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- EMDB-72071: Bacterial ribosomal 2'-O-methyltransferase RsmI in complex with t... -

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Basic information

Entry
Database: EMDB / ID: EMD-72071
TitleBacterial ribosomal 2'-O-methyltransferase RsmI in complex with the small ribosomal subunit
Map dataFull map of the RsmI-30S complex
Sample
  • Complex: Complex of bacterial 2'-O-methyltransferase RsmI and the small ribosomal subunit.
    • Complex: RsmI (16S rRNA 2'-O-methyltransferase)
      • Protein or peptide: x 1 types
    • Complex: E. coli 30S subunit
      • RNA: x 1 types
      • Protein or peptide: x 20 types
  • Ligand: x 3 types
KeywordsRibosome / methylation / 30S subunit / helix 44 / methyltransferase
Function / homology
Function and homology information


16S rRNA (cytidine1402-2'-O)-methyltransferase / rRNA (cytosine-2'-O-)-methyltransferase activity / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / four-way junction DNA binding / negative regulation of translational initiation / regulation of mRNA stability ...16S rRNA (cytidine1402-2'-O)-methyltransferase / rRNA (cytosine-2'-O-)-methyltransferase activity / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / four-way junction DNA binding / negative regulation of translational initiation / regulation of mRNA stability / mRNA regulatory element binding translation repressor activity / positive regulation of RNA splicing / regulation of DNA-templated transcription elongation / transcription elongation factor complex / DNA endonuclease activity / transcription antitermination / DNA-templated transcription termination / maintenance of translational fidelity / mRNA 5'-UTR binding / regulation of translation / ribosome biogenesis / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / small ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / cytoplasmic translation / tRNA binding / negative regulation of translation / rRNA binding / structural constituent of ribosome / ribosome / translation / response to antibiotic / mRNA binding / RNA binding / zinc ion binding / membrane / cytoplasm / cytosol
Similarity search - Function
: / RsmI C-terminal HTH domain / SAM-dependent methyltransferase RsmI, conserved site / RsmI AdoMet-dependent methyltransferase protein family signature. / rRNA small subunit methyltransferase I / Tetrapyrrole methylase, subdomain 2 / Tetrapyrrole methylase / Tetrapyrrole (Corrin/Porphyrin) Methylases / Tetrapyrrole methylase, subdomain 1 / Tetrapyrrole methylase superfamily ...: / RsmI C-terminal HTH domain / SAM-dependent methyltransferase RsmI, conserved site / RsmI AdoMet-dependent methyltransferase protein family signature. / rRNA small subunit methyltransferase I / Tetrapyrrole methylase, subdomain 2 / Tetrapyrrole methylase / Tetrapyrrole (Corrin/Porphyrin) Methylases / Tetrapyrrole methylase, subdomain 1 / Tetrapyrrole methylase superfamily / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein S14, bacterial/plastid / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / Ribosomal protein S3, bacterial-type / Ribosomal protein S19, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / 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 S9, bacterial/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 S2 signature 2. / Ribosomal protein S3, conserved site / Ribosomal protein S3 signature. / Ribosomal protein S10, conserved site / Ribosomal protein S10 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 S5, N-terminal, conserved site / Ribosomal protein S5 signature. / Ribosomal protein S19/S15 / Ribosomal protein S19/S15, superfamily / Ribosomal protein S19 / Ribosomal protein S2, conserved site / Ribosomal protein S2 / Ribosomal protein S2, flavodoxin-like domain superfamily / Ribosomal protein S2 / Ribosomal protein S7, conserved site / Ribosomal protein S7 signature. / : / K homology domain superfamily, prokaryotic type / Ribosomal protein S5 / S5 double stranded RNA-binding domain profile. / Ribosomal protein S5, N-terminal / Ribosomal protein S5, C-terminal / Ribosomal protein S5, N-terminal domain / Ribosomal protein S5, C-terminal domain / Ribosomal protein S17, conserved site / Ribosomal protein S17 signature. / 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 S14 / Ribosomal protein S14p/S29e / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S4/S9 N-terminal domain / Ribosomal protein S4/S9, N-terminal
Similarity search - Domain/homology
Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein bS20 ...Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein uS15 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Ribosomal RNA small subunit methyltransferase I / Small ribosomal subunit protein bS21
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli BW25113 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.42 Å
AuthorsBarmada MI / Conn GL
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI088025 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)F31 AI186518 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM135060 United States
CitationJournal: bioRxiv / Year: 2025
Title: Mechanism of 30S subunit recognition and modification by the conserved bacterial ribosomal RNA methyltransferase RsmI.
Authors: Mohamed I Barmada / Erin N McGinity / Suparno Nandi / Debayan Dey / Natalia Zelinskaya / George M Harris / Lindsay R Comstock / Christine M Dunham / Graeme L Conn /
Abstract: Ribosomal RNA (rRNA) modifications are important for ribosome function and can influence bacterial susceptibility to ribosome-targeting antibiotics. The universally conserved 16S rRNA nucleotide ...Ribosomal RNA (rRNA) modifications are important for ribosome function and can influence bacterial susceptibility to ribosome-targeting antibiotics. The universally conserved 16S rRNA nucleotide C1402, for example, is the only 2'--methylated nucleotide in the bacterial small (30S) ribosomal subunit and this modification fine tunes the shape and structure of the peptidyl tRNA binding site. The Cm1402 modification is incorporated by the conserved bacterial 16S rRNA methyltransferase RsmI, but it is unclear how RsmI is able to recognize its 30S substrate and specifically modify its buried target nucleotide. We determined a 2.42 Å resolution cryo-EM structure of the RsmI-30S complex and, with accompanying functional analyses, show that RsmI anchors itself to the 30S subunit through multiple contacts with a conserved 16S rRNA tertiary surface present only in the assembled subunit. This positions RsmI to induce an extensive h44 distortion to access C1402 that is unprecedented among 16S rRNA methyltransferases characterized to date. These analyses also reveal an essential contribution to 30S subunit interaction made by the previously structurally uncharacterized RsmI C-terminal domain, RsmI-induced RNA-RNA interactions with C1402, and an unappreciated dependence on a divalent metal ion for catalysis, marking RsmI as the first of a distinct class of metal- and SAM-dependent RNA -methyltransferases. This study significantly expands our mechanistic understanding of how intrinsic bacterial methyltransferases like RsmI modify their rRNA targets. Further, recognition of distant ribosome features and extensive unfolding of a critical rRNA functional center point to a potential role in accurate 30S subunit biogenesis.
History
DepositionAug 11, 2025-
Header (metadata) releaseSep 24, 2025-
Map releaseSep 24, 2025-
UpdateSep 24, 2025-
Current statusSep 24, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_72071.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map of the RsmI-30S complex
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 448 pix.
= 369.6 Å
0.83 Å/pix.
x 448 pix.
= 369.6 Å
0.83 Å/pix.
x 448 pix.
= 369.6 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density
Contour LevelBy AUTHOR: 0.035
Minimum - Maximum-0.11109048 - 0.40037102
Average (Standard dev.)0.00045314583 (±0.010223618)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 369.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_72071_msk_1.map
Projections & Slices
AxesZYX

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Additional map: truncated, focused map of RsmI and its 30S binding site.

Fileemd_72071_additional_1.map
Annotationtruncated, focused map of RsmI and its 30S binding site.
Projections & Slices
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Additional map: half map B of truncated focused map of...

Fileemd_72071_additional_2.map
Annotationhalf map B of truncated focused map of RsmI and its 30S binding surface.
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AxesZYX

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Additional map: half map A of truncated focused map of...

Fileemd_72071_additional_3.map
Annotationhalf map A of truncated focused map of RsmI and its 30S binding surface.
Projections & Slices
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Additional map: DeepEMenhancer-sharpened version of the truncated map of RsmI...

Fileemd_72071_additional_4.map
AnnotationDeepEMenhancer-sharpened version of the truncated map of RsmI and its 30S binding surface.
Projections & Slices
AxesZYX

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Additional map: DeepEMenhancer-sharpened version of the full map of the...

Fileemd_72071_additional_5.map
AnnotationDeepEMenhancer-sharpened version of the full map of the RsmI-30S complex
Projections & Slices
AxesZYX

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Additional map: low-resolution version of the full map of the RsmI-30S complex.

Fileemd_72071_additional_6.map
Annotationlow-resolution version of the full map of the RsmI-30S complex.
Projections & Slices
AxesZYX

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Half map: halfmap B of the full of the RsmI-30S complex

Fileemd_72071_half_map_1.map
Annotationhalfmap B of the full of the RsmI-30S complex
Projections & Slices
AxesZYX

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Half map: halfmap A of the full map of the RsmI-30S complex

Fileemd_72071_half_map_2.map
Annotationhalfmap A of the full map of the RsmI-30S complex
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Sample components

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Entire : Complex of bacterial 2'-O-methyltransferase RsmI and the small ri...

EntireName: Complex of bacterial 2'-O-methyltransferase RsmI and the small ribosomal subunit.
Components
  • Complex: Complex of bacterial 2'-O-methyltransferase RsmI and the small ribosomal subunit.
    • Complex: RsmI (16S rRNA 2'-O-methyltransferase)
      • Protein or peptide: Ribosomal RNA small subunit methyltransferase I
    • Complex: E. coli 30S subunit
      • RNA: 16S rRNA
      • Protein or peptide: Small ribosomal subunit protein uS2
      • Protein or peptide: Small ribosomal subunit protein uS3
      • Protein or peptide: Small ribosomal subunit protein uS4
      • Protein or peptide: Small ribosomal subunit protein uS5
      • Protein or peptide: Small ribosomal subunit protein bS6, fully modified isoform
      • Protein or peptide: Small ribosomal subunit protein uS7
      • Protein or peptide: Small ribosomal subunit protein uS8
      • Protein or peptide: Small ribosomal subunit protein uS9
      • Protein or peptide: Small ribosomal subunit protein uS10
      • Protein or peptide: Small ribosomal subunit protein uS11
      • Protein or peptide: Small ribosomal subunit protein uS12
      • Protein or peptide: Small ribosomal subunit protein uS13
      • Protein or peptide: Small ribosomal subunit protein uS14
      • Protein or peptide: Small ribosomal subunit protein uS15
      • Protein or peptide: Small ribosomal subunit protein bS16
      • Protein or peptide: Small ribosomal subunit protein uS17
      • Protein or peptide: Small ribosomal subunit protein bS18
      • Protein or peptide: Small ribosomal subunit protein uS19
      • Protein or peptide: Small ribosomal subunit protein bS20
      • Protein or peptide: Small ribosomal subunit protein bS21
  • Ligand: 5'-{[(3S)-3-amino-3-carboxypropyl](ethyl)amino}-5'-deoxyadenosine
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Complex of bacterial 2'-O-methyltransferase RsmI and the small ri...

SupramoleculeName: Complex of bacterial 2'-O-methyltransferase RsmI and the small ribosomal subunit.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#22
Source (natural)Organism: Escherichia coli (E. coli) / Strain: delta rsmI
Molecular weightTheoretical: 850 KDa

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Supramolecule #2: RsmI (16S rRNA 2'-O-methyltransferase)

SupramoleculeName: RsmI (16S rRNA 2'-O-methyltransferase) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #22
Details: RsmI dimer, modified ribose 2'OH of 16S rRNA nt C1402
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: E. coli 30S subunit

SupramoleculeName: E. coli 30S subunit / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#21 / Details: E. coli 30S subunit lacking Cm1402 modification
Source (natural)Organism: Escherichia coli (E. coli) / Strain: delta rsmI

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Macromolecule #1: 16S rRNA

MacromoleculeName: 16S rRNA / type: rna / ID: 1
Details: 16S rRNA with residue m4C1402 covalently linked to the S'-adenosyl-methionine analogue NM6.
Number of copies: 1
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 499.760156 KDa
SequenceString: AAAUUGAAGA GUUUGAUCAU GGCUCAGAUU GAACGCUGGC GGCAGGCCUA ACACAUGCAA GUCGAACGGU AACAGGAAGA AGCUUGCUU CUUUGCUGAC GAGUGGCGGA CGGGUGAGUA AUGUCUGGGA AACUGCCUGA UGGAGGGGGA UAACUACUGG A AACGGUAG ...String:
AAAUUGAAGA GUUUGAUCAU GGCUCAGAUU GAACGCUGGC GGCAGGCCUA ACACAUGCAA GUCGAACGGU AACAGGAAGA AGCUUGCUU CUUUGCUGAC GAGUGGCGGA CGGGUGAGUA AUGUCUGGGA AACUGCCUGA UGGAGGGGGA UAACUACUGG A AACGGUAG CUAAUACCGC AUAACGUCGC AAGACCAAAG AGGGGGACCU UCGGGCCUCU UGCCAUCGGA UGUGCCCAGA UG GGAUUAG CUAGUAGGUG GGGUAACGGC UCACCUAGGC GACGAUCCCU AGCUGGUCUG AGAGGAUGAC CAGCCACACU GGA ACUGAG ACACGGUCCA GACUCCUACG GGAGGCAGCA GUGGGGAAUA UUGCACAAUG GGCGCAAGCC UGAUGCAGCC AUGC CGCGU GUAUGAAGAA GGCCUUCGGG UUGUAAAGUA CUUUCAGCGG GGAGGAAGGG AGUAAAGUUA AUACCUUUGC UCAUU GACG UUACCCGCAG AAGAAGCACC GGCUAACUCC GUGCCAGCAG CCGCGGUAAU ACGGAGGGUG CAAGCGUUAA UCGGAA UUA CUGGGCGUAA AGCGCACGCA GGCGGUUUGU UAAGUCAGAU GUGAAAUCCC CGGGCUCAAC CUGGGAACUG CAUCUGA UA CUGGCAAGCU UGAGUCUCGU AGAGGGGGGU AGAAUUCCAG GUGUAGCGGU GAAAUGCGUA GAGAUCUGGA GGAAUACC G GUGGCGAAGG CGGCCCCCUG GACGAAGACU GACGCUCAGG UGCGAAAGCG UGGGGAGCAA ACAGGAUUAG AUACCCUGG UAGUCCACGC CGUAAACGAU GUCGACUUGG AGGUUGUGCC CUUGAGGCGU GGCUUCCGGA GCUAACGCGU UAAGUCGACC GCCUGGGGA GUACGGCCGC AAGGUUAAAA CUCAAAUGAA UUGACGGGGG CCCGCACAAG CGGUGGAGCA UGUGGUUUAA U UCGAUGCA ACGCGAAGAA CCUUACCUGG UCUUGACAUC CACGGAAGUU UUCAGAGAUG AGAAUGUGCC UUCGGGAACC GU GAGACAG GUGCUGCAUG GCUGUCGUCA GCUCGUGUUG UGAAAUGUUG GGUUAAGUCC CGCAACGAGC GCAACCCUUA UCC UUUGUU GCCAGCGGUC CGGCCGGGAA CUCAAAGGAG ACUGCCAGUG AUAAACUGGA GGAAGGUGGG GAUGACGUCA AGUC AUCAU GGCCCUUACG ACCAGGGCUA CACACGUGCU ACAAUGGCGC AUACAAAGAG AAGCGACCUC GCGAGAGCAA GCGGA CCUC AUAAAGUGCG UCGUAGUCCG GAUUGGAGUC UGCAACUCGA CUCCAUGAAG UCGGAAUCGC UAGUAAUCGU GGAUCA GAA UGCCACGGUG AAUACGUUCC CGGGCCUUGU ACACACCG(B8T)C CGUCACACCA UGGGAGUGGG UUGCAAAAGA AGU AGGUAG CUUAACCUUC GGGAGGGCGC UUACCACUUU GUGAUUCAUG ACUGGGGUGA AGUCGUAACA AGGUAACCGU AGGG G(MA6)(MA6)CC UGCGGUUGGA UCACCUCCUU A

GENBANK: GENBANK: CP047127.1

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Macromolecule #2: Small ribosomal subunit protein uS2

MacromoleculeName: Small ribosomal subunit protein uS2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 26.78167 KDa
SequenceString: MATVSMRDML KAGVHFGHQT RYWNPKMKPF IFGARNKVHI INLEKTVPMF NEALAELNKI ASRKGKILFV GTKRAASEAV KDAALSCDQ FFVNHRWLGG MLTNWKTVRQ SIKRLKDLET QSQDGTFDKL TKKEALMRTR ELEKLENSLG GIKDMGGLPD A LFVIDADH ...String:
MATVSMRDML KAGVHFGHQT RYWNPKMKPF IFGARNKVHI INLEKTVPMF NEALAELNKI ASRKGKILFV GTKRAASEAV KDAALSCDQ FFVNHRWLGG MLTNWKTVRQ SIKRLKDLET QSQDGTFDKL TKKEALMRTR ELEKLENSLG GIKDMGGLPD A LFVIDADH EHIAIKEANN LGIPVFAIVD TNSDPDGVDF VIPGNDDAIR AVTLYLGAVA ATVREGRSQD LASQAEESFV EA E

UniProtKB: Small ribosomal subunit protein uS2

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Macromolecule #3: Small ribosomal subunit protein uS3

MacromoleculeName: Small ribosomal subunit protein uS3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 26.031316 KDa
SequenceString: MGQKVHPNGI RLGIVKPWNS TWFANTKEFA DNLDSDFKVR QYLTKELAKA SVSRIVIERP AKSIRVTIHT ARPGIVIGKK GEDVEKLRK VVADIAGVPA QINIAEVRKP ELDAKLVADS ITSQLERRVM FRRAMKRAVQ NAMRLGAKGI KVEVSGRLGG A EIARTEWY ...String:
MGQKVHPNGI RLGIVKPWNS TWFANTKEFA DNLDSDFKVR QYLTKELAKA SVSRIVIERP AKSIRVTIHT ARPGIVIGKK GEDVEKLRK VVADIAGVPA QINIAEVRKP ELDAKLVADS ITSQLERRVM FRRAMKRAVQ NAMRLGAKGI KVEVSGRLGG A EIARTEWY REGRVPLHTL RADIDYNTSE AHTTYGVIGV KVWIFKGEIL GGMAAVEQPE KPAAQPKKQQ RKGRK

UniProtKB: Small ribosomal subunit protein uS3

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Macromolecule #4: Small ribosomal subunit protein uS4

MacromoleculeName: Small ribosomal subunit protein uS4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 23.514199 KDa
SequenceString: MARYLGPKLK LSRREGTDLF LKSGVRAIDT KCKIEQAPGQ HGARKPRLSD YGVQLREKQK VRRIYGVLER QFRNYYKEAA RLKGNTGEN LLALLEGRLD NVVYRMGFGA TRAEARQLVS HKAIMVNGRV VNIASYQVSP NDVVSIREKA KKQSRVKAAL E LAEQREKP ...String:
MARYLGPKLK LSRREGTDLF LKSGVRAIDT KCKIEQAPGQ HGARKPRLSD YGVQLREKQK VRRIYGVLER QFRNYYKEAA RLKGNTGEN LLALLEGRLD NVVYRMGFGA TRAEARQLVS HKAIMVNGRV VNIASYQVSP NDVVSIREKA KKQSRVKAAL E LAEQREKP TWLEVDAGKM EGTFKRKPER SDLSADINEH LIVELYSK

UniProtKB: Small ribosomal subunit protein uS4

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Macromolecule #5: Small ribosomal subunit protein uS5

MacromoleculeName: Small ribosomal subunit protein uS5 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 17.629398 KDa
SequenceString:
MAHIEKQAGE LQEKLIAVNR VSKTVKGGRI FSFTALTVVG DGNGRVGFGY GKAREVPAAI QKAMEKARRN MINVALNNGT LQHPVKGVH TGSRVFMQPA SEGTGIIAGG AMRAVLEVAG VHNVLAKAYG STNPINVVRA TIDGLENMNS PEMVAAKRGK S VEEILGK

UniProtKB: Small ribosomal subunit protein uS5

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Macromolecule #6: Small ribosomal subunit protein bS6, fully modified isoform

MacromoleculeName: Small ribosomal subunit protein bS6, fully modified isoform
type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 15.727512 KDa
SequenceString:
MRHYEIVFMV HPDQSEQVPG MIERYTAAIT GAEGKIHRLE DWGRRQLAYP INKLHKAHYV LMNVEAPQEV IDELETTFRF NDAVIRSMV MRTKHAVTEA SPMVKAKDER RERRDDFANE TADDAEAGDS EEEEEE

UniProtKB: Small ribosomal subunit protein bS6

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Macromolecule #7: Small ribosomal subunit protein uS7

MacromoleculeName: Small ribosomal subunit protein uS7 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 20.055156 KDa
SequenceString:
MPRRRVIGQR KILPDPKFGS ELLAKFVNIL MVDGKKSTAE SIVYSALETL AQRSGKSELE AFEVALENVR PTVEVKSRRV GGSTYQVPV EVRPVRRNAL AMRWIVEAAR KRGDKSMALR LANELSDAAE NKGTAVKKRE DVHRMAEANK AFAHYRWLSL R SFSHQAGA SSKQPALGYL N

UniProtKB: Small ribosomal subunit protein uS7

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Macromolecule #8: Small ribosomal subunit protein uS8

MacromoleculeName: Small ribosomal subunit protein uS8 / type: protein_or_peptide / ID: 8 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 14.146557 KDa
SequenceString:
MSMQDPIADM LTRIRNGQAA NKAAVTMPSS KLKVAIANVL KEEGFIEDFK VEGDTKPELE LTLKYFQGKA VVESIQRVSR PGLRIYKRK DELPKVMAGL GIAVVSTSKG VMTDRAARQA GLGGEIICYV A

UniProtKB: Small ribosomal subunit protein uS8

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Macromolecule #9: Small ribosomal subunit protein uS9

MacromoleculeName: Small ribosomal subunit protein uS9 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 14.88627 KDa
SequenceString:
MAENQYYGTG RRKSSAARVF IKPGNGKIVI NQRSLEQYFG RETARMVVRQ PLELVDMVEK LDLYITVKGG GISGQAGAIR HGITRALME YDESLRSELR KAGFVTRDAR QVERKKVGLR KARRRPQFSK R

UniProtKB: Small ribosomal subunit protein uS9

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Macromolecule #10: Small ribosomal subunit protein uS10

MacromoleculeName: Small ribosomal subunit protein uS10 / type: protein_or_peptide / ID: 10 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 11.755597 KDa
SequenceString:
MQNQRIRIRL KAFDHRLIDQ ATAEIVETAK RTGAQVRGPI PLPTRKERFT VLISPHVNKD ARDQYEIRTH LRLVDIVEPT EKTVDALMR LDLAAGVDVQ ISLG

UniProtKB: Small ribosomal subunit protein uS10

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Macromolecule #11: Small ribosomal subunit protein uS11

MacromoleculeName: Small ribosomal subunit protein uS11 / type: protein_or_peptide / ID: 11 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 13.870975 KDa
SequenceString:
MAKAPIRARK RVRKQVSDGV AHIHASFNNT IVTITDRQGN ALGWATAGGS GFRGSRKSTP FAAQVAAERC ADAVKEYGIK NLEVMVKGP GPGRESTIRA LNAAGFRITN ITDVTPIPHN GCRPPKKRRV

UniProtKB: Small ribosomal subunit protein uS11

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Macromolecule #12: Small ribosomal subunit protein uS12

MacromoleculeName: Small ribosomal subunit protein uS12 / type: protein_or_peptide / ID: 12 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 13.768157 KDa
SequenceString:
MATVNQLVRK PRARKVAKSN VPALEACPQK RGVCTRVYTT TPKKPNSALR KVCRVRLTNG FEVTSYIGGE GHNLQEHSVI LIRGGRVKD LPGVRYHTVR GALDCSGVKD RKQARSKYGV KRPKA

UniProtKB: Small ribosomal subunit protein uS12

+
Macromolecule #13: Small ribosomal subunit protein uS13

MacromoleculeName: Small ribosomal subunit protein uS13 / type: protein_or_peptide / ID: 13 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 13.128467 KDa
SequenceString:
MARIAGINIP DHKHAVIALT SIYGVGKTRS KAILAAAGIA EDVKISELSE GQIDTLRDEV AKFVVEGDLR REISMSIKRL MDLGCYRGL RHRRGLPVRG QRTKTNARTR KGPRKPIKK

UniProtKB: Small ribosomal subunit protein uS13

+
Macromolecule #14: Small ribosomal subunit protein uS14

MacromoleculeName: Small ribosomal subunit protein uS14 / type: protein_or_peptide / ID: 14 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 11.60656 KDa
SequenceString:
MAKQSMKARE VKRVALADKY FAKRAELKAI ISDVNASDED RWNAVLKLQT LPRDSSPSRQ RNRCRQTGRP HGFLRKFGLS RIKVREAAM RGEIPGLKKA SW

UniProtKB: Small ribosomal subunit protein uS14

+
Macromolecule #15: Small ribosomal subunit protein uS15

MacromoleculeName: Small ribosomal subunit protein uS15 / type: protein_or_peptide / ID: 15 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 10.290816 KDa
SequenceString:
MSLSTEATAK IVSEFGRDAN DTGSTEVQVA LLTAQINHLQ GHFAEHKKDH HSRRGLLRMV SQRRKLLDYL KRKDVARYTQ LIERLGLRR

UniProtKB: Small ribosomal subunit protein uS15

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Macromolecule #16: Small ribosomal subunit protein bS16

MacromoleculeName: Small ribosomal subunit protein bS16 / type: protein_or_peptide / ID: 16 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 9.207572 KDa
SequenceString:
MVTIRLARHG AKKRPFYQVV VADSRNARNG RFIERVGFFN PIASEKEEGT RLDLDRIAHW VGQGATISDR VAALIKEVNK AA

UniProtKB: Small ribosomal subunit protein bS16

+
Macromolecule #17: Small ribosomal subunit protein uS17

MacromoleculeName: Small ribosomal subunit protein uS17 / type: protein_or_peptide / ID: 17 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 9.724491 KDa
SequenceString:
MTDKIRTLQG RVVSDKMEKS IVVAIERFVK HPIYGKFIKR TTKLHVHDEN NECGIGDVVE IRECRPLSKT KSWTLVRVVE KAVL

UniProtKB: Small ribosomal subunit protein uS17

+
Macromolecule #18: Small ribosomal subunit protein bS18

MacromoleculeName: Small ribosomal subunit protein bS18 / type: protein_or_peptide / ID: 18 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 9.005472 KDa
SequenceString:
MARYFRRRKF CRFTAEGVQE IDYKDIATLK NYITESGKIV PSRITGTRAK YQRQLARAIK RARYLSLLPY TDRHQ

UniProtKB: Small ribosomal subunit protein bS18

+
Macromolecule #19: Small ribosomal subunit protein uS19

MacromoleculeName: Small ribosomal subunit protein uS19 / type: protein_or_peptide / ID: 19 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 10.455355 KDa
SequenceString:
MPRSLKKGPF IDLHLLKKVE KAVESGDKKP LRTWSRRSTI FPNMIGLTIA VHNGRQHVPV FVTDEMVGHK LGEFAPTRTY RGHAADKKA KKK

UniProtKB: Small ribosomal subunit protein uS19

+
Macromolecule #20: Small ribosomal subunit protein bS20

MacromoleculeName: Small ribosomal subunit protein bS20 / type: protein_or_peptide / ID: 20 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 9.708464 KDa
SequenceString:
MANIKSAKKR AIQSEKARKH NASRRSMMRT FIKKVYAAIE AGDKAAAQKA FNEMQPIVDR QAAKGLIHKN KAARHKANLT AQINKLA

UniProtKB: Small ribosomal subunit protein bS20

+
Macromolecule #21: Small ribosomal subunit protein bS21

MacromoleculeName: Small ribosomal subunit protein bS21 / type: protein_or_peptide / ID: 21 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 8.524039 KDa
SequenceString:
MPVIKVRENE PFDVALRRFK RSCEKAGVLA EVRRREFYEK PTTERKRAKA SAVKRHAKKL ARENARRTRL Y

UniProtKB: Small ribosomal subunit protein bS21

+
Macromolecule #22: Ribosomal RNA small subunit methyltransferase I

MacromoleculeName: Ribosomal RNA small subunit methyltransferase I / type: protein_or_peptide / ID: 22
Details: his-tagged construct with a TEV protease cleavage site preceding the start of the open reading frame with the S'-adenosyl-methionine (SAM) analogue NM6 (PDB code AN6) present in the SAM binding site.
Number of copies: 2 / Enantiomer: LEVO / EC number: 16S rRNA (cytidine1402-2'-O)-methyltransferase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 33.313023 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHHHASG LVPRGSHMKQ HQSADNSQGQ LYIVPTPIGN LADITQRALE VLQAVDLIAA EDTRHTGLLL QHFGINARLF ALHDHNEQQ KAETLLAKLQ EGQNIALVSD AGTPLINDPG YHLVRTCREA GIRVVPLPGP CAAITALSAA GLPSDRFCYE G FLPAKSKG ...String:
MHHHHHHASG LVPRGSHMKQ HQSADNSQGQ LYIVPTPIGN LADITQRALE VLQAVDLIAA EDTRHTGLLL QHFGINARLF ALHDHNEQQ KAETLLAKLQ EGQNIALVSD AGTPLINDPG YHLVRTCREA GIRVVPLPGP CAAITALSAA GLPSDRFCYE G FLPAKSKG RRDALKAIEA EPRTLIFYES THRLLDSLED IVAVLGESRY VVLARELTKT WETIHGAPVG ELLAWVKEDE NR RKGEMVL IVEGHKAQEE DLPADALRTL ALLQAELPLK KAAALAAEIH GVKKNALYKY ALEQQG

UniProtKB: Ribosomal RNA small subunit methyltransferase I

+
Macromolecule #23: 5'-{[(3S)-3-amino-3-carboxypropyl](ethyl)amino}-5'-deoxyadenosine

MacromoleculeName: 5'-{[(3S)-3-amino-3-carboxypropyl](ethyl)amino}-5'-deoxyadenosine
type: ligand / ID: 23
Details: 16S rRNA with residue m4C1402 covalently linked to the S'-adenosyl-methionine analogue NM6.
Number of copies: 2 / Formula: AN6
Source (natural)Organism: Escherichia coli BW25113 (bacteria)
Molecular weightTheoretical: 395.414 Da
Chemical component information

ChemComp-AN6:
5'-{[(3S)-3-amino-3-carboxypropyl](ethyl)amino}-5'-deoxyadenosine

+
Macromolecule #24: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 24 / Number of copies: 79 / Formula: MG
Molecular weightTheoretical: 24.305 Da

+
Macromolecule #25: water

MacromoleculeName: water / type: ligand / ID: 25 / Number of copies: 170 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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: 7.6
Details: 10 mM HEPES, 10 mM MgCl2, 100 mM NH4Cl, 6 mM Beta-mercaptoethanol
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsRecombinant RsmI (1.2 uM), delta-rsmI 30S (0.4 uM), and the SAM analog NM6 (24 uM) were incubated for 30 minutes at 37C to prepare the complex.

-
Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 8164 / Average exposure time: 1.8 sec. / Average electron dose: 49.27 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.9 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

+
Image processing

Particle selectionNumber selected: 3781314
CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model generated by CryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.42 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 74553
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

source_name: PDB, initial_model_type: experimental model

5wh4
PDB Unreleased entry

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9pzg:
Bacterial ribosomal 2'-O-methyltransferase RsmI in complex with the small ribosomal subunit

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