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- PDB-6c4h: Conformation of methylated GGQ in the peptidyl transferase center... -

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Basic information

Entry
Database: PDB / ID: 6c4h
TitleConformation of methylated GGQ in the peptidyl transferase center during translation termination (PTC region)
Components
  • (50S ribosomal protein ...) x 4
  • 23S rRNA23S ribosomal RNA
  • P-site tRNA fMet
  • Peptide chain release factor RF2
KeywordsRIBOSOMAL PROTEIN/RNA / nonstop / termination / ArfA / RF2 / methylation / RIBOSOMAL PROTEIN-RNA complex
Function / homology
Function and homology information


translation release factor activity, codon specific / positive regulation of ribosome biogenesis / translational termination / DnaA-L2 complex / negative regulation of DNA-templated DNA replication initiation / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / regulation of cell growth / ribosomal large subunit assembly / ribosome binding ...translation release factor activity, codon specific / positive regulation of ribosome biogenesis / translational termination / DnaA-L2 complex / negative regulation of DNA-templated DNA replication initiation / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / regulation of cell growth / ribosomal large subunit assembly / ribosome binding / cytoplasmic translation / cytosolic large ribosomal subunit / transferase activity / tRNA binding / rRNA binding / structural constituent of ribosome / RNA binding / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
Peptide chain release factor 2 / Peptide chain release factor / PCRF domain / PCRF / Peptide chain release factor class I superfamily / Prokaryotic-type class I peptide chain release factors signature. / Double Stranded RNA Binding Domain - #20 / Peptide chain release factor class I / RF-1 domain / Double Stranded RNA Binding Domain ...Peptide chain release factor 2 / Peptide chain release factor / PCRF domain / PCRF / Peptide chain release factor class I superfamily / Prokaryotic-type class I peptide chain release factors signature. / Double Stranded RNA Binding Domain - #20 / Peptide chain release factor class I / RF-1 domain / Double Stranded RNA Binding Domain / Ribosomal protein L16 signature 1. / Ribosomal protein L16, conserved site / Ribosomal protein L16 signature 2. / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L16 / Ribosomal protein L27 / Ribosomal L27 protein / Ribosomal protein L3, bacterial/organelle-type / : / Ribosomal protein L2, conserved site / Ribosomal protein L2 signature. / Ribosomal protein L10e/L16 / Ribosomal protein L10e/L16 superfamily / Ribosomal protein L16p/L10e / Ribosomal protein L2, domain 3 / Ribosomal Proteins L2, C-terminal domain / Ribosomal protein L2, C-terminal / Ribosomal Proteins L2, C-terminal domain / Ribosomal Proteins L2, RNA binding domain / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein L2 / Ribosomal Proteins L2, RNA binding domain / Ribosomal protein L3, conserved site / Ribosomal protein L3 / Ribosomal protein L3 / Ribosomal protein L3 signature. / Translation protein SH3-like domain superfamily / Translation protein, beta-barrel domain superfamily / Ribosomal protein L2, domain 2 / Nucleic acid-binding, OB-fold / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
: / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Peptide chain release factor RF2 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsZeng, F. / Jin, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM120552 United States
CitationJournal: Sci Rep / Year: 2018
Title: Conformation of methylated GGQ in the Peptidyl Transferase Center during Translation Termination.
Authors: Fuxing Zeng / Hong Jin /
Abstract: The universally conserved Gly-Gly-Gln (GGQ) tripeptide in release factors or release factor-like surveillance proteins is required to catalyze the release of nascent peptide in the ribosome. The ...The universally conserved Gly-Gly-Gln (GGQ) tripeptide in release factors or release factor-like surveillance proteins is required to catalyze the release of nascent peptide in the ribosome. The glutamine of the GGQ is methylated post-translationally at the N position in vivo; this covalent modification is essential for optimal cell growth and efficient translation termination. However, the precise conformation of the methylated-GGQ tripeptide in the ribosome remains unknown. Using cryoEM and X-ray crystallography, we report the conformation of methylated-GGQ in the peptidyl transferase center of the ribosome during canonical translational termination and co-translation quality control. It has been suggested that the GGQ motif arose independently through convergent evolution among otherwise unrelated proteins that catalyze peptide release. The requirement for this tripeptide in the highly conserved peptidyl transferase center suggests that the conformation reported here is likely shared during termination of protein synthesis in all domains of life.
History
DepositionJan 12, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 21, 2018Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2018Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 2.0Jan 8, 2020Group: Author supporting evidence / Polymer sequence / Category: entity_poly / pdbx_audit_support
Item: _entity_poly.pdbx_seq_one_letter_code_can / _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: 23S rRNA
C: 50S ribosomal protein L2
D: 50S ribosomal protein L3
N: 50S ribosomal protein L16
X: 50S ribosomal protein L27
x: P-site tRNA fMet
v: Peptide chain release factor RF2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,087,47741
Polymers1,086,6507
Non-polymers82634
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area31690 Å2
ΔGint-496 kcal/mol
Surface area89540 Å2

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Components

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RNA chain , 2 types, 2 molecules Ax

#1: RNA chain 23S rRNA / 23S ribosomal RNA


Mass: 941818.625 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: GenBank: 687670942
#6: RNA chain P-site tRNA fMet


Mass: 24786.785 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: GenBank: 817573384

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50S ribosomal protein ... , 4 types, 4 molecules CDNX

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


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


Mass: 22277.535 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P60438
#4: Protein 50S ribosomal protein L16 / / Large ribosomal subunit protein uL16


Mass: 15312.269 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0ADY7
#5: 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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Protein / Non-polymers , 2 types, 35 molecules v

#7: Protein Peptide chain release factor RF2 / RF-2


Mass: 43384.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: prfB, supK, b2891, JW5847 / Production host: Escherichia coli (E. coli) / References: UniProt: P07012
#8: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 34 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

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

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1nonstop ribosomal complex with ArfA/RF2RIBOSOMEnonstop termination complex with 70S, ArfA, RF2, tRNA and nonstop mRNA#1-#70MULTIPLE SOURCES
250S subunitProkaryotic large ribosomal subunitCOMPLEX#1-#51NATURAL
3P-site tRNA fMetORGANELLE OR CELLULAR COMPONENT#61RECOMBINANT
4Peptide chain release factor 2ORGANELLE OR CELLULAR COMPONENT#71RECOMBINANT
523S rRNA23S ribosomal RNAORGANELLE OR CELLULAR COMPONENT#12NATURAL
650S ribosomal protein L2ORGANELLE OR CELLULAR COMPONENT#22NATURAL
750S ribosomal protein L3ORGANELLE OR CELLULAR COMPONENT#32NATURAL
850S ribosomal protein L16ORGANELLE OR CELLULAR COMPONENT#42NATURAL
950S ribosomal protein L27ORGANELLE OR CELLULAR COMPONENT#52NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
44Escherichia coli (E. coli)562
55Escherichia coli (E. coli)562
66Escherichia coli (E. coli)562
77Escherichia coli (E. coli)562
88Escherichia coli (E. coli)562
99Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
23Escherichia coli (E. coli)562
34Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidC8H18N2O4S1
215 mMmagnesium acetateMg(CH3COO)21
3150 mMpotassium acetateCH3CO2K1
44 mMbeta-mercapthoethanolHOCH2CH2SH1
52 mMspermidineC7H19N31
60.05 mMspermineC10H26N41
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: C-flat-2/0.5 4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Grids were blotted for 3.5 seconds.

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Electron microscopy imaging

MicroscopyModel: JEOL 3200FS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 83822 X
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 20 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1RELION1.4particle selection
2Leginonimage acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.11model fitting
8Coot0.8.3model fitting
10REFMAC5.8model refinement
11RELION2initial Euler assignment
12RELION2final Euler assignment
13RELION2classification
14RELION23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 143372 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: RECIPROCAL / Target criteria: Average FSC

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