EMDB-31659: T.thermophilus 30S ribosome with KsgA, class K5

Basic information

Entry

Database: EMDB / ID: EMD-31659

• Title: T.thermophilus 30S ribosome with KsgA, class K5
• Map data: B-factor sharpened map from the postprocess routine in Relion

Sample

• Complex: 30S ribosomal with bound KsgA, a RNA methyltransferase
  • Complex: 30S ribosomal subunit
    • RNA: x 1 types
    • Protein or peptide: x 20 types
  • Complex: KsgA
    • Protein or peptide: x 1 types
• Ligand: x 1 types

• Keywords: 30S subunit / KsgA / rRNA methyltransferase / RIBOSOME

Function / homology

Function:

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 / ribonucleoprotein complex / translation / mRNA binding / RNA binding / zinc ion binding / metal ion binding / cytosol / cytoplasm

Domain/homology:

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 S5, N-terminal, conserved site / Ribosomal protein S5 signature. / Ribosomal protein S2, conserved site / 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

Component:

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

• Biological species: Thermus thermophilus HB8 (bacteria) / Bacillus subtilis (strain 168) (bacteria)
• Method: single particle reconstruction / cryo EM / Resolution: 3.3 Å
• Authors: Raina R / Singh J

Funding support

India, 3 items

Organization	Grant number	Country
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

• Citation: Journal: 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-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

Deposition	Aug 9, 2021	-
Header (metadata) release	Apr 6, 2022	-
Map release	Apr 6, 2022	-
Update	Jun 12, 2024	-
Current status	Jun 12, 2024	Processing site: PDBj / Status: Released

Map

• File: Download / File: emd_31659.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: B-factor sharpened map from the postprocess routine in Relion
• Voxel size: X=Y=Z: 1.38 Å

Density

Contour Level	By AUTHOR: 0.075
Minimum - Maximum	-0.1484187 - 0.46798885
Average (Standard dev.)	-0.0000017484523 (±0.014882398)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 320 320 320 Spacing 320 320 320
Cell	A=B=C: 441.6 Å α=β=γ: 90.0 °

Supplemental data

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

• File: emd_31659_additional_1.map
• Annotation: One of the maps from multibody refinement from Relion. This map was used in model building.

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

• File: emd_31659_additional_2.map
• Annotation: One of the maps from multibody refinement from Relion. This map was used in model building.

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

• File: emd_31659_additional_3.map
• Annotation: One of the maps from multibody refinement from Relion. This map was used in model building.

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

• File: emd_31659_additional_4.map
• Annotation: This map was calculated using LocSpiral. This map was used in model building.

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

• File: emd_31659_additional_5.map
• Annotation: Composite map of the multibody refinement from Phenix software. This map was used in model building.

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

• File: emd_31659_half_map_1.map
• Annotation: One of the half maps from 3D refine, used in the postprocess routine to obtain the primary map.

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

• File: emd_31659_half_map_2.map
• Annotation: One of the half maps from 3D refine, used in the postprocess routine to obtain the primary map.

Sample components

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

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

Components

• Complex: 30S ribosomal with bound KsgA, a RNA methyltransferase
  • Complex: 30S 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 Z
    • 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 Thx
  • Complex: KsgA
    • Protein or peptide: Ribosomal RNA small subunit methyltransferase A
• Ligand: ZINC ION

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

• Supramolecule: Name: 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 weight: Theoretical: 720 KDa

Supramolecule #2: 30S ribosomal subunit

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

Supramolecule #3: KsgA

• Supramolecule: Name: KsgA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #21
• Source (natural): Organism: Bacillus subtilis (strain 168) (bacteria)

Macromolecule #1: 16s ribosomal RNA

• Macromolecule: Name: 16s ribosomal RNA / type: rna / ID: 1 / Number of copies: 1
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 493.958281 KDa

Sequence

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

GENBANK: GENBANK: M26923.1

Macromolecule #2: 30S ribosomal protein S2

• Macromolecule: Name: 30S ribosomal protein S2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 29.317703 KDa

Sequence

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

UniProtKB: Small ribosomal subunit protein uS2

Macromolecule #3: 30S ribosomal protein S3

• Macromolecule: Name: 30S ribosomal protein S3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 26.751076 KDa

Sequence

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

UniProtKB: Small ribosomal subunit protein uS3

Macromolecule #4: 30S ribosomal protein S4

• Macromolecule: Name: 30S ribosomal protein S4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 24.373447 KDa

Sequence

String:

MGRYIGPVCR LCRREGVKLY LKGERCYSPK CAMERRPYPP GQHGQKRARR PSDYAVRLRE KQKLRRIYGI SERQFRNLFE EASKKKGVT GSVFLGLLES RLDNVVYRLG FAVSRRQARQ LVRHGHITVN GRRVDLPSYR VRPGDEIAVA EKSRNLELIR Q NLEAMKGR KVGPWLSLDV EGMKGKFLRL PDREDLALPV NEQLVIEFYS R

UniProtKB: Small ribosomal subunit protein uS4

Macromolecule #5: 30S ribosomal protein S5

• Macromolecule: Name: 30S ribosomal protein S5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 17.583416 KDa

Sequence

String:

MPETDFEEKM ILIRRTARMQ AGGRRFRFGA LVVVGDRQGR VGLGFGKAPE VPLAVQKAGY YARRNMVEVP LQNGTIPHEI EVEFGASKI VLKPAAPGTG VIAGAVPRAI LELAGVTDIL TKELGSRNPI NIAYATMEAL RQLRTKADVE RLRKGEAHAQ A QG

UniProtKB: Small ribosomal subunit protein uS5

Macromolecule #6: 30S ribosomal protein S6

• Macromolecule: Name: 30S ribosomal protein S6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 11.988753 KDa

Sequence

String:

MRRYEVNIVL NPNLDQSQLA LEKEIIQRAL ENYGARVEKV EELGLRRLAY PIAKDPQGYF LWYQVEMPED RVNDLARELR IRDNVRRVM VVKSQEPFLA NA

UniProtKB: Small ribosomal subunit protein bS6

Macromolecule #7: 30S ribosomal protein S7

• Macromolecule: Name: 30S ribosomal protein S7 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 18.050973 KDa

Sequence

String:

MARRRRAEVR QLQPDLVYGD VLVTAFINKI MRDGKKNLAA RIFYDACKII QEKTGQEPLK VFKQAVENVK PRMEVRSRRV GGANYQVPM EVSPRRQQSL ALRWLVQAAN QRPERRAAVR IAHELMDAAE GKGGAVKKKE DVERMAEANR AYAHYRW

UniProtKB: Small ribosomal subunit protein uS7

Macromolecule #8: 30S ribosomal protein S8

• Macromolecule: Name: 30S ribosomal protein S8 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 15.86857 KDa

Sequence

String:

MLTDPIADML TRIRNATRVY KESTDVPASR FKEEILRILA REGFIKGYER VDVDGKPYLR VYLKYGPRRQ GPDPRPEQVI HHIRRISKP GRRVYVGVKE IPRVRRGLGI AILSTSKGVL TDREARKLGV GGELICEVW

UniProtKB: Small ribosomal subunit protein uS8

Macromolecule #9: 30S ribosomal protein S9

• Macromolecule: Name: 30S ribosomal protein S9 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 14.410614 KDa

Sequence

String:

MEQYYGTGRR KEAVARVFLR PGNGKVTVNG QDFNEYFQGL VRAVAALEPL RAVDALGHFD AYITVRGGGK SGQIDAIKLG IARALVQYN PDYRAKLKPL GFLTRDARVV ERKKYGKHKA RRAPQYSKR

UniProtKB: Small ribosomal subunit protein uS9

Macromolecule #10: 30S ribosomal protein S10

• Macromolecule: Name: 30S ribosomal protein S10 / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 11.954968 KDa

Sequence

String:

MPKIRIKLRG FDHKTLDASA QKIVEAARRS GAQVSGPIPL PTRVRRFTVI RGPFKHKDSR EHFELRTHNR LVDIINPNRK TIEQLMTLD LPTGVEIEIK TVGGGR

UniProtKB: Small ribosomal subunit protein uS10

Macromolecule #11: 30S ribosomal protein S11

• Macromolecule: Name: 30S ribosomal protein S11 / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 13.737868 KDa

Sequence

String:

MAKKPSKKKV KRQVASGRAY IHASYNNTIV TITDPDGNPI TWSSGGVIGY KGSRKGTPYA AQLAALDAAK KAMAYGMQSV DVIVRGTGA GREQAIRALQ ASGLQVKSIV DDTPVPHNGC RPKKKFRKAS

UniProtKB: Small ribosomal subunit protein uS11

Macromolecule #12: 30S ribosomal protein S12

• Macromolecule: Name: 30S ribosomal protein S12 / type: protein_or_peptide / ID: 12 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 14.637384 KDa

Sequence

String:

MPTINQLVRK GREKVRKKSK VPALKGAPFR RGVCTVVRTV TPKKPNSALR KVAKVRLTSG YEVTAYIPGE GHNLQEHSVV LIRGGRVKD LPGVRYHIVR GVYDAAGVKD RKKSRSKYGT KKPKEAAKTA AKK

UniProtKB: Small ribosomal subunit protein uS12

Macromolecule #13: 30S ribosomal protein S13

• Macromolecule: Name: 30S ribosomal protein S13 / type: protein_or_peptide / ID: 13 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 14.338861 KDa

Sequence

String:

MARIAGVEIP RNKRVDVALT YIYGIGKARA KEALEKTGIN PATRVKDLTE AEVVRLREYV ENTWKLEGEL RAEVAANIKR LMDIGCYRG LRHRRGLPVR GQRTRTNART RKGPRKTVAG KKKAPRK

UniProtKB: Small ribosomal subunit protein uS13

Macromolecule #14: 30S ribosomal protein S14 type Z

• Macromolecule: Name: 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 weight: Theoretical: 7.158725 KDa

Sequence

String:

MARKALIEKA KRTPKFKVRA YTRCVRCGRA RSVYRFFGLC RICLRELAHK GQLPGVRKAS W

UniProtKB: Small ribosomal subunit protein uS14

Macromolecule #15: 30S ribosomal protein S15

• Macromolecule: Name: 30S ribosomal protein S15 / type: protein_or_peptide / ID: 15 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 10.578407 KDa

Sequence

String:

MPITKEEKQK VIQEFARFPG DTGSTEVQVA LLTLRINRLS EHLKVHKKDH HSHRGLLMMV GQRRRLLRYL QREDPERYRA LIEKLGIRG

UniProtKB: Small ribosomal subunit protein uS15

Macromolecule #16: 30S ribosomal protein S16

• Macromolecule: Name: 30S ribosomal protein S16 / type: protein_or_peptide / ID: 16 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 10.409983 KDa

Sequence

String:

MVKIRLARFG SKHNPHYRIV VTDARRKRDG KYIEKIGYYD PRKTTPDWLK VDVERARYWL SVGAQPTDTA RRLLRQAGVF RQEAREGA

UniProtKB: Small ribosomal subunit protein bS16

Macromolecule #17: 30S ribosomal protein S17

• Macromolecule: Name: 30S ribosomal protein S17 / type: protein_or_peptide / ID: 17 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 12.325655 KDa

Sequence

String:

MPKKVLTGVV VSDKMQKTVT VLVERQFPHP LYGKVIKRSK KYLAHDPEEK YKLGDVVEII ESRPISKRKR FRVLRLVESG RMDLVEKYL IRRQNYESLS KRGGKA

UniProtKB: Small ribosomal subunit protein uS17

Macromolecule #18: 30S ribosomal protein S18

• Macromolecule: Name: 30S ribosomal protein S18 / type: protein_or_peptide / ID: 18 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 10.258299 KDa

Sequence

String:

MSTKNAKPKK EAQRRPSRKA KVKATLGEFD LRDYRNVEVL KRFLSETGKI LPRRRTGLSA KEQRILAKTI KRARILGLLP FTEKLVRK

UniProtKB: Small ribosomal subunit protein bS18

Macromolecule #19: 30S ribosomal protein S19

• Macromolecule: Name: 30S ribosomal protein S19 / type: protein_or_peptide / ID: 19 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 10.605464 KDa

Sequence

String:

MPRSLKKGVF VDDHLLEKVL ELNAKGEKRL IKTWSRRSTI VPEMVGHTIA VYNGKQHVPV YITENMVGHK LGEFAPTRTY RGHGKEAKA TKKK

UniProtKB: Small ribosomal subunit protein uS19

Macromolecule #20: 30S ribosomal protein S20

• Macromolecule: Name: 30S ribosomal protein S20 / type: protein_or_peptide / ID: 20 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 11.736143 KDa

Sequence

String:

MAQKKPKRNL SALKRHRQSL KRRLRNKAKK SAIKTLSKKA IQLAQEGKAE EALKIMRKAE SLIDKAAKGS TLHKNAAARR KSRLMRKVR QLLEAAGAPL IGGGLSA

UniProtKB: Small ribosomal subunit protein bS20

Macromolecule #21: Ribosomal RNA small subunit methyltransferase A

• Macromolecule: Name: Ribosomal RNA small subunit methyltransferase A / type: protein_or_peptide / ID: 21 / Number of copies: 1 / Enantiomer: LEVO; EC number: 16S rRNA (adenine1518-N6/adenine1519-N6)-dimethyltransferase
• Source (natural): Organism: Bacillus subtilis (strain 168) (bacteria)
• Molecular weight: Theoretical: 33.588812 KDa
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

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

UniProtKB: Ribosomal RNA small subunit methyltransferase A

Macromolecule #22: 30S ribosomal protein Thx

• Macromolecule: Name: 30S ribosomal protein Thx / type: protein_or_peptide / ID: 22 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Thermus thermophilus HB8 (bacteria)
• Molecular weight: Theoretical: 3.35003 KDa

Sequence

String:

MGKGDRRTRR GKIWRGTYGK YRPRKKK

UniProtKB: Small ribosomal subunit protein bTHX

Macromolecule #23: ZINC ION

• Macromolecule: Name: ZINC ION / type: ligand / ID: 23 / Number of copies: 2 / Formula: ZN
• Molecular weight: Theoretical: 65.409 Da

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

Buffer

pH: 7.5
Component:

Concentration	Name
50.0 mM	Hepes
40.0 mM	Ammonium chloride
4.0 mM	Magensium acetate
6.0 mM	Beta mercaptoethanol

• Grid: Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
• Vitrification: Cryogen 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

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Image recording: Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Average exposure time: 2.0 sec. / Average electron dose: 43.0 e/Ų; Details: Total number of frames is 30. Each frame had 1.43 e/A2
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 50.0 µm / Calibrated magnification: 101449 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 59000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Startup model: Type of model: OTHER
• Final reconstruction: Number classes used: 1 / Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 102297
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD
• Final angle assignment: Type: MAXIMUM LIKELIHOOD
• Final 3D classification: Software - Name: RELION (ver. 3.1)

Atomic model buiding 1

Initial model

PDB ID:

4b3r

Chain - Source name: PDB / Chain - Initial model type: experimental model

• Details: Phenix was used in real space for initial refinement. The final refinements was performed with REFMAC within ccpem
• Refinement: Space: RECIPROCAL / Protocol: OTHER / Overall B value: 75.2

Output model

PDB-7v2p:
T.thermophilus 30S ribosome with KsgA, class K5