Kutti R Vinothkumar / Ruchi Anand /
Abstract: Methylation of specific nucleotides is integral for ribosomal biogenesis and also serves as a common mechanism to confer antibiotic resistance by pathogenic bacteria. Here, by determining the high- ...Methylation of specific nucleotides is integral for ribosomal biogenesis and also serves as a common mechanism to confer antibiotic resistance by pathogenic bacteria. Here, by determining the high-resolution structure of the 30S-KsgA complex by cryo-electron microscopy, a state was captured, where KsgA juxtaposes between helices h44 and h45 of the 30S ribosome, separating them, thereby enabling remodeling of the surrounded rRNA and allowing the cognate site to enter the methylation pocket. With the structure as a guide, several mutant versions of the ribosomes, where interacting bases in the catalytic helix h45 and surrounding helices h44, h24, and h27, were mutated and evaluated for their methylation efficiency revealing factors that direct the enzyme to its cognate site with high fidelity. The biochemical studies show that the three-dimensional environment of the ribosome enables the interaction of select loop regions in KsgA with the ribosome helices paramount to maintain selectivity.
History
DepositionAug 9, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 6, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 16s ribosomal RNA
B: 30S ribosomal protein S2
C: 30S ribosomal protein S3
D: 30S ribosomal protein S4
E: 30S ribosomal protein S5
F: 30S ribosomal protein S6
G: 30S ribosomal protein S7
H: 30S ribosomal protein S8
I: 30S ribosomal protein S9
J: 30S ribosomal protein S10
K: 30S ribosomal protein S11
L: 30S ribosomal protein S12
M: 30S ribosomal protein S13
N: 30S ribosomal protein S14 type Z
O: 30S ribosomal protein S15
P: 30S ribosomal protein S16
Q: 30S ribosomal protein S17
R: 30S ribosomal protein S18
S: 30S ribosomal protein S19
T: 30S ribosomal protein S20
U: Ribosomal RNA small subunit methyltransferase A
V: 30S ribosomal protein Thx
W: Ribosomal RNA small subunit methyltransferase A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)850,70325
Polymers850,57223
Non-polymers1312
Water00
1


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

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Components

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

#2: Protein 30S ribosomal protein S2


Mass: 29317.703 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P80371
#3: Protein 30S ribosomal protein S3


Mass: 26751.076 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P80372
#4: Protein 30S ribosomal protein S4


Mass: 24373.447 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P80373
#5: Protein 30S ribosomal protein S5


Mass: 17583.416 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SHQ5
#6: Protein 30S ribosomal protein S6 / TS9


Mass: 11988.753 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SLP8
#7: Protein 30S ribosomal protein S7


Mass: 18050.973 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P17291
#8: Protein 30S ribosomal protein S8


Mass: 15868.570 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P0DOY9
#9: Protein 30S ribosomal protein S9


Mass: 14410.614 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P80374
#10: Protein 30S ribosomal protein S10


Mass: 11954.968 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SHN7
#11: Protein 30S ribosomal protein S11


Mass: 13737.868 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P80376
#12: Protein 30S ribosomal protein S12


Mass: 14637.384 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SHN3
#13: Protein 30S ribosomal protein S13


Mass: 14338.861 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P80377
#14: Protein 30S ribosomal protein S14 type Z


Mass: 7158.725 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P0DOY6
#15: Protein 30S ribosomal protein S15


Mass: 10578.407 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SJ76
#16: Protein 30S ribosomal protein S16


Mass: 10409.983 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SJH3
#17: Protein 30S ribosomal protein S17


Mass: 12325.655 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P0DOY7
#18: Protein 30S ribosomal protein S18


Mass: 10258.299 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SLQ0
#19: Protein 30S ribosomal protein S19


Mass: 10605.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SHP2
#20: Protein 30S ribosomal protein S20


Mass: 11736.143 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: P80380
#22: Protein/peptide 30S ribosomal protein Thx / S31


Mass: 3350.030 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SIH3

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RNA chain / Protein / Non-polymers , 3 types, 5 molecules AUW

#1: RNA chain 16s ribosomal RNA


Mass: 493958.281 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: GenBank: M26923.1
#21: Protein Ribosomal RNA small subunit methyltransferase A / 16S rRNA (adenine(1518)-N(6)/adenine(1519)-N(6))-dimethyltransferase / 16S rRNA dimethyladenosine ...16S rRNA (adenine(1518)-N(6)/adenine(1519)-N(6))-dimethyltransferase / 16S rRNA dimethyladenosine transferase / 16S rRNA dimethylase / S-adenosylmethionine-6-N' / N'-adenosyl(rRNA) dimethyltransferase


Mass: 33588.812 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (strain 168) (bacteria)
Gene: rsmA, ksgA, BSU00420 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P37468, 16S rRNA (adenine1518-N6/adenine1519-N6)-dimethyltransferase
#23: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN

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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
IDNameTypeDetailsEntity IDParent-IDSource
130S ribosomal with bound KsgA, a RNA methyltransferaseRIBOSOME30S ribosomal subunit purified from delta KsgA strain and KsgA recombinantly purified has been added after isolation for complex formation#1-#220MULTIPLE SOURCES
230S ribosomal subunitRIBOSOME#1-#20, #221NATURAL
3KsgACOMPLEX#211RECOMBINANT
Molecular weightValue: 0.771 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Thermus thermophilus HB8 (bacteria)300852
23Bacillus subtilis (strain 168) (bacteria)224308
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
150 mMHepes1
240 mMAmmonium chloride1
34 mMMagensium acetate1
46 mMBeta mercaptoethanol1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: ~2 micromolar concentration of ribosome+KsgA was used during freezing
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: After application of the complex, 15 seconds wait time was given before blotting.

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 59000 X / Calibrated magnification: 101449 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 43 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1
Details: Total number of frames is 30. Each frame had 1.43 e/A2
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails
1Gautomatchparticle selection
2EPUimage acquisition
4Gctf1.06CTF correctionDetermine CTF value
5RELION3.1CTF correctionCTF correction
8Coot0.9.5model fitting
9UCSF Chimera1.15model fitting
11PHENIX1.19.2-4158model refinement
12REFMAC5.8.0267model refinement
15RELION3.1classification
16RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50689 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 102.6 / Protocol: OTHER / Space: RECIPROCAL
Details: Phenix was used in real space for initial refinement. The final refinements was performed with REFMAC within ccpem
Atomic model buildingPDB-ID: 4B3R

4b3r


Accession code: 4B3R / Source name: PDB / Type: experimental model

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