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- PDB-8rcm: Escherichia coli paused disome complex (Non-rotated disome interf... -

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Basic information

Entry
Database: PDB / ID: 8rcm
TitleEscherichia coli paused disome complex (Non-rotated disome interface class 2)
Components
  • (30S ribosomal protein ...) x 7
  • (50S ribosomal protein ...) x 12
  • (Large ribosomal subunit protein ...) x 2
  • (Small ribosomal subunit protein ...) x 14
  • 16S ribosomal RNA
  • 23S ribosomal RNA
  • 5S ribosomal RNA
  • Nascent chain
  • messenger RNA
  • tRNA-Ala (A-site)
  • tRNA-Arg (E-site)
  • tRNA-Phe (P-site)
  • tRNA-Trp (P-site)
  • tRNA-Val (A-site)
KeywordsRIBOSOME / polysome / tranlsation / elongation / pausing / disome / collision / PURE system
Function / homology
Function and homology information


RNA secondary structure unwinding / positive regulation of cytoplasmic translation / 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 / positive regulation of ribosome biogenesis ...RNA secondary structure unwinding / positive regulation of cytoplasmic translation / 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 / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / DnaA-L2 complex / four-way junction DNA binding / negative regulation of translational initiation / translational initiation / negative regulation of DNA-templated DNA replication initiation / regulation of mRNA stability / mRNA regulatory element binding translation repressor activity / assembly of large subunit precursor of preribosome / positive regulation of RNA splicing / DNA endonuclease activity / transcription elongation factor complex / cytosolic ribosome assembly / regulation of DNA-templated transcription elongation / transcription antitermination / regulation of cell growth / maintenance of translational fidelity / DNA-templated transcription termination / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / ribosomal large subunit assembly / small ribosomal subunit rRNA binding / ribosomal small subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / ribosome binding / ribosome biogenesis / regulation of translation / cytoplasmic translation / small ribosomal subunit / 5S rRNA binding / cytosolic large ribosomal subunit / transferase activity / tRNA binding / negative regulation of translation / single-stranded RNA binding / rRNA binding / molecular adaptor activity / ribosome / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Ribosomal protein S1 / Ribosomal protein S1-like / Ribosomal protein L1, bacterial-type / S1 domain profile. / Ribosomal protein L1, conserved site / Ribosomal protein L1 / Ribosomal protein L1 signature. / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. ...Ribosomal protein S1 / Ribosomal protein S1-like / Ribosomal protein L1, bacterial-type / S1 domain profile. / Ribosomal protein L1, conserved site / Ribosomal protein L1 / Ribosomal protein L1 signature. / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L1p/L10e family / Ribosomal protein S1-like RNA-binding domain / Ribosomal protein L31 type A / S1 RNA binding domain / Ribosomal protein S21 superfamily / Ribosomal protein S21 / S1 domain / 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 L16 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 L28/L24 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 L18, bacterial-type / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein S3, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein S19, bacterial-type / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S4, bacterial-type / Ribosomal protein L34, conserved site / Ribosomal protein L34 signature. / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-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. / L28p-like / Ribosomal protein S16 / Ribosomal protein S16 / Ribosomal protein S16 domain superfamily / Ribosomal protein L27 / Ribosomal L27 protein / Ribosomal protein S15, bacterial-type / Ribosomal protein S2 signature 2. / Ribosomal protein S6 / Ribosomal protein S6 / Ribosomal protein S6 superfamily / Ribosomal protein S12, bacterial-type / Ribosomal protein L34 / Ribosomal protein L34 / Translation elongation factor EF1B/ribosomal protein S6 / Ribosomal protein L15, bacterial-type
Similarity search - Domain/homology
ALANINE / 1,4-DIAMINOBUTANE / : / : / : / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) ...ALANINE / 1,4-DIAMINOBUTANE / : / : / : / : / 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 uL1 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein bL31 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / 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 uL16 / Small ribosomal subunit protein uS15 / Large ribosomal subunit protein uL30 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Small ribosomal subunit protein bS1 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL5 / Small ribosomal subunit protein bS21
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.59 Å
AuthorsFluegel, T. / Schacherl, M.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG) Germany
CitationJournal: Nat Commun / Year: 2024
Title: Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM.
Authors: Timo Flügel / Magdalena Schacherl / Anett Unbehaun / Birgit Schroeer / Marylena Dabrowski / Jörg Bürger / Thorsten Mielke / Thiemo Sprink / Christoph A Diebolder / Yollete V Guillén ...Authors: Timo Flügel / Magdalena Schacherl / Anett Unbehaun / Birgit Schroeer / Marylena Dabrowski / Jörg Bürger / Thorsten Mielke / Thiemo Sprink / Christoph A Diebolder / Yollete V Guillén Schlippe / Christian M T Spahn /
Abstract: Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex ...Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9's CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts.
History
DepositionDec 6, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_1 ..._chem_comp_atom.atom_id / _chem_comp_bond.atom_id_1 / _chem_comp_bond.atom_id_2 / _chem_comp_bond.pdbx_aromatic_flag / _chem_comp_bond.value_order

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
12: 50S ribosomal protein L28
32: 50S ribosomal protein L30
4: Large ribosomal subunit protein bL31A
62: 50S ribosomal protein L35
71: 23S ribosomal RNA
72: 23S ribosomal RNA
82: 5S ribosomal RNA
A1: 16S ribosomal RNA
A2: 16S ribosomal RNA
B: Small ribosomal subunit protein uS2
B2: Small ribosomal subunit protein uS2
C1: Small ribosomal subunit protein uS3
C2: Small ribosomal subunit protein uS3
D1: Small ribosomal subunit protein uS4
D2: Small ribosomal subunit protein uS4
E1: Small ribosomal subunit protein uS5
E2: Small ribosomal subunit protein uS5
F1: 30S ribosomal protein S6
F2: 30S ribosomal protein S6
G1: 30S ribosomal protein S7
G2: 30S ribosomal protein S7
H1: Small ribosomal subunit protein uS8
H2: Small ribosomal subunit protein uS8
I1: Small ribosomal subunit protein uS9
I2: Small ribosomal subunit protein uS9
J1: 30S ribosomal protein S10
J2: 30S ribosomal protein S10
K1: Small ribosomal subunit protein uS11
K2: Small ribosomal subunit protein uS11
L1: Small ribosomal subunit protein uS12
L2: Small ribosomal subunit protein uS12
M1: Small ribosomal subunit protein uS13
M2: Small ribosomal subunit protein uS13
N1: Small ribosomal subunit protein uS14
N2: Small ribosomal subunit protein uS14
O1: 30S ribosomal protein S15
O2: 30S ribosomal protein S15
P1: 30S ribosomal protein S16
Q1: Small ribosomal subunit protein uS17
Q2: Small ribosomal subunit protein uS17
R1: Small ribosomal subunit protein bS18
R2: Small ribosomal subunit protein bS18
S1: Small ribosomal subunit protein uS19
S2: Small ribosomal subunit protein uS19
T1: Small ribosomal subunit protein bS20
U1: 30S ribosomal protein S21
U2: 30S ribosomal protein S21
V2: messenger RNA
W: tRNA-Trp (P-site)
W1: tRNA-Phe (P-site)
X2: tRNA-Arg (E-site)
Y1: tRNA-Val (A-site)
Y2: tRNA-Ala (A-site)
Z1: 30S ribosomal protein S1
a2: Large ribosomal subunit protein uL1
b2: 50S ribosomal protein L2
e2: 50S ribosomal protein L5
g2: 50S ribosomal protein L33
h2: 50S ribosomal protein L16
i2: 50S ribosomal protein L9
l2: 50S ribosomal protein L34
o2: 50S ribosomal protein L15
p: Nascent chain
r2: 50S ribosomal protein L18
z2: 50S ribosomal protein L27
hetero molecules


Theoretical massNumber of molelcules
Total (without water)3,882,660366
Polymers3,875,07065
Non-polymers7,590301
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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50S ribosomal protein ... , 12 types, 12 molecules 123262b2e2g2h2i2l2o2r2z2

#1: Protein 50S ribosomal protein L28 / / ribosomal protein bL28


Mass: 9027.551 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7M2
#2: Protein 50S ribosomal protein L30 / / ribosomal protein uL30


Mass: 6554.820 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0AG51
#4: Protein 50S ribosomal protein L35 / / Ribosomal protein A / ribosomal protein bL35


Mass: 7313.032 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7Q1
#36: Protein 50S ribosomal protein L2 / / ribosomal protein uL2


Mass: 29923.619 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P60422
#37: Protein 50S ribosomal protein L5 / / ribosomal protein uL5


Mass: 20333.611 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P62399
#38: Protein 50S ribosomal protein L33 / / ribosomal protein bL33


Mass: 6388.631 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7N9
#39: Protein 50S ribosomal protein L16 / / ribosomal protein uL16


Mass: 15312.269 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0ADY7
#40: Protein 50S ribosomal protein L9 / / ribosomal protein bL9


Mass: 15789.020 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7R1
#41: Protein/peptide 50S ribosomal protein L34 / / ribosomal protein bL34


Mass: 5397.463 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7P5
#42: Protein 50S ribosomal protein L15 / / ribosomal protein uL15


Mass: 15008.471 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P02413
#44: Protein 50S ribosomal protein L18 / / ribosomal protein uL18


Mass: 12794.668 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0C018
#45: 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 (E. coli) / References: UniProt: P0A7L8

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Large ribosomal subunit protein ... , 2 types, 2 molecules 4a2

#3: Protein Large ribosomal subunit protein bL31A / 50S ribosomal protein L31


Mass: 7887.117 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7M9
#35: Protein Large ribosomal subunit protein uL1 / 50S ribosomal protein L1


Mass: 24765.660 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7L0

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RNA chain , 9 types, 11 molecules 717282A1A2V2WW1X2Y1Y2

#5: RNA chain 23S ribosomal RNA /


Mass: 941812.562 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#6: RNA chain 5S ribosomal RNA /


Mass: 38790.090 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: GenBank: CP035706.1
#7: RNA chain 16S ribosomal RNA /


Mass: 499873.406 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: GenBank: 1388194076
#28: RNA chain messenger RNA /


Mass: 20786.527 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#29: RNA chain tRNA-Trp (P-site)


Mass: 24628.865 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#30: RNA chain tRNA-Phe (P-site)


Mass: 24651.904 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#31: RNA chain tRNA-Arg (E-site)


Mass: 24978.115 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#32: RNA chain tRNA-Val (A-site)


Mass: 24617.768 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: GenBank: 1847302804
#33: RNA chain tRNA-Ala (A-site)


Mass: 24555.654 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: GenBank: 1845258627

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Small ribosomal subunit protein ... , 14 types, 27 molecules BB2C1C2D1D2E1E2H1H2I1I2K1K2L1L2M1M2N1N2Q1Q2R1R2S1S2T1

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


Mass: 26781.670 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7V0
#9: Protein Small ribosomal subunit protein uS3 / 30S ribosomal protein S3


Mass: 26031.316 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7V3
#10: Protein Small ribosomal subunit protein uS4 / 30S ribosomal protein S4


Mass: 23514.199 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7V8
#11: Protein Small ribosomal subunit protein uS5 / 30S ribosomal protein S5


Mass: 17629.398 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7W1
#14: Protein Small ribosomal subunit protein uS8 / 30S ribosomal protein S8


Mass: 14146.557 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7W7
#15: Protein Small ribosomal subunit protein uS9 / 30S ribosomal protein S9


Mass: 14886.270 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7X3
#17: Protein Small ribosomal subunit protein uS11 / 30S ribosomal protein S11


Mass: 13870.975 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7R9
#18: Protein Small ribosomal subunit protein uS12 / 30S ribosomal protein S12


Mass: 13768.157 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7S3
#19: Protein Small ribosomal subunit protein uS13 / 30S ribosomal protein S13


Mass: 13128.467 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7S9
#20: Protein Small ribosomal subunit protein uS14 / 30S ribosomal protein S14


Mass: 11606.560 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0AG59
#23: Protein Small ribosomal subunit protein uS17 / 30S ribosomal protein S17


Mass: 9724.491 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0AG63
#24: Protein Small ribosomal subunit protein bS18 / 30S ribosomal protein S18


Mass: 9005.472 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7T7
#25: Protein Small ribosomal subunit protein uS19 / 30S ribosomal protein S19


Mass: 10455.355 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7U3
#26: Protein Small ribosomal subunit protein bS20 / 30S ribosomal protein S20


Mass: 9708.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7U7

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30S ribosomal protein ... , 7 types, 12 molecules F1F2G1G2J1J2O1O2P1U1U2Z1

#12: Protein 30S ribosomal protein S6 / / Small ribosomal subunit protein bS6


Mass: 15727.512 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P02358
#13: Protein 30S ribosomal protein S7 / / Small ribosomal subunit protein uS7


Mass: 20055.156 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P02359
#16: Protein 30S ribosomal protein S10 / / Small ribosomal subunit protein uS10 / Transcription termination/antitermination protein NusE


Mass: 11755.597 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7R5
#21: Protein 30S ribosomal protein S15 / / Small ribosomal subunit protein uS15


Mass: 10290.816 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0ADZ4
#22: 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 (E. coli) / References: UniProt: P0A7T3
#27: Protein 30S ribosomal protein S21 / / Small ribosomal subunit protein bS21


Mass: 8524.039 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P68679
#34: Protein 30S ribosomal protein S1 / / Bacteriophage Q beta RNA-directed RNA polymerase subunit I / Small ribosomal subunit protein bS1


Mass: 61238.887 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0AG67

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Protein/peptide , 1 types, 1 molecules p

#43: Protein/peptide Nascent chain


Mass: 1087.235 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)

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Non-polymers , 4 types, 301 molecules

#46: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#47: Chemical ChemComp-PUT / 1,4-DIAMINOBUTANE / PUTRESCINE / Putrescine


Mass: 88.151 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H12N2
#48: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 296 / Source method: obtained synthetically / Formula: Mg
#49: Chemical ChemComp-ALA / ALANINE / Alanine


Type: L-peptide linking / Mass: 89.093 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H7NO2

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Details

Has ligand of interestN

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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

ComponentName: Escherichia coli paused disome complex / Type: RIBOSOME
Details: Structure of the non-rotated disome interface class 2
Entity ID: #1-#32, #34-#45 / Source: NATURAL
Molecular weightValue: 2.270379 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE 600
Buffer solutionpH: 7.6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: In vitro translation reactions were performed in the PURE translation system using the PURExpress delta ribosome kit (NEB, #E3313S). Translation reactions were supplemented with 0.8 U/uL ...Details: In vitro translation reactions were performed in the PURE translation system using the PURExpress delta ribosome kit (NEB, #E3313S). Translation reactions were supplemented with 0.8 U/uL RNAsin Plus RNase Inhibitor (Promega, N261B). SolA, factor mix, and RNAsin Plus were combined on ice, followed by a preincubation at 37 degrees Celcius for 2 min, and added directly to polysomes (700 nM final concentration) that had been preincubated at 37 degrees Celsius for 2 min. After 1 min reaction time, 4 uL of the reaction mixture were withdrawn for plunge freezing.
Specimen supportDetails: Operated at 15 mA, easiGlow Discharge Cleaning system (Pelco)
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 75 % / Chamber temperature: 277 K
Details: Withdrawn samples were spotted directly onto freshly glow-discharged holey carbon grids, blotted for 1-2 s, and flash frozen in liquid ethane using a Vitrobot Mark IV plunger (ThermoFisher ...Details: Withdrawn samples were spotted directly onto freshly glow-discharged holey carbon grids, blotted for 1-2 s, and flash frozen in liquid ethane using a Vitrobot Mark IV plunger (ThermoFisher Scientific) after a wait time of 40 s at 4 degrees Celcius.

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 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 / Temperature (max): 82 K / Temperature (min): 80 K
Image recordingAverage exposure time: 1.13 sec. / Electron dose: 45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8982

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Processing

EM software
IDNameVersionCategoryDetails
1Gautomatch0.56particle selection
2EPU2.8.1image acquisition
4Gctf0.53CTF correction
7Coot0.9.6model fitting
9PHENIX1.20.1_4487model refinement
10ERRASERmodel refinementphenix/1.14 and phenix/rosetta
11LIBGccp4-8.0model refinement
12cryoSPARC3.3initial Euler assignment
13cryoSPARC3.3final Euler assignment
14cryoSPARC3.3classification
15cryoSPARC3.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1883839
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29998 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: The atomic model of the E. coli 70S ribosome (PDB ID: 7N1P) was initially docked into the postprocessed density maps with UCSF Chimera and manually adjusted in Coot. All models were refined ...Details: The atomic model of the E. coli 70S ribosome (PDB ID: 7N1P) was initially docked into the postprocessed density maps with UCSF Chimera and manually adjusted in Coot. All models were refined over multiple rounds using the module phenix.real_space_refine in PHENIX and interactive model building and refinement in Coot, using libG restraints for the RNAs.
Atomic model buildingPDB-ID: 7N1P

7n1p


Accession code: 7N1P / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002207579
ELECTRON MICROSCOPYf_angle_d0.516312976
ELECTRON MICROSCOPYf_dihedral_angle_d16.47485368
ELECTRON MICROSCOPYf_chiral_restr0.03140184
ELECTRON MICROSCOPYf_plane_restr0.00715180

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