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- PDB-3deg: Complex of elongating Escherichia coli 70S ribosome and EF4(LepA)... -

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Basic information

Entry
Database: PDB / ID: 3deg
TitleComplex of elongating Escherichia coli 70S ribosome and EF4(LepA)-GMPPNP
Components
  • (30S RNA helix ...) x 2
  • (50S RNA helix ...) x 4
  • 30S ribosomal protein S12
  • 50S ribosomal protein L11
  • A/L-tRNA
  • GTP-binding protein lepA
  • P-tRNA
KeywordsRIBOSOME / translation / LepA / EF4 / GTP-binding / Membrane / Nucleotide-binding / Antibiotic resistance / Ribonucleoprotein / Ribosomal protein / RNA-binding / rRNA-binding / tRNA-binding / Methylation
Function / homology
Function and homology information


: / response to pH / guanosine tetraphosphate binding / stringent response / ribosomal large subunit binding / ribosomal small subunit binding / misfolded RNA binding / Group I intron splicing / RNA folding / translational termination ...: / response to pH / guanosine tetraphosphate binding / stringent response / ribosomal large subunit binding / ribosomal small subunit binding / misfolded RNA binding / Group I intron splicing / RNA folding / translational termination / translation elongation factor activity / response to salt stress / response to cold / positive regulation of RNA splicing / positive regulation of translation / maintenance of translational fidelity / ribosomal small subunit biogenesis / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / ribosome binding / cytoplasmic translation / cytosolic large ribosomal subunit / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / GTPase activity / GTP binding / identical protein binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Elongation factor 4 / GTP-binding protein LepA, C-terminal / Elongation factor 4, domain IV / LepA, C-terminal domain superfamily / GTP-binding protein LepA C-terminus / Elongation factor EFG, domain V-like / Elongation factor G C-terminus / EF-G domain III/V-like / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. ...Elongation factor 4 / GTP-binding protein LepA, C-terminal / Elongation factor 4, domain IV / LepA, C-terminal domain superfamily / GTP-binding protein LepA C-terminus / Elongation factor EFG, domain V-like / Elongation factor G C-terminus / EF-G domain III/V-like / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Ribosomal protein L11, bacterial-type / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L11, N-terminal / 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, N-terminal domain / Ribosomal protein L11, RNA binding domain / Ribosomal protein S12, bacterial-type / Ribosomal protein S12/S23 / Ribosomal protein S12/S23 / Small GTP-binding protein domain / Ribosomal protein S12 signature. / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RNA / RNA (> 10) / Large ribosomal subunit protein uL11 / Small ribosomal subunit protein uS12 / Elongation factor 4
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 10.9 Å
AuthorsConnell, S.R. / Topf, M. / Qin, Y. / Wilson, D.N. / Mielke, T. / Fucini, P. / Nierhaus, K.H. / Spahn, C.M.T.
CitationJournal: Nat Struct Mol Biol / Year: 2008
Title: A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation.
Authors: Sean R Connell / Maya Topf / Yan Qin / Daniel N Wilson / Thorsten Mielke / Paola Fucini / Knud H Nierhaus / Christian M T Spahn /
Abstract: EF4 (LepA) is an almost universally conserved translational GTPase in eubacteria. It seems to be essential under environmental stress conditions and has previously been shown to back-translocate the ...EF4 (LepA) is an almost universally conserved translational GTPase in eubacteria. It seems to be essential under environmental stress conditions and has previously been shown to back-translocate the tRNAs on the ribosome, thereby reverting the canonical translocation reaction. In the current work, EF4 was directly visualized in the process of back-translocating tRNAs by single-particle cryo-EM. Using flexible fitting methods, we built a model of ribosome-bound EF4 based on the cryo-EM map and a recently published unbound EF4 X-ray structure. The cryo-EM map establishes EF4 as a noncanonical elongation factor that interacts not only with the elongating ribosome, but also with the back-translocated tRNA in the A-site region, which is present in a previously unseen, intermediate state and deviates markedly from the position of a canonical A-tRNA. Our results, therefore, provide insight into the underlying structural principles governing back-translocation.
History
DepositionJun 10, 2008Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Aug 19, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Non-polymer description / Version format compliance
Revision 1.2May 30, 2012Group: Refinement description
Revision 1.3Apr 16, 2014Group: Database references
Revision 1.4Dec 11, 2019Group: Data collection / Database references / Derived calculations
Category: database_2 / em_image_scans ...database_2 / em_image_scans / em_software / struct_conn
Item: _em_software.image_processing_id / _em_software.name / _struct_conn.pdbx_leaving_atom_flag
Revision 1.5Mar 20, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

Movie
  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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  • Superimposition on EM map
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: A/L-tRNA
B: P-tRNA
E: 30S RNA helix 8
F: 30S RNA helix 14
G: 50S RNA helix 42-44
I: 50S RNA helix 95
J: 50S RNA helix 71
K: 50S RNA helix 92
C: GTP-binding protein lepA
D: 30S ribosomal protein S12
H: 50S ribosomal protein L11


Theoretical massNumber of molelcules
Total (without water)190,05911
Polymers190,05911
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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RNA chain , 2 types, 2 molecules AB

#1: RNA chain A/L-tRNA


Mass: 24890.121 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#2: RNA chain P-tRNA


Mass: 24816.811 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)

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30S RNA helix ... , 2 types, 2 molecules EF

#3: RNA chain 30S RNA helix 8


Mass: 5161.144 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#4: RNA chain 30S RNA helix 14


Mass: 3827.336 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)

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50S RNA helix ... , 4 types, 4 molecules GIJK

#5: RNA chain 50S RNA helix 42-44


Mass: 22580.502 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#6: RNA chain 50S RNA helix 95


Mass: 9394.642 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#7: RNA chain 50S RNA helix 71


Mass: 5765.451 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#8: RNA chain 50S RNA helix 92


Mass: 4745.833 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)

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Protein , 3 types, 3 molecules CDH

#9: Protein GTP-binding protein lepA


Mass: 60476.660 Da / Num. of mol.: 1 / Fragment: EF4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: lepA, b2569, JW2553 / Plasmid: pET14b / Production host: Escherichia coli (E. coli) / Strain (production host): Bl21(DE3) / References: UniProt: P60785
#10: Protein 30S ribosomal protein S12 /


Mass: 13636.961 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: K12 / References: UniProt: P0A7S3
#11: Protein 50S ribosomal protein L11 /


Mass: 14763.165 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: K12 / References: UniProt: P0A7J7

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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: Complex of elongating Escherichia coli 70S ribosome and EF4(LepA)-GMPPNP
Type: COMPLEX
Buffer solutionDetails: 20 mM HEPES-KOH (pH 7.6), 4.5 mM Mg(CH3COO)2, 150 mM NH4CH3COO, 4 mM B-mercaptoethanol, 2 mM spermidine, and 0.05 mM spermine
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 39000 X / Nominal defocus max: 3900 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 20 e/Å2 / Film or detector model: KODAK SO-163 FILM

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Processing

EM software
IDNameCategory
1Flex-EMmodel fitting
2Mod-EMmodel fitting
3Situsmodel fitting
4SPIDER3D reconstruction
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 10.9 Å / Num. of particles: 41294 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Details: REFINEMENT PROTOCOL--rigid body and flexible fitting
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
12I2P

2i2p
PDB Unreleased entry

12I2P1PDBexperimental model
22I2T

2i2t
PDB Unreleased entry

12I2T2PDBexperimental model
33CB4

3cb4

13CB43PDBexperimental model
41GIX

1gix
PDB Unreleased entry

11GIX4PDBexperimental model
52J01

2j01
PDB Unreleased entry

12J015PDBexperimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms6229 6721 0 0 12950

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