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- EMDB-31656: T.thermophilus 30S ribosome with KsgA, class K1k4 -

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Basic information

Entry
Database: EMDB / ID: EMD-31656
TitleT.thermophilus 30S ribosome with KsgA, class K1k4
Map dataB-factor sharpened map from the postprocess routine in Relion
Sample
  • Complex: 30S ribosomal with bound KsgA, a RNA methyltransferase
    • Complex: 30S ribosomal subunitProkaryotic small ribosomal subunit
      • RNA: x 1 types
      • Protein or peptide: x 20 types
    • Complex: KsgA
      • Protein or peptide: x 1 types
  • Ligand: x 1 types
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 / small ribosomal subunit / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / ribonucleoprotein complex ...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 / small ribosomal subunit / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / ribonucleoprotein complex / translation / 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 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. / 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 S14, 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 S14 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 / Ribosomal protein S5, N-terminal domain / Ribosomal protein S8
Similarity search - Domain/homology
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 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 speciesThermus thermophilus HB8 (bacteria) / Bacillus subtilis (strain 168) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsRaina R / Singh J / Anand R / Vinothkumar KR
Funding support India, 3 items
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, 2021-
Header (metadata) releaseApr 6, 2022-
Map releaseApr 6, 2022-
UpdateApr 27, 2022-
Current statusApr 27, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_31656.png

Downloads & links

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Map

FileDownload / File: emd_31656.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationB-factor sharpened map from the postprocess routine in Relion
Voxel sizeX=Y=Z: 1.38 Å
Density
Contour LevelBy AUTHOR: 0.078
Minimum - Maximum-0.13385887 - 0.5619429
Average (Standard dev.)6.567384e-06 (±0.01568623)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 441.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: One of the maps from multibody refinement from...

Fileemd_31656_additional_1.map
AnnotationOne of the maps from multibody refinement from Relion. This map was used in model building.
Projections & Slices
AxesZYX

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Additional map: One of the maps from multibody refinement from...

Fileemd_31656_additional_2.map
AnnotationOne of the maps from multibody refinement from Relion. This map was used in model building.
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: This map was calculated using LocSpiral. This map...

Fileemd_31656_additional_3.map
AnnotationThis map was calculated using LocSpiral. This map was used in model building.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: One of the maps from multibody refinement from...

Fileemd_31656_additional_4.map
AnnotationOne of the maps from multibody refinement from Relion. This map was used in model building.
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Additional map: Composite map of the multibody refinement from Phenix...

Fileemd_31656_additional_5.map
AnnotationComposite map of the multibody refinement from Phenix software. This map was used in model building.
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: One of the half maps from 3D refine,...

Fileemd_31656_half_map_1.map
AnnotationOne of the half maps from 3D refine, used in the postprocess routine to obtain the primary map.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: One of the half maps from 3D refine,...

Fileemd_31656_half_map_2.map
AnnotationOne of the half maps from 3D refine, used in the postprocess routine to obtain the primary map.
Projections & Slices
AxesZYX

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Sample components

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Entire : 30S ribosomal with bound KsgA, a RNA methyltransferase

EntireName: 30S ribosomal with bound KsgA, a RNA methyltransferase
Components
  • Complex: 30S ribosomal with bound KsgA, a RNA methyltransferase
    • Complex: 30S ribosomal subunitProkaryotic small ribosomal subunit
      • RNA: 16s ribosomal RNA
      • Protein or peptide: 30S ribosomal protein S2
      • Protein or peptide: 30S ribosomal protein S3
      • Protein or peptide: 30S ribosomal protein S4
      • Protein or peptide: 30S ribosomal protein S5
      • Protein or peptide: 30S ribosomal protein S6
      • Protein or peptide: 30S ribosomal protein S7
      • Protein or peptide: 30S ribosomal protein S8
      • Protein or peptide: 30S ribosomal protein S9
      • Protein or peptide: 30S ribosomal protein S10
      • Protein or peptide: 30S ribosomal protein S11
      • Protein or peptide: 30S ribosomal protein S12
      • Protein or peptide: 30S ribosomal protein S13
      • Protein or peptide: 30S ribosomal protein S14 type ZRibosome
      • Protein or peptide: 30S ribosomal protein S15
      • Protein or peptide: 30S ribosomal protein S16
      • Protein or peptide: 30S ribosomal protein S17
      • Protein or peptide: 30S ribosomal protein S18
      • Protein or peptide: 30S ribosomal protein S19
      • Protein or peptide: 30S ribosomal protein S20
      • Protein or peptide: 30S ribosomal protein ThxRibosome
    • Complex: KsgA
      • Protein or peptide: Ribosomal RNA small subunit methyltransferase A
  • Ligand: ZINC ION

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Supramolecule #1: 30S ribosomal with bound KsgA, a RNA methyltransferase

SupramoleculeName: 30S ribosomal with bound KsgA, a RNA methyltransferase
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#22
Details: 30S ribosomal subunit purified from delta KsgA strain and KsgA recombinantly purified has been added after isolation for complex formation
Molecular weightTheoretical: 771 KDa

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Supramolecule #2: 30S ribosomal subunit

SupramoleculeName: 30S ribosomal subunit / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#20, #22
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)

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Supramolecule #3: KsgA

SupramoleculeName: KsgA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #21
Source (natural)Organism: Bacillus subtilis (strain 168) (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: 16s ribosomal RNA

MacromoleculeName: 16s ribosomal RNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 493.958281 KDa
SequenceString: UUUGUUGGAG AGUUUGAUCC UGGCUCAGGG UGAACGCUGG CGGCGUGCCU AAGACAUGCA AGUCGUGCGG GCCGCGGGGU UUUACUCCG UGGUCAGCGG CGGACGGGUG AGUAACGCGU GGGUGACCUA CCCGGAAGAG GGGGACAACC CGGGGAAACU C GGGCUAAU ...String:
UUUGUUGGAG AGUUUGAUCC UGGCUCAGGG UGAACGCUGG CGGCGUGCCU AAGACAUGCA AGUCGUGCGG GCCGCGGGGU UUUACUCCG UGGUCAGCGG CGGACGGGUG AGUAACGCGU GGGUGACCUA CCCGGAAGAG GGGGACAACC CGGGGAAACU C GGGCUAAU CCCCCAUGUG GACCCGCCCC UUGGGGUGUG UCCAAAGGGC UUUGCCCGCU UCCGGAUGGG CCCGCGUCCC AU CAGCUAG UUGGUGGGGU AAUGGCCCAC CAAGGCGACG ACGGGUAGCC GGUCUGAGAG GAUGGCCGGC CACAGGGGCA CUG AGACAC GGGCCCCACU CCUACGGGAG GCAGCAGUUA GGAAUCUUCC GCAAUGGGCG CAAGCCUGAC GGAGCGACGC CGCU UGGAG GAAGAAGCCC UUCGGGGUGU AAACUCCUGA ACCCGGGACG AAACCCCCGA CGAGGGGACU GACGGUACCG GGGUA AUAG CGCCGGCCAA CUCCGUGCCA GCAGCCGCGG UAAUACGGAG GGCGCGAGCG UUACCCGGAU UCACUGGGCG UAAAGG GCG UGUAGGCGGC CUGGGGCGUC CCAUGUGAAA GACCACGGCU CAACCGUGGG GGAGCGUGGG AUACGCUCAG GCUAGAC GG UGGGAGAGGG UGGUGGAAUU CCCGGAGUAG CGGUGAAAUG CGCAGAUACC GGGAGGAACG CCGAUGGCGA AGGCAGCC A CCUGGUCCAC CCGUGACGCU GAGGCGCGAA AGCGUGGGGA GCAAACCGGA UUAGAUACCC GGGUAGUCCA CGCCCUAAA CGAUGCGCGC UAGGUCUCUG GGUCUCCUGG GGGCCGAAGC UAACGCGUUA AGCGCGCCGC CUGGGGAGUA CGGCCGCAAG GCUGAAACU CAAAGGAAUU GACGGGGGCC CGCACAAGCG GUGGAGCAUG UGGUUUAAUU CGAAGCAACG CGAAGAACCU U ACCAGGCC UUGACAUGCU AGGGAACCCG GGUGAAAGCC UGGGGUGCCC CGCGAGGGGA GCCCUAGCAC AGGUGCUGCA UG GCCGUCG UCAGCUCGUG CCGUGAGGUG UUGGGUUAAG UCCCGCAACG AGCGCAACCC CCGCCGUUAG UUGCCAGCGG UUC GGCCGG GCACUCUAAC GGGACUGCCC GCGAAAGCGG GAGGAAGGAG GGGACGACGU CUGGUCAGCA UGGCCCUUAC GGCC UGGGC GACACACGUG CUACAAUGCC CACUACAAAG CGAUGCCACC CGGCAACGGG GAGCUAAUCG CAAAAAGGUG GGCCC AGUU CGGAUUGGGG UCUGCAACCC GACCCCAUGA AGCCGGAAUC GCUAGUAAUC GCGGAUCAGC CAUGCCGCGG UGAAUA CGU UCCCGGGCCU UGUACACACC GCCCGUCACG CCAUGGGAGC GGGCUCUACC CGAAGUCGCC GGGAGCCUAC GGGCAGG CG CCGAGGGUAG GGCCCGUGAC UGGGGCGAAG UCGUAACAAG GUAGCUGUAC CGGAAGGUGC GGCUGGAUCA CCUCCUUU C U

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Macromolecule #2: 30S ribosomal protein S2

MacromoleculeName: 30S ribosomal protein S2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 29.317703 KDa
SequenceString: MPVEITVKEL LEAGVHFGHE RKRWNPKFAR YIYAERNGIH IIDLQKTMEE LERTFRFIED LAMRGGTILF VGTKKQAQDI VRMEAERAG MPYVNQRWLG GMLTNFKTIS QRVHRLEELE ALFASPEIEE RPKKEQVRLK HELERLQKYL SGFRLLKRLP D AIFVVDPT ...String:
MPVEITVKEL LEAGVHFGHE RKRWNPKFAR YIYAERNGIH IIDLQKTMEE LERTFRFIED LAMRGGTILF VGTKKQAQDI VRMEAERAG MPYVNQRWLG GMLTNFKTIS QRVHRLEELE ALFASPEIEE RPKKEQVRLK HELERLQKYL SGFRLLKRLP D AIFVVDPT KEAIAVREAR KLFIPVIALA DTDSDPDLVD YIIPGNDDAI RSIQLILSRA VDLIIQARGG VVEPSPSYAL VQ EAEATET PEGESEVEA

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Macromolecule #3: 30S ribosomal protein S3

MacromoleculeName: 30S ribosomal protein S3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 26.751076 KDa
SequenceString: MGNKIHPIGF RLGITRDWES RWYAGKKQYR HLLLEDQRIR GLLEKELYSA GLARVDIERA ADNVAVTVHV AKPGVVIGRG GERIRVLRE ELAKLTGKNV ALNVQEVQNP NLSAPLVAQR VAEQIERRFA VRRAIKQAVQ RVMESGAKGA KVIVSGRIGG A EQARTEWA ...String:
MGNKIHPIGF RLGITRDWES RWYAGKKQYR HLLLEDQRIR GLLEKELYSA GLARVDIERA ADNVAVTVHV AKPGVVIGRG GERIRVLRE ELAKLTGKNV ALNVQEVQNP NLSAPLVAQR VAEQIERRFA VRRAIKQAVQ RVMESGAKGA KVIVSGRIGG A EQARTEWA AQGRVPLHTL RANIDYGFAL ARTTYGVLGV KAYIFLGEVI GGQKPKARPE LPKAEERPRR RRPAVRVKKE E

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Macromolecule #4: 30S ribosomal protein S4

MacromoleculeName: 30S ribosomal protein S4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 24.373447 KDa
SequenceString: MGRYIGPVCR LCRREGVKLY LKGERCYSPK CAMERRPYPP GQHGQKRARR PSDYAVRLRE KQKLRRIYGI SERQFRNLFE EASKKKGVT GSVFLGLLES RLDNVVYRLG FAVSRRQARQ LVRHGHITVN GRRVDLPSYR VRPGDEIAVA EKSRNLELIR Q NLEAMKGR ...String:
MGRYIGPVCR LCRREGVKLY LKGERCYSPK CAMERRPYPP GQHGQKRARR PSDYAVRLRE KQKLRRIYGI SERQFRNLFE EASKKKGVT GSVFLGLLES RLDNVVYRLG FAVSRRQARQ LVRHGHITVN GRRVDLPSYR VRPGDEIAVA EKSRNLELIR Q NLEAMKGR KVGPWLSLDV EGMKGKFLRL PDREDLALPV NEQLVIEFYS R

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Macromolecule #5: 30S ribosomal protein S5

MacromoleculeName: 30S ribosomal protein S5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 17.583416 KDa
SequenceString:
MPETDFEEKM ILIRRTARMQ AGGRRFRFGA LVVVGDRQGR VGLGFGKAPE VPLAVQKAGY YARRNMVEVP LQNGTIPHEI EVEFGASKI VLKPAAPGTG VIAGAVPRAI LELAGVTDIL TKELGSRNPI NIAYATMEAL RQLRTKADVE RLRKGEAHAQ A QG

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Macromolecule #6: 30S ribosomal protein S6

MacromoleculeName: 30S ribosomal protein S6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 11.988753 KDa
SequenceString:
MRRYEVNIVL NPNLDQSQLA LEKEIIQRAL ENYGARVEKV EELGLRRLAY PIAKDPQGYF LWYQVEMPED RVNDLARELR IRDNVRRVM VVKSQEPFLA NA

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Macromolecule #7: 30S ribosomal protein S7

MacromoleculeName: 30S ribosomal protein S7 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 18.050973 KDa
SequenceString:
MARRRRAEVR QLQPDLVYGD VLVTAFINKI MRDGKKNLAA RIFYDACKII QEKTGQEPLK VFKQAVENVK PRMEVRSRRV GGANYQVPM EVSPRRQQSL ALRWLVQAAN QRPERRAAVR IAHELMDAAE GKGGAVKKKE DVERMAEANR AYAHYRW

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Macromolecule #8: 30S ribosomal protein S8

MacromoleculeName: 30S ribosomal protein S8 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 15.86857 KDa
SequenceString:
MLTDPIADML TRIRNATRVY KESTDVPASR FKEEILRILA REGFIKGYER VDVDGKPYLR VYLKYGPRRQ GPDPRPEQVI HHIRRISKP GRRVYVGVKE IPRVRRGLGI AILSTSKGVL TDREARKLGV GGELICEVW

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Macromolecule #9: 30S ribosomal protein S9

MacromoleculeName: 30S ribosomal protein S9 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 14.410614 KDa
SequenceString:
MEQYYGTGRR KEAVARVFLR PGNGKVTVNG QDFNEYFQGL VRAVAALEPL RAVDALGHFD AYITVRGGGK SGQIDAIKLG IARALVQYN PDYRAKLKPL GFLTRDARVV ERKKYGKHKA RRAPQYSKR

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Macromolecule #10: 30S ribosomal protein S10

MacromoleculeName: 30S ribosomal protein S10 / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 11.954968 KDa
SequenceString:
MPKIRIKLRG FDHKTLDASA QKIVEAARRS GAQVSGPIPL PTRVRRFTVI RGPFKHKDSR EHFELRTHNR LVDIINPNRK TIEQLMTLD LPTGVEIEIK TVGGGR

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Macromolecule #11: 30S ribosomal protein S11

MacromoleculeName: 30S ribosomal protein S11 / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 13.737868 KDa
SequenceString:
MAKKPSKKKV KRQVASGRAY IHASYNNTIV TITDPDGNPI TWSSGGVIGY KGSRKGTPYA AQLAALDAAK KAMAYGMQSV DVIVRGTGA GREQAIRALQ ASGLQVKSIV DDTPVPHNGC RPKKKFRKAS

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Macromolecule #12: 30S ribosomal protein S12

MacromoleculeName: 30S ribosomal protein S12 / type: protein_or_peptide / ID: 12 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 14.637384 KDa
SequenceString:
MPTINQLVRK GREKVRKKSK VPALKGAPFR RGVCTVVRTV TPKKPNSALR KVAKVRLTSG YEVTAYIPGE GHNLQEHSVV LIRGGRVKD LPGVRYHIVR GVYDAAGVKD RKKSRSKYGT KKPKEAAKTA AKK

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Macromolecule #13: 30S ribosomal protein S13

MacromoleculeName: 30S ribosomal protein S13 / type: protein_or_peptide / ID: 13 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 14.338861 KDa
SequenceString:
MARIAGVEIP RNKRVDVALT YIYGIGKARA KEALEKTGIN PATRVKDLTE AEVVRLREYV ENTWKLEGEL RAEVAANIKR LMDIGCYRG LRHRRGLPVR GQRTRTNART RKGPRKTVAG KKKAPRK

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Macromolecule #14: 30S ribosomal protein S14 type Z

MacromoleculeName: 30S ribosomal protein S14 type Z / type: protein_or_peptide / ID: 14 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 7.158725 KDa
SequenceString:
MARKALIEKA KRTPKFKVRA YTRCVRCGRA RSVYRFFGLC RICLRELAHK GQLPGVRKAS W

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Macromolecule #15: 30S ribosomal protein S15

MacromoleculeName: 30S ribosomal protein S15 / type: protein_or_peptide / ID: 15 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 10.578407 KDa
SequenceString:
MPITKEEKQK VIQEFARFPG DTGSTEVQVA LLTLRINRLS EHLKVHKKDH HSHRGLLMMV GQRRRLLRYL QREDPERYRA LIEKLGIRG

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Macromolecule #16: 30S ribosomal protein S16

MacromoleculeName: 30S ribosomal protein S16 / type: protein_or_peptide / ID: 16 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 10.409983 KDa
SequenceString:
MVKIRLARFG SKHNPHYRIV VTDARRKRDG KYIEKIGYYD PRKTTPDWLK VDVERARYWL SVGAQPTDTA RRLLRQAGVF RQEAREGA

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Macromolecule #17: 30S ribosomal protein S17

MacromoleculeName: 30S ribosomal protein S17 / type: protein_or_peptide / ID: 17 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 12.325655 KDa
SequenceString:
MPKKVLTGVV VSDKMQKTVT VLVERQFPHP LYGKVIKRSK KYLAHDPEEK YKLGDVVEII ESRPISKRKR FRVLRLVESG RMDLVEKYL IRRQNYESLS KRGGKA

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Macromolecule #18: 30S ribosomal protein S18

MacromoleculeName: 30S ribosomal protein S18 / type: protein_or_peptide / ID: 18 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 10.258299 KDa
SequenceString:
MSTKNAKPKK EAQRRPSRKA KVKATLGEFD LRDYRNVEVL KRFLSETGKI LPRRRTGLSA KEQRILAKTI KRARILGLLP FTEKLVRK

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Macromolecule #19: 30S ribosomal protein S19

MacromoleculeName: 30S ribosomal protein S19 / type: protein_or_peptide / ID: 19 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 10.605464 KDa
SequenceString:
MPRSLKKGVF VDDHLLEKVL ELNAKGEKRL IKTWSRRSTI VPEMVGHTIA VYNGKQHVPV YITENMVGHK LGEFAPTRTY RGHGKEAKA TKKK

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Macromolecule #20: 30S ribosomal protein S20

MacromoleculeName: 30S ribosomal protein S20 / type: protein_or_peptide / ID: 20 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 11.736143 KDa
SequenceString:
MAQKKPKRNL SALKRHRQSL KRRLRNKAKK SAIKTLSKKA IQLAQEGKAE EALKIMRKAE SLIDKAAKGS TLHKNAAARR KSRLMRKVR QLLEAAGAPL IGGGLSA

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Macromolecule #21: Ribosomal RNA small subunit methyltransferase A

MacromoleculeName: Ribosomal RNA small subunit methyltransferase A / type: protein_or_peptide / ID: 21 / Number of copies: 2 / Enantiomer: LEVO
EC number: 16S rRNA (adenine1518-N6/adenine1519-N6)-dimethyltransferase
Source (natural)Organism: Bacillus subtilis (strain 168) (bacteria)
Molecular weightTheoretical: 33.588812 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: HHHHHHMNKD IATPIRTKEI LKKYGFSFKK SLGQNFLIDT NILNRIVDHA EVTEKTGVIE IGPGIGALTE QLAKRAKKVV AFEIDQRLL PILKDTLSPY ENVTVIHQDV LKADVKSVIE EQFQDCDEIM VVANLPYYVT TPIIMKLLEE HLPLKGIVVM L QKEVAERM ...String:
HHHHHHMNKD IATPIRTKEI LKKYGFSFKK SLGQNFLIDT NILNRIVDHA EVTEKTGVIE IGPGIGALTE QLAKRAKKVV AFEIDQRLL PILKDTLSPY ENVTVIHQDV LKADVKSVIE EQFQDCDEIM VVANLPYYVT TPIIMKLLEE HLPLKGIVVM L QKEVAERM AADPSSKEYG SLSIAVQFYT EAKTVMIVPK TVFVPQPNVD SAVIRLILRD GPAVDVENES FFFQLIKASF AQ RRKTLLN NLVNNLPEGK AQKSTIEQVL EETNIDGKRR GESLSIEEFA ALSNGLYKAL F

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Macromolecule #22: 30S ribosomal protein Thx

MacromoleculeName: 30S ribosomal protein Thx / type: protein_or_peptide / ID: 22 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 3.35003 KDa
SequenceString:
MGKGDRRTRR GKIWRGTYGK YRPRKKK

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Macromolecule #23: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 23 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationName
50.0 mMHepes
40.0 mMAmmonium chloride
4.0 mMMagensium acetate
6.0 mMBeta mercaptoethanol
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: After application of the complex, 15 seconds wait time was given before blotting..
Details~2 micromolar concentration of ribosome+KsgA was used during freezing

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 101449 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 14.0 µm / Number grids imaged: 1 / Average exposure time: 2.0 sec. / Average electron dose: 43.0 e/Å2
Details: Total number of frames is 30. Each frame had 1.43 e/A2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware:
Namedetails
Gctf (ver. 1.06)Determine CTF value
RELION (ver. 3.1)CTF correction
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 50689
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4b3r

DetailsPhenix was used in real space for initial refinement. The final refinements was performed with REFMAC within ccpem
RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 102.6
Output model

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

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