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- PDB-8bh4: Elongating E. coli 70S ribosome containing deacylated tRNA(iMet) ... -

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Basic information

Entry
Database: PDB / ID: 8bh4
TitleElongating E. coli 70S ribosome containing deacylated tRNA(iMet) in the P-site and AAm6A mRNA codon with cognate dipeptidyl-tRNA(Lys) in the A-site
Components
  • (30S ribosomal protein ...) x 20
  • (50S ribosomal protein ...) x 28
  • 16S rRNA RRSB-RRNA
  • 23S rRNA RRLG-RRNA
  • 5S rRNA
  • Deacylated P-site tRNA(fMet)
  • Dipeptidyl A-site tRNA(Lys)
  • fMet-Lys dipeptide
  • mRNA
KeywordsTRANSLATION / m6A / mRNA modification / tRNA recognition
Function / homology
Function and homology information


ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation ...ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / DnaA-L2 complex / four-way junction DNA binding / translation repressor activity / negative regulation of translational initiation / negative regulation of DNA-templated DNA replication initiation / regulation of mRNA stability / mRNA regulatory element binding translation repressor activity / ribosome assembly / assembly of large subunit precursor of preribosome / positive regulation of RNA splicing / cytosolic ribosome assembly / transcription elongation factor complex / regulation of DNA-templated transcription elongation / DNA endonuclease activity / response to reactive oxygen species / transcription antitermination / regulation of cell growth / DNA-templated transcription termination / maintenance of translational fidelity / response to radiation / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / large ribosomal subunit / ribosome biogenesis / ribosome binding / regulation of translation / ribosomal small subunit assembly / large ribosomal subunit rRNA binding / 5S rRNA binding / transferase activity / small ribosomal subunit / cytosolic small ribosomal subunit / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit / tRNA binding / molecular adaptor activity / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / negative regulation of DNA-templated transcription / mRNA binding / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form / 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 S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form / 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 L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L16 signature 1. / : / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L16, conserved site / Ribosomal protein L16 signature 2. / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L17 signature. / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L28/L24 superfamily / Ribosomal protein L36 signature. / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L32p, bacterial type / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / : / : / Ribosomal protein L28 / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L5, bacterial-type / Ribosomal protein L18, bacterial-type / Ribosomal protein L6, bacterial-type / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein S3, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein L20 signature. / Ribosomal protein S6 signature. / Ribosomal protein S19, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / 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 L14P, bacterial-type / Ribosomal protein L34, conserved site / Ribosomal protein L34 signature. / Ribosomal protein L22, bacterial/chloroplast-type / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L35 / Ribosomal protein L35 superfamily / Ribosomal protein S2, bacteria/mitochondria/plastid / Ribosomal protein L35 / Ribosomal L28 family / Ribosomal protein L33 / Ribosomal protein L33 / Ribosomal protein L28/L24 / Ribosomal protein L30, bacterial-type / Ribosomal protein L18 / Ribosomal L18 of archaea, bacteria, mitoch. and chloroplast / Ribosomal protein L33 superfamily / Ribosomal protein L16 / Ribosomal protein S18, conserved site / Ribosomal protein S18 signature. / : / L28p-like / Ribosomal protein L20 / Ribosomal protein S16 / Ribosomal protein S16 domain superfamily / Ribosomal protein S16 / Ribosomal protein L20 / Ribosomal protein L20, C-terminal / Ribosomal protein L27 / Ribosomal L27 protein
Similarity search - Domain/homology
: / : / : / : / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Small ribosomal subunit protein bS6 ...: / : / : / : / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL36A / Large ribosomal subunit protein bL9 / 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 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL23 / Small ribosomal subunit protein uS15 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL5 / Small ribosomal subunit protein bS21 / Large ribosomal subunit protein bL25
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.62 Å
AuthorsKoziej, L. / Glatt, S.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)101001394European Union
CitationJournal: Nat Commun / Year: 2023
Title: Modulation of translational decoding by mA modification of mRNA.
Authors: Sakshi Jain / Lukasz Koziej / Panagiotis Poulis / Igor Kaczmarczyk / Monika Gaik / Michal Rawski / Namit Ranjan / Sebastian Glatt / Marina V Rodnina /
Abstract: N-methyladenosine (mA) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. mA in the mRNA coding regions inhibits translation elongation. Here, we show how ...N-methyladenosine (mA) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. mA in the mRNA coding regions inhibits translation elongation. Here, we show how mA modulates decoding in the bacterial translation system using a combination of rapid kinetics, smFRET and single-particle cryo-EM. We show that, while the modification does not impair the initial binding of aminoacyl-tRNA to the ribosome, in the presence of mA fewer ribosomes complete the decoding process due to the lower stability of the complexes and enhanced tRNA drop-off. The mRNA codon adopts a π-stacked codon conformation that is remodeled upon aminoacyl-tRNA binding. mA does not exclude canonical codon-anticodon geometry, but favors alternative more dynamic conformations that are rejected by the ribosome. These results highlight how modifications outside the Watson-Crick edge can still interfere with codon-anticodon base pairing and complex recognition by the ribosome, thereby modulating the translational efficiency of modified mRNAs.
History
DepositionOct 29, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 16, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 50S ribosomal protein L2
B: 50S ribosomal protein L3
C: 50S ribosomal protein L4
D: 50S ribosomal protein L5
E: 50S ribosomal protein L6
F: 50S ribosomal protein L9
G: 50S ribosomal protein L13
H: 50S ribosomal protein L14
I: 50S ribosomal protein L15
J: 50S ribosomal protein L16
K: 50S ribosomal protein L17
L: 50S ribosomal protein L18
M: 50S ribosomal protein L19
N: 50S ribosomal protein L20
O: 50S ribosomal protein L21
P: 50S ribosomal protein L22
Q: 50S ribosomal protein L23
R: 50S ribosomal protein L24
S: 50S ribosomal protein L25
T: 50S ribosomal protein L27
U: 50S ribosomal protein L28
V: 50S ribosomal protein L29
W: 50S ribosomal protein L30
a: 50S ribosomal protein L32
b: 50S ribosomal protein L33
c: 50S ribosomal protein L34
d: 50S ribosomal protein L35
e: 50S ribosomal protein L36
f: 30S ribosomal protein S2
g: 30S ribosomal protein S3
h: 30S ribosomal protein S4
i: 30S ribosomal protein S5
j: 30S ribosomal protein S6
k: 30S ribosomal protein S7
l: 30S ribosomal protein S8
m: 30S ribosomal protein S9
n: 30S ribosomal protein S10
o: 30S ribosomal protein S11
p: 30S ribosomal protein S12
q: 30S ribosomal protein S13
r: 30S ribosomal protein S14
s: 30S ribosomal protein S15
t: 30S ribosomal protein S16
u: 30S ribosomal protein S17
v: 30S ribosomal protein S18
w: 30S ribosomal protein S19
x: 30S ribosomal protein S20
y: 30S ribosomal protein S21
0: 23S rRNA RRLG-RRNA
1: 5S rRNA
2: 16S rRNA RRSB-RRNA
3: mRNA
4: Deacylated P-site tRNA(fMet)
5: Dipeptidyl A-site tRNA(Lys)
6: fMet-Lys dipeptide


Theoretical massNumber of molelcules
Total (without water)2,191,15055
Polymers2,191,15055
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area235090 Å2
ΔGint-1913 kcal/mol
Surface area868820 Å2

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Components

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50S ribosomal protein ... , 28 types, 28 molecules ABCDEFGHIJKLMNOPQRSTUVWabcde

#1: Protein 50S ribosomal protein L2 / Large ribosomal subunit protein uL2


Mass: 29923.619 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P60422
#2: Protein 50S ribosomal protein L3 / Large ribosomal subunit protein uL3


Mass: 22277.535 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P60438
#3: Protein 50S ribosomal protein L4 / Large ribosomal subunit protein uL4


Mass: 22121.566 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P60723
#4: Protein 50S ribosomal protein L5 / Large ribosomal subunit protein uL5


Mass: 20333.611 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P62399
#5: Protein 50S ribosomal protein L6 / Large ribosomal subunit protein uL6


Mass: 18932.791 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0AG55
#6: Protein 50S ribosomal protein L9 / Large ribosomal subunit protein bL9


Mass: 15789.020 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: UniProt: P0A7R1
#7: Protein 50S ribosomal protein L13 / Large ribosomal subunit protein uL13


Mass: 16050.606 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0AA10
#8: Protein 50S ribosomal protein L14 / Large ribosomal subunit protein uL14


Mass: 13565.067 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0ADY3
#9: Protein 50S ribosomal protein L15 / Large ribosomal subunit protein uL15


Mass: 15008.471 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P02413
#10: Protein 50S ribosomal protein L16 / Large ribosomal subunit protein uL16


Mass: 15312.269 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0ADY7
#11: Protein 50S ribosomal protein L17 / Large ribosomal subunit protein bL17


Mass: 14393.657 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0AG44
#12: Protein 50S ribosomal protein L18 / Large ribosomal subunit protein uL18


Mass: 12794.668 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0C018
#13: Protein 50S ribosomal protein L19 / Large ribosomal subunit protein bL19


Mass: 13159.278 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7K6
#14: Protein 50S ribosomal protein L20 / Large ribosomal subunit protein bL20


Mass: 13528.024 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7L3
#15: Protein 50S ribosomal protein L21 / Large ribosomal subunit protein bL21


Mass: 11586.374 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0AG48
#16: Protein 50S ribosomal protein L22 / Large ribosomal subunit protein uL22


Mass: 12253.359 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P61175
#17: Protein 50S ribosomal protein L23 / Large ribosomal subunit protein uL23


Mass: 11222.160 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0ADZ0
#18: Protein 50S ribosomal protein L24 / Large ribosomal subunit protein uL24


Mass: 11339.250 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P60624
#19: Protein 50S ribosomal protein L25 / Large ribosomal subunit protein bL25


Mass: 10713.465 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P68919
#20: Protein 50S ribosomal protein L27 / Large ribosomal subunit protein bL27


Mass: 9146.540 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7L8
#21: Protein 50S ribosomal protein L28 / Large ribosomal subunit protein bL28


Mass: 9027.551 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7M2
#22: Protein 50S ribosomal protein L29 / Large ribosomal subunit protein uL29


Mass: 7286.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7M6
#23: Protein 50S ribosomal protein L30 / Large ribosomal subunit protein uL30


Mass: 6554.820 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0AG51
#24: Protein 50S ribosomal protein L32 / Large ribosomal subunit protein bL32


Mass: 6463.445 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7N4
#25: Protein 50S ribosomal protein L33 / Large ribosomal subunit protein bL33


Mass: 6388.631 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7N9
#26: Protein/peptide 50S ribosomal protein L34 / Large ribosomal subunit protein bL34


Mass: 5397.463 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7P5
#27: Protein 50S ribosomal protein L35 / Large ribosomal subunit protein bL35 / Ribosomal protein A


Mass: 7313.032 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7Q1
#28: Protein/peptide 50S ribosomal protein L36 / Large ribosomal subunit protein bL36-A / Ribosomal protein B


Mass: 4377.390 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7Q6

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

#29: Protein 30S ribosomal protein S2 / Small ribosomal subunit protein uS2


Mass: 26781.670 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7V0
#30: Protein 30S ribosomal protein S3 / Small ribosomal subunit protein uS3


Mass: 26031.316 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7V3
#31: Protein 30S ribosomal protein S4 / Small ribosomal subunit protein uS4


Mass: 23514.199 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7V8
#32: Protein 30S ribosomal protein S5 / Small ribosomal subunit protein uS5


Mass: 17629.398 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7W1
#33: Protein 30S ribosomal protein S6 / Small ribosomal subunit protein bS6


Mass: 15211.058 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P02358
#34: Protein 30S ribosomal protein S7 / Small ribosomal subunit protein uS7


Mass: 20055.156 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P02359
#35: Protein 30S ribosomal protein S8 / Small ribosomal subunit protein uS8


Mass: 14146.557 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7W7
#36: Protein 30S ribosomal protein S9 / Small ribosomal subunit protein uS9


Mass: 14886.270 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7X3
#37: Protein 30S ribosomal protein S10 / Small ribosomal subunit protein uS10 / Transcription termination/antitermination protein NusE


Mass: 11755.597 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7R5
#38: Protein 30S ribosomal protein S11 / Small ribosomal subunit protein uS11


Mass: 13870.975 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7R9
#39: Protein 30S ribosomal protein S12 / Small ribosomal subunit protein uS12


Mass: 13768.157 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7S3
#40: Protein 30S ribosomal protein S13 / Small ribosomal subunit protein uS13


Mass: 13128.467 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7S9
#41: Protein 30S ribosomal protein S14 / Small ribosomal subunit protein uS14


Mass: 11606.560 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0AG59
#42: Protein 30S ribosomal protein S15 / Small ribosomal subunit protein uS15


Mass: 10290.816 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0ADZ4
#43: Protein 30S ribosomal protein S16 / Small ribosomal subunit protein bS16


Mass: 9207.572 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7T3
#44: Protein 30S ribosomal protein S17 / Small ribosomal subunit protein uS17


Mass: 9724.491 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0AG63
#45: Protein 30S ribosomal protein S18 / Small ribosomal subunit protein bS18


Mass: 9005.472 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7T7
#46: Protein 30S ribosomal protein S19 / Small ribosomal subunit protein uS19


Mass: 10455.355 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7U3
#47: Protein 30S ribosomal protein S20 / Small ribosomal subunit protein bS20


Mass: 9708.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P0A7U7
#48: Protein 30S ribosomal protein S21 / Small ribosomal subunit protein bS21


Mass: 8524.039 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / Strain: K-12 / References: UniProt: P68679

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RNA chain , 6 types, 6 molecules 012345

#49: RNA chain 23S rRNA RRLG-RRNA


Mass: 941612.375 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: GenBank: 1063812051
#50: RNA chain 5S rRNA


Mass: 38790.090 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: GenBank: 1845258627
#51: RNA chain 16S rRNA RRSB-RRNA


Mass: 499690.031 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: GenBank: 1789840096
#52: RNA chain mRNA


Mass: 9739.893 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#53: RNA chain Deacylated P-site tRNA(fMet)


Mass: 24846.902 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: GenBank: 1848851804
#54: RNA chain Dipeptidyl A-site tRNA(Lys)


Mass: 24602.729 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria) / References: GenBank: 1848841577

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Protein/peptide , 1 types, 1 molecules 6

#55: Protein/peptide fMet-Lys dipeptide


Mass: 306.401 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli K-12 (bacteria)

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Elongating E. coli 70S ribosome with deacylated tRNA(iMet) in the P-site and mRNA containing AAm6A codon and dipeptidyl-tRNA(Lys) in the A-siteRIBOSOMEall0NATURAL
250S subunitCOMPLEX#1-#28, #49-#501NATURAL
330S subunitCOMPLEX#29-#48, #511NATURAL
423S and 5S rRNACOMPLEX#49-#502NATURAL
516S rRNACOMPLEX#513NATURAL
6mRNA and tRNACOMPLEX#52-#551NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
112.7 MDaNO
211.6 MDaNO
310.9 MDaNO
44
55
66
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli K-12 (bacteria)83333
32Escherichia coli K-12 (bacteria)83333
43Escherichia coli K-12 (bacteria)83333
54Escherichia coli K-12 (bacteria)83333
65Escherichia coli K-12 (bacteria)83333
76Escherichia coli K-12 (bacteria)83333
Buffer solutionpH: 7.5 / Details: 1x TAKM7 buffer
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMtris(hydroxymethyl)aminomethaneTris-HCl1
270 mMammonium chlorideNH4Cl1
330 mMpotassium chlorideKCl1
47 mMmagnesium chlorideMgCl21
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Initiation complex containing: 1x TAKM7 buffer 1 mM DTT (dithiothreitol) 1 mM GTP (guanosine 5'-triphosphate) 2.0 uM 70S ribosomes 3.0 uM IF1, IF2, IF3 mix 6.0 uM A-site AAm6A mRNA 3.0 uM ...Details: Initiation complex containing: 1x TAKM7 buffer 1 mM DTT (dithiothreitol) 1 mM GTP (guanosine 5'-triphosphate) 2.0 uM 70S ribosomes 3.0 uM IF1, IF2, IF3 mix 6.0 uM A-site AAm6A mRNA 3.0 uM fMet-tRNAfMet Mixed 1:1 with a ternary complex containing: 1x TAKM7 buffer 3 mM PEP (phosphoenolpyruvate) 1 mM GTP (guanosine 5'-triphosphate) 1 mM DTT (dithiothreitol) 1% PK (pyruvate kinase) 0.02 uM EF-Ts 1.5 uM EF-Tu 0.3 uM Lys-tRNALys Incubated on ice for 60 seconds Final concentration of the sample: 20 A260 (absorbance at 260 nm, 10 mm path), 10 A280 (absorbance at 280 nm, 10 mm path)
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

-
Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 105000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.82 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7207
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetailsFitting-ID
1cryoSPARC3.3.0particle selection
3EPU2.1image acquisition
5cryoSPARC3.3.0CTF correction
8UCSF ChimeraX1.2.5model fittingRigid body fit1
12cryoSPARC3.3.0initial Euler assignmentAb-initio
15cryoSPARC3.3.0classification3D Class
17cryoSPARC3.3.03D reconstructionHomogenous Refinement
19PHENIXOnlinemodel refinementhttps://namdinator.au.dk/namdinator/1
20ISOLDE1.4model fitting2
21PHENIX1.19.2-4158model refinement2
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 500
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 271987 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model building
IDProtocolSpaceDetails
1RIGID BODY FITREALFollowing rigid body fitting we used online Namdiantor tool to perform initial flexible fitting: https://namdinator.au.dk/namdinator/
2FLEXIBLE FITREAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004154579
ELECTRON MICROSCOPYf_angle_d0.617231364
ELECTRON MICROSCOPYf_dihedral_angle_d15.10672223
ELECTRON MICROSCOPYf_chiral_restr0.0429571
ELECTRON MICROSCOPYf_plane_restr0.00512322

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