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- PDB-8p8b: Mycoplasma pneumoniae large ribosomal subunit in chloramphenicol-... -

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Basic information

Entry
Database: PDB / ID: 8p8b
TitleMycoplasma pneumoniae large ribosomal subunit in chloramphenicol-treated cells
Components
  • (50S ribosomal protein ...) x 29
  • 16S ribosomal RNA
  • 23S ribosomal RNA
  • 5S ribosomal RNA
  • Trigger factor
  • mRNA
  • nascent peptide
  • tRNA-Ala (E-site)
  • tRNA-Asp (P-site)
  • tRNA-Lys (A-site)
KeywordsTRANSLATION / In situ / Ribosome / Chloramphenicol / cryo-ET
Function / homology
Function and homology information


RNA polymerase II CTD heptapeptide repeat P3 isomerase activity / RNA polymerase II CTD heptapeptide repeat P6 isomerase activity / peptidylprolyl isomerase / protein transport / protein folding / large ribosomal subunit / transferase activity / 5S rRNA binding / ribosomal large subunit assembly / large ribosomal subunit rRNA binding ...RNA polymerase II CTD heptapeptide repeat P3 isomerase activity / RNA polymerase II CTD heptapeptide repeat P6 isomerase activity / peptidylprolyl isomerase / protein transport / protein folding / large ribosomal subunit / transferase activity / 5S rRNA binding / ribosomal large subunit assembly / large ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / ribonucleoprotein complex / cell division / response to antibiotic / mRNA binding / cytoplasm
Similarity search - Function
Trigger factor / Trigger factor, C-terminal / Trigger factor, ribosome-binding, bacterial / Trigger factor ribosome-binding domain superfamily / Bacterial trigger factor protein (TF) / Bacterial trigger factor protein (TF) C-terminus / Trigger factor, C-terminal domain superfamily / Trigger factor/SurA domain superfamily / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. ...Trigger factor / Trigger factor, C-terminal / Trigger factor, ribosome-binding, bacterial / Trigger factor ribosome-binding domain superfamily / Bacterial trigger factor protein (TF) / Bacterial trigger factor protein (TF) C-terminus / Trigger factor, C-terminal domain superfamily / Trigger factor/SurA domain superfamily / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / : / : / FKBP-type peptidyl-prolyl cis-trans isomerase domain profile. / FKBP-type peptidyl-prolyl cis-trans isomerase / FKBP-type peptidyl-prolyl cis-trans isomerase domain / Peptidyl-prolyl cis-trans isomerase domain superfamily / Ribosomal protein L11, bacterial-type / Ribosomal protein L31 type A / Ribosomal protein L31 signature. / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L10-like domain superfamily / Ribosomal protein L10P / Ribosomal protein L10 / Ribosomal protein L16 signature 1. / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L16 signature 2. / Ribosomal protein L16, conserved site / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / : / 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 L11, N-terminal / Ribosomal protein L11, N-terminal domain / Ribosomal protein L17 signature. / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, RNA binding domain / Ribosomal protein L36 signature. / Ribosomal protein L32p, bacterial type / 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 L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L28 / 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 L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein L20 signature. / Ribosomal protein L22, bacterial/chloroplast-type / Ribosomal protein L14P, bacterial-type / Ribosomal protein L34, conserved site / Ribosomal protein L34 signature. / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L35 / Ribosomal protein L35 superfamily / Ribosomal protein L35 / Ribosomal protein L33 / Ribosomal protein L33 / Ribosomal L28 family / Ribosomal protein L33 superfamily / Ribosomal protein L16 / Ribosomal protein L28/L24 / Ribosomal protein L18 / Ribosomal L18 of archaea, bacteria, mitoch. and chloroplast / : / L28p-like / Ribosomal protein L27 / Ribosomal L27 protein / Ribosomal protein L20 / Ribosomal L32p protein family / Ribosomal protein L19
Similarity search - Domain/homology
CHLORAMPHENICOL / : / LYSINE / PENTANE-1,5-DIAMINE / 1,4-DIAMINOBUTANE / SPERMIDINE / SPERMINE / : / RNA / RNA (> 10) ...CHLORAMPHENICOL / : / LYSINE / PENTANE-1,5-DIAMINE / 1,4-DIAMINOBUTANE / SPERMIDINE / SPERMINE / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein bL36 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein bL35 / Trigger factor / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL9 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL33A / Large ribosomal subunit protein bL31 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein uL5 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL17
Similarity search - Component
Biological speciesMycoplasmoides pneumoniae M129 (bacteria)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 2.9 Å
AuthorsSchacherl, M. / Xue, L. / Spahn, C.M.T. / Mahamid, J.
Funding support United States, Germany, 2items
OrganizationGrant numberCountry
Chan Zuckerberg InitiativeVisual Proteomics Imaging United States
German Research Foundation (DFG) Germany
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Structural insights into context-dependent inhibitory mechanisms of chloramphenicol in cells.
Authors: Liang Xue / Christian M T Spahn / Magdalena Schacherl / Julia Mahamid /
Abstract: Ribosome-targeting antibiotics represent an important class of antimicrobial drugs. Chloramphenicol (Cm) is a well-studied ribosomal peptidyl transferase center (PTC) binder and growing evidence ...Ribosome-targeting antibiotics represent an important class of antimicrobial drugs. Chloramphenicol (Cm) is a well-studied ribosomal peptidyl transferase center (PTC) binder and growing evidence suggests that its inhibitory action depends on the sequence of the nascent peptide. How such selective inhibition on the molecular scale manifests on the cellular level remains unclear. Here, we use cryo-electron tomography to analyze the impact of Cm inside the bacterium Mycoplasma pneumoniae. By resolving the Cm-bound ribosomes to 3.0 Å, we elucidate Cm's coordination with natural nascent peptides and transfer RNAs in the PTC. We find that Cm leads to the accumulation of a number of translation elongation states, indicating ongoing futile accommodation cycles, and to extensive ribosome collisions. We, thus, suggest that, beyond its direct inhibition of protein synthesis, the action of Cm may involve the activation of cellular stress responses. This work exemplifies how in-cell structural biology can expand the understanding of mechanisms of action for extensively studied antibiotics.
History
DepositionMay 31, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 20, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Feb 26, 2025Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.3Mar 12, 2025Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Item: _chem_comp_atom.atom_id / _chem_comp_atom.pdbx_aromatic_flag ..._chem_comp_atom.atom_id / _chem_comp_atom.pdbx_aromatic_flag / _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
0: 50S ribosomal protein L34
1: 50S ribosomal protein L35
2: 50S ribosomal protein L36
3: 23S ribosomal RNA
4: 5S ribosomal RNA
5: 16S ribosomal RNA
6: tRNA-Ala (E-site)
7: tRNA-Asp (P-site)
8: tRNA-Lys (A-site)
X: Trigger factor
Y: mRNA
Z: nascent peptide
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 L10
h: 50S ribosomal protein L11
i: 50S ribosomal protein L13
j: 50S ribosomal protein L14
k: 50S ribosomal protein L15
l: 50S ribosomal protein L16
m: 50S ribosomal protein L17
n: 50S ribosomal protein L18
o: 50S ribosomal protein L19
p: 50S ribosomal protein L20
q: 50S ribosomal protein L21
r: 50S ribosomal protein L22
s: 50S ribosomal protein L23
t: 50S ribosomal protein L24
u: 50S ribosomal protein L27
v: 50S ribosomal protein L28
w: 50S ribosomal protein L29
x: 50S ribosomal protein L31
y: 50S ribosomal protein L32
z: 50S ribosomal protein L33 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,044,186304
Polymers2,033,79238
Non-polymers10,394266
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area214420 Å2
ΔGint-3496 kcal/mol
Surface area530660 Å2

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Components

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50S ribosomal protein ... , 29 types, 29 molecules 012abcdefghijklmnopqrstuvwxyz

#1: Protein/peptide 50S ribosomal protein L34


Mass: 5616.773 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P78006
#2: Protein 50S ribosomal protein L35


Mass: 6793.135 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75447
#3: Protein/peptide 50S ribosomal protein L36


Mass: 4400.488 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P52864
#13: Protein 50S ribosomal protein L2


Mass: 31966.871 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75577
#14: Protein 50S ribosomal protein L3


Mass: 31298.172 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75580
#15: Protein 50S ribosomal protein L4


Mass: 23632.572 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75579
#16: Protein 50S ribosomal protein L5


Mass: 20270.834 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: Q50306
#17: Protein 50S ribosomal protein L6


Mass: 20624.062 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: Q50303
#18: Protein 50S ribosomal protein L9


Mass: 17185.037 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75540
#19: Protein 50S ribosomal protein L10


Mass: 17645.719 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75240
#20: Protein 50S ribosomal protein L11


Mass: 14803.610 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75550
#21: Protein 50S ribosomal protein L13


Mass: 16821.477 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75178
#22: Protein 50S ribosomal protein L14


Mass: 13478.906 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: Q50308
#23: Protein 50S ribosomal protein L15


Mass: 16768.812 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: Q50300
#24: Protein 50S ribosomal protein L16


Mass: 15646.479 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P41204
#25: Protein 50S ribosomal protein L17


Mass: 14270.727 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: Q59547
#26: Protein 50S ribosomal protein L18


Mass: 13073.256 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: Q50302
#27: Protein 50S ribosomal protein L19


Mass: 13870.327 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75133
#28: Protein 50S ribosomal protein L20


Mass: 14742.431 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P78023
#29: Protein 50S ribosomal protein L21


Mass: 11664.824 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P78026
#30: Protein 50S ribosomal protein L22


Mass: 17355.402 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75575
#31: Protein 50S ribosomal protein L23


Mass: 25892.740 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75578
#32: Protein 50S ribosomal protein L24


Mass: 12461.865 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: Q50307
#33: Protein 50S ribosomal protein L27


Mass: 11527.338 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75458
#34: Protein 50S ribosomal protein L28


Mass: 7549.902 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75171
#35: Protein 50S ribosomal protein L29


Mass: 13003.328 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: Q50310
#36: Protein 50S ribosomal protein L31


Mass: 10980.657 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P78020
#37: Protein 50S ribosomal protein L32


Mass: 6641.897 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75238
#38: Protein 50S ribosomal protein L33 1


Mass: 6298.612 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P78015

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RNA chain , 7 types, 7 molecules 345678Y

#4: RNA chain 23S ribosomal RNA


Mass: 940953.500 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: GenBank: 26117688
#5: RNA chain 5S ribosomal RNA


Mass: 34796.676 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: GenBank: 26117688
#6: RNA chain 16S ribosomal RNA


Mass: 491327.844 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: Sequence comprises 16S rRNA ribonucleotides 1374-1387 and 1463-1480 only.
Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: GenBank: 26117688
#7: RNA chain tRNA-Ala (E-site)


Mass: 24440.492 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: GenBank: 26117688
#8: RNA chain tRNA-Asp (P-site)


Mass: 24136.244 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: GenBank: 26117688
#9: RNA chain tRNA-Lys (A-site)


Mass: 24401.449 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: GenBank: 26117688
#11: RNA chain mRNA


Mass: 2894.807 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria)

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Protein / Protein/peptide , 2 types, 2 molecules XZ

#10: Protein Trigger factor / TF / PPIase


Mass: 51427.031 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria) / References: UniProt: P75454, peptidylprolyl isomerase
#12: Protein/peptide nascent peptide


Mass: 3127.794 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mycoplasmoides pneumoniae M129 (bacteria)

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Non-polymers , 9 types, 266 molecules

#39: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#40: Chemical ChemComp-CLM / CHLORAMPHENICOL


Mass: 323.129 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C11H12Cl2N2O5 / Feature type: SUBJECT OF INVESTIGATION / Comment: antibiotic*YM
#41: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K
#42: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 229 / Source method: obtained synthetically / Formula: Mg
#43: Chemical
ChemComp-PUT / 1,4-DIAMINOBUTANE / PUTRESCINE


Mass: 88.151 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C4H12N2
#44: Chemical
ChemComp-SPM / SPERMINE


Mass: 202.340 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H26N4
#45: Chemical
ChemComp-SPD / SPERMIDINE / N-(2-AMINO-PROPYL)-1,4-DIAMINOBUTANE / PA(34)


Mass: 145.246 Da / Num. of mol.: 16 / Source method: obtained synthetically / Formula: C7H19N3
#46: Chemical ChemComp-N2P / PENTANE-1,5-DIAMINE


Mass: 102.178 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C5H14N2
#47: Chemical ChemComp-LYS / LYSINE


Type: L-peptide linking / Mass: 147.195 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H15N2O2

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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: CELL / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: Large ribosomal subunit with some elements of the small ribosomal subunit. Focused refinement on 50S subunit of the in situ 70S subunit map.
Type: CELL
Details: Mycoplasma pneumoniae M129 cells treated with chloramphenicol
Entity ID: #1-#8, #10-#38 / Source: NATURAL
Source (natural)Organism: Mycoplasmoides pneumoniae M129 (bacteria)
Buffer solutionpH: 7.4
Details: The modified Hayflick medium: 14.7g/L Difco PPLO(Becton Dickinson), 20% (v/v) Gibco horse serum(New Zealand origin), 100 mM HEPES-Na; pH 7.4, 1% (w/w) glucose, 0.002% (w/w) phenol red, 1000 U/mL penicillin G.
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Mycoplasma pneumoniae M129 cells were grown on gold Quantifoil grids at 37 Celsius in the modified Hayflick medium. Treatment with chloramphenicol at the final concentration of 0.2 mg/ml was ...Details: Mycoplasma pneumoniae M129 cells were grown on gold Quantifoil grids at 37 Celsius in the modified Hayflick medium. Treatment with chloramphenicol at the final concentration of 0.2 mg/ml was performed for about 15 minutes before plunge freezing.
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE-PROPANE
Details: Back-side blotting for 2-3 seconds before plunging using a manual plunger without an environmental control chamber.

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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 FIELD / Nominal magnification: 64000 X / Nominal defocus max: 3250 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 3.34 e/Å2 / Avg electron dose per subtomogram: 137 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 1
Details: Gatan K3 camera in non-CDS counting mode, targeted dose rate on camera ~20 e/pixel/second, 10 frames per tilt image, constant exposure time for each tilt, pixel size 1.329A
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1PyTom0.9.7.1volume selection
2Warp1.0.9volume selection
3SerialEM3.9image acquisition
5Warp1.0.9CTF correction
8Coot0.9.6model fittinginteractive model building and refinement
9Coot0.9.8.5model fittinginteractive model building and refinement
10CCP4 package8model fittinglibG restraints
13RELION3.0.8final Euler assignment
14Warp1.0.9final Euler assignment
15RELION3.0.8classification
16Warp1.0.93D reconstructionWarp/M
17PHENIX1.20.1_4487model refinementreal space refine
CTF correctionDetails: CTF estimation and 3D CTF correction are done in Warp
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30774 / Algorithm: FOURIER SPACE / Details: half maps from Warp/M / Symmetry type: POINT
EM volume selectionNum. of tomograms: 139 / Num. of volumes extracted: 30774
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
17OOD

7ood

17OOD1PDBexperimental model
27OOC

7ooc

17OOC2PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004105536
ELECTRON MICROSCOPYf_angle_d0.597158059
ELECTRON MICROSCOPYf_dihedral_angle_d16.15949646
ELECTRON MICROSCOPYf_chiral_restr0.02920300
ELECTRON MICROSCOPYf_plane_restr0.0058031

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Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

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