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- PDB-6szs: Release factor-dependent ribosome rescue by BrfA in the Gram-posi... -

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Basic information

Entry
Database: PDB / ID: 6szs
TitleRelease factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis
Components
  • (30S ribosomal protein ...) x 20
  • (50S ribosomal protein ...) x 29
  • 16S ribosomal RNA
  • 23S ribosomal RNA
  • 5S ribosomal RNA
  • P-site tRNA-Pro(CGG)
  • Peptide chain release factor 2
  • Uncharacterized protein YqkK
  • mRNAMessenger RNA
KeywordsRIBOSOME / Cryo-EM / bacterial ribosome rescue / Bacillus ribosome rescue factor A / Bacillus subtilis peptide chain release factor 2
Function / homology
Function and homology information


translation release factor activity, codon specific / negative regulation of cytoplasmic translational initiation / mRNA base-pairing translational repressor activity / ornithine decarboxylase inhibitor activity / misfolded RNA binding / transcription antitermination factor activity, RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity ...translation release factor activity, codon specific / negative regulation of cytoplasmic translational initiation / mRNA base-pairing translational repressor activity / ornithine decarboxylase inhibitor activity / misfolded RNA binding / transcription antitermination factor activity, 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 / translational initiation / negative regulation of DNA-templated DNA replication initiation / regulation of mRNA stability / ribosome assembly / mRNA regulatory element binding translation repressor activity / response to reactive oxygen species / assembly of large subunit precursor of preribosome / transcription elongation factor complex / positive regulation of RNA splicing / DNA endonuclease activity / : / cytosolic ribosome assembly / regulation of DNA-templated transcription elongation / transcription antitermination / regulation of cell growth / maintenance of translational fidelity / DNA-templated transcription termination / response to radiation / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / ribosomal small subunit assembly / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / ribosome binding / large ribosomal subunit / ribosome biogenesis / regulation of translation / small ribosomal subunit / 5S rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / transferase activity / tRNA binding / negative regulation of translation / rRNA binding / molecular adaptor activity / ribosome / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / negative regulation of DNA-templated transcription / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Peptide chain release factor 2 / Peptide chain release factor / PCRF domain / PCRF / Prokaryotic-type class I peptide chain release factors signature. / Peptide chain release factor class I / RF-1 domain / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form ...Peptide chain release factor 2 / Peptide chain release factor / PCRF domain / PCRF / Prokaryotic-type class I peptide chain release factors signature. / Peptide chain release factor class I / RF-1 domain / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L31 type A / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein L31 signature. / Ribosomal protein S21 / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / 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 L17 signature. / 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 L25p family / Ribosomal protein L25 / Ribosomal protein L28/L24 superfamily / Ribosomal protein L36 signature. / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L32p, bacterial type / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / 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 L6, bacterial-type / Ribosomal protein L18, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein L20 signature. / Ribosomal protein S3, bacterial-type / Ribosomal protein L27, conserved site / Ribosomal protein S6, conserved site / Ribosomal protein L27 signature. / Ribosomal protein S6 signature. / Ribosomal protein S19, bacterial-type / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein S9, bacterial/plastid / Ribosomal protein L14P, bacterial-type / Ribosomal protein S4, bacterial-type / Ribosomal protein L34, conserved site / Ribosomal protein L34 signature. / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein L22, bacterial/chloroplast-type / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein L35 / Ribosomal protein L35 superfamily / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L35 / Ribosomal protein S2, bacteria/mitochondria/plastid / Ribosomal L28 family / Ribosomal protein L33 / Ribosomal protein L33 / Ribosomal protein L28/L24 / Ribosomal protein L33 superfamily / : / Ribosomal protein L30, bacterial-type / Ribosomal protein L16 / Ribosomal protein L18 / Ribosomal L18 of archaea, bacteria, mitoch. and chloroplast / Ribosomal protein S18, conserved site / Ribosomal protein S18 signature.
Similarity search - Domain/homology
: / : / : / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 ...: / : / : / : / 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 bL31 / 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 / Peptide chain release factor 2 / Uncharacterized protein YqkK / 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 (E. coli)
Escherichia coli K-12 (bacteria)
Bacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsMuller, C. / Beckert, B. / Wilson, D.N.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research FoundationSPP1879-WI3285/5-2 Germany
CitationJournal: Nat Commun / Year: 2019
Title: Release factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis.
Authors: Naomi Shimokawa-Chiba / Claudia Müller / Keigo Fujiwara / Bertrand Beckert / Koreaki Ito / Daniel N Wilson / Shinobu Chiba /
Abstract: Rescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some ...Rescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop-codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named BrfA. Genetic analysis shows that B. subtilis requires the function of either trans-translation or BrfA for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-electron microscopy (cryo-EM) characterization demonstrates that BrfA binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although BrfA is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.
History
DepositionOct 2, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 4, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 11, 2019Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
0: 50S ribosomal protein L32
1: 50S ribosomal protein L33
2: 50S ribosomal protein L34
3: 50S ribosomal protein L35
4: 50S ribosomal protein L36
5: 50S ribosomal protein L31
A: 23S ribosomal RNA
B: 5S ribosomal RNA
C: 50S ribosomal protein L2
D: 50S ribosomal protein L3
E: 50S ribosomal protein L4
F: 50S ribosomal protein L5
G: 50S ribosomal protein L6
H: 50S ribosomal protein L9
J: 50S ribosomal protein L13
K: 50S ribosomal protein L14
L: 50S ribosomal protein L15
M: 50S ribosomal protein L16
N: 50S ribosomal protein L17
O: 50S ribosomal protein L18
P: 50S ribosomal protein L19
Q: 50S ribosomal protein L20
R: 50S ribosomal protein L21
S: 50S ribosomal protein L22
T: 50S ribosomal protein L23
U: 50S ribosomal protein L24
V: 50S ribosomal protein L25
W: 50S ribosomal protein L27
X: 50S ribosomal protein L28
Y: 50S ribosomal protein L29
Z: 50S ribosomal protein L30
a: 16S ribosomal RNA
b: 30S ribosomal protein S2
c: 30S ribosomal protein S3
d: 30S ribosomal protein S4
e: 30S ribosomal protein S5
f: 30S ribosomal protein S6
g: 30S ribosomal protein S7
h: 30S ribosomal protein S8
i: 30S ribosomal protein S9
j: 30S ribosomal protein S10
k: 30S ribosomal protein S11
l: 30S ribosomal protein S12
m: 30S ribosomal protein S13
n: 30S ribosomal protein S14
o: 30S ribosomal protein S15
p: 30S ribosomal protein S16
q: 30S ribosomal protein S17
r: 30S ribosomal protein S18
s: 30S ribosomal protein S19
t: 30S ribosomal protein S20
u: 30S ribosomal protein S21
v: mRNA
x: P-site tRNA-Pro(CGG)
y: Uncharacterized protein YqkK
z: Peptide chain release factor 2


Theoretical massNumber of molelcules
Total (without water)2,215,58156
Polymers2,215,58156
Non-polymers00
Water0
1


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

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Components

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50S ribosomal protein ... , 29 types, 29 molecules 012345CDEFGHJKLMNOPQRSTUVWXYZ

#1: Protein 50S ribosomal protein L32 / / Large ribosomal subunit protein bL32


Mass: 6463.445 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpmF, b1089, JW1075 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7N4
#2: Protein 50S ribosomal protein L33 / / Large ribosomal subunit protein bL33


Mass: 6388.631 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpmG, b3636, JW3611 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7N9
#3: Protein/peptide 50S ribosomal protein L34 / / Large ribosomal subunit protein bL34


Mass: 5397.463 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpmH, rimA, ssaF, b3703, JW3680 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7P5
#4: 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 genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpmI, b1717, JW1707 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7Q1
#5: 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 genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpmJ, b3299, JW3261 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7Q6
#6: Protein 50S ribosomal protein L31 / / Large ribosomal subunit protein bL31-A


Mass: 7887.117 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpmE, b3936, JW3907 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7M9
#9: Protein 50S ribosomal protein L2 / / Large ribosomal subunit protein uL2


Mass: 29923.619 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplB, b3317, JW3279 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P60422
#10: Protein 50S ribosomal protein L3 / / Large ribosomal subunit protein uL3


Mass: 22277.535 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplC, b3320, JW3282 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P60438
#11: Protein 50S ribosomal protein L4 / / Large ribosomal subunit protein uL4


Mass: 22121.566 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplD, eryA, b3319, JW3281 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P60723
#12: Protein 50S ribosomal protein L5 / / Large ribosomal subunit protein uL5


Mass: 20333.611 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplE, b3308, JW3270 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P62399
#13: Protein 50S ribosomal protein L6 / / Large ribosomal subunit protein uL6


Mass: 18932.791 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplF, b3305, JW3267 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0AG55
#14: Protein 50S ribosomal protein L9 / / Large ribosomal subunit protein bL9


Mass: 15789.020 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplI, b4203, JW4161 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7R1
#15: Protein 50S ribosomal protein L13 / / Large ribosomal subunit protein uL13


Mass: 16050.606 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplM, b3231, JW3200 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0AA10
#16: Protein 50S ribosomal protein L14 / / Large ribosomal subunit protein uL14


Mass: 13565.067 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplN, b3310, JW3272 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0ADY3
#17: Protein 50S ribosomal protein L15 / / Large ribosomal subunit protein uL15


Mass: 15008.471 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplO, b3301, JW3263 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P02413
#18: Protein 50S ribosomal protein L16 / / Large ribosomal subunit protein uL16


Mass: 15312.269 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplP, b3313, JW3275 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0ADY7
#19: Protein 50S ribosomal protein L17 / / Large ribosomal subunit protein bL17


Mass: 14393.657 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplQ, b3294, JW3256 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0AG44
#20: Protein 50S ribosomal protein L18 / / Large ribosomal subunit protein uL18


Mass: 12794.668 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplR, b3304, JW3266 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0C018
#21: Protein 50S ribosomal protein L19 / / Large ribosomal subunit protein bL19


Mass: 13159.278 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplS, b2606, JW2587 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7K6
#22: Protein 50S ribosomal protein L20 / / Large ribosomal subunit protein bL20


Mass: 13528.024 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplT, pdzA, b1716, JW1706 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7L3
#23: Protein 50S ribosomal protein L21 / / Large ribosomal subunit protein bL21


Mass: 11586.374 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplU, b3186, JW3153 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0AG48
#24: Protein 50S ribosomal protein L22 / / Large ribosomal subunit protein uL22


Mass: 12253.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplV, eryB, b3315, JW3277 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P61175
#25: Protein 50S ribosomal protein L23 / / Large ribosomal subunit protein uL23


Mass: 11222.160 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplW, b3318, JW3280 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0ADZ0
#26: Protein 50S ribosomal protein L24 / / Large ribosomal subunit protein uL24


Mass: 11339.250 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplX, b3309, JW3271 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P60624
#27: Protein 50S ribosomal protein L25 / / Large ribosomal subunit protein bL25


Mass: 10713.465 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rplY, b2185, JW2173 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P68919
#28: Protein 50S ribosomal protein L27 / / Large ribosomal subunit protein bL27


Mass: 9146.540 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpmA, b3185, JW3152 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7L8
#29: Protein 50S ribosomal protein L28 / / Large ribosomal subunit protein bL28


Mass: 9027.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpmB, b3637, JW3612 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7M2
#30: Protein 50S ribosomal protein L29 / / Large ribosomal subunit protein uL29


Mass: 7286.464 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpmC, b3312, JW3274 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7M6
#31: Protein 50S ribosomal protein L30 / / Large ribosomal subunit protein uL30


Mass: 6554.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpmD, b3302, JW3264 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0AG51

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RNA chain , 5 types, 5 molecules ABavx

#7: RNA chain 23S ribosomal RNA /


Mass: 941306.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Production host: Escherichia coli K-12 (bacteria) / References: GenBank: 802133627
#8: RNA chain 5S ribosomal RNA /


Mass: 38790.090 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Production host: Escherichia coli K-12 (bacteria) / References: GenBank: 1706539273
#32: RNA chain 16S ribosomal RNA /


Mass: 499054.625 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Production host: Escherichia coli K-12 (bacteria) / References: GenBank: 1476653947
#53: RNA chain mRNA / Messenger RNA


Mass: 1866.181 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Production host: Escherichia coli K-12 (bacteria)
#54: RNA chain P-site tRNA-Pro(CGG)


Mass: 24846.746 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Production host: Escherichia coli K-12 (bacteria) / References: GenBank: 1727528259

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

#33: Protein 30S ribosomal protein S2 /


Mass: 26781.670 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsB, b0169, JW0164 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7V0
#34: Protein 30S ribosomal protein S3 / / Small ribosomal subunit protein uS3


Mass: 26031.316 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsC, b3314, JW3276 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7V3
#35: Protein 30S ribosomal protein S4 / / Small ribosomal subunit protein uS4


Mass: 23514.199 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsD, ramA, b3296, JW3258 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7V8
#36: Protein 30S ribosomal protein S5 /


Mass: 17629.398 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsE, spc, b3303, JW3265 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7W1
#37: Protein 30S ribosomal protein S6 /


Mass: 15727.512 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsF, b4200, JW4158 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P02358
#38: Protein 30S ribosomal protein S7 /


Mass: 20055.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsG, b3341, JW3303 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P02359
#39: Protein 30S ribosomal protein S8 / / Small ribosomal subunit protein uS8


Mass: 14146.557 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsH, b3306, JW3268 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7W7
#40: Protein 30S ribosomal protein S9 / / Small ribosomal subunit protein uS9


Mass: 14886.270 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsI, b3230, JW3199 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7X3
#41: Protein 30S ribosomal protein S10 / / Small ribosomal subunit protein uS10


Mass: 11755.597 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsJ, nusE, b3321, JW3283 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7R5
#42: Protein 30S ribosomal protein S11 / / Small ribosomal subunit protein uS11


Mass: 13870.975 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsK, b3297, JW3259 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7R9
#43: Protein 30S ribosomal protein S12 / / Small ribosomal subunit protein uS12


Mass: 13768.157 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsL, strA, b3342, JW3304 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7S3
#44: Protein 30S ribosomal protein S13 / / Small ribosomal subunit protein uS13


Mass: 13128.467 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsM, b3298, JW3260 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7S9
#45: Protein 30S ribosomal protein S14 / / Small ribosomal subunit protein uS14


Mass: 11606.560 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsN, b3307, JW3269 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0AG59
#46: Protein 30S ribosomal protein S15 / / Small ribosomal subunit protein uS15


Mass: 10290.816 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsO, secC, b3165, JW3134 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0ADZ4
#47: Protein 30S ribosomal protein S16 / / Small ribosomal subunit protein bS16


Mass: 9207.572 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsP, b2609, JW2590 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7T3
#48: Protein 30S ribosomal protein S17 / / Small ribosomal subunit protein uS17


Mass: 9724.491 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsQ, neaA, b3311, JW3273 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0AG63
#49: Protein 30S ribosomal protein S18 / / Small ribosomal subunit protein bS18


Mass: 9005.472 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsR, b4202, JW4160 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7T7
#50: Protein 30S ribosomal protein S19 / / Small ribosomal subunit protein uS19


Mass: 10455.355 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsS, b3316, JW3278 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7U3
#51: Protein 30S ribosomal protein S20 / / Small ribosomal subunit protein bS20


Mass: 9708.464 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsT, b0023, JW0022 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A7U7
#52: Protein 30S ribosomal protein S21 / / Small ribosomal subunit protein bS21


Mass: 8524.039 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: rpsU, b3065, JW3037 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P68679

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Protein , 2 types, 2 molecules yz

#55: Protein Uncharacterized protein YqkK


Mass: 8113.376 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (strain 168) (bacteria)
Gene: yqkK, BSU23540 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P54573
#56: Protein Peptide chain release factor 2 / RF-2


Mass: 41638.559 Da / Num. of mol.: 1 / Mutation: Q251P
Source method: isolated from a genetically manipulated source
Details: -Q251P mutation (hydrolysis defective release factor) -low resolution regions were only the backbone was traced were kept as ALA
Source: (gene. exp.) Bacillus subtilis (strain 168) (bacteria)
Gene: prfB, BSU35290 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P28367

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Experimental details

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Experiment

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

-
Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1E. coli 70S stalled on truncated mRNA in complex BsBrfA and BsRF2-Q251PRIBOSOMEE. coli 70S stalled on truncated mRNA in complex with Bacillus ribosome rescue factor A (BrfA) and a hydrolysis deminished mutant of Bacillus peptide chain release factor 2 (RF2-Q251P)all0MULTIPLE SOURCES
2Escherichia coli ribosome with P-site tRNA-Pro(CGG, carrying the nascent chainCOMPLEX#1-#541RECOMBINANT
3Bacillus subtilis ribosome rescue factor (BrfA; former YqkK) and peptide chain release factor 2 (RF2)COMPLEX#55-#561RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli K-12 (bacteria)83333
23Bacillus subtilis (strain 168) (bacteria)224308
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli K-12 (bacteria)83333
23Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingElectron dose: 83 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

EM software
IDNameVersionCategory
7Cootmodel fitting
12RELION33D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 154405 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 5MGP

5mgp

RefinementStereochemistry target values: CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0081159953
ELECTRON MICROSCOPYf_angle_d0.9311238970
ELECTRON MICROSCOPYf_chiral_restr0.052630476
ELECTRON MICROSCOPYf_plane_restr0.006613031
ELECTRON MICROSCOPYf_dihedral_angle_d15.307385659

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