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- PDB-4cxh: Regulation of the mammalian elongation cycle by 40S subunit rolli... -

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Entry
Database: PDB / ID: 4cxh
TitleRegulation of the mammalian elongation cycle by 40S subunit rolling: a eukaryotic-specific ribosome rearrangement
Components
  • 18S RRNA - H44
  • 18S RRNA - H5-H14
  • 18S RRNA - H8
  • 28S RRNA - H89
  • 28S RRNA - H95
  • 40S RIBOSOMAL PROTEIN US12
  • ELONGATION FACTOR 1A
  • MESSENGER RNA
  • TRANSFER RNA
KeywordsTRANSLATION / MAMMALIAN 80S RIBOSOME / ELONGATION CYCLE / TRNA SELECTION / EUKARYOTIC TERNARY COMPLEX / ELONGATION FACTOR EEF1A
Function / homologyTranslation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Ribosomal protein S23, eukaryotic/archaeal / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Transcription factor, GTP-binding domain / Translation elongation factor EFTu/EF1A, C-terminal / Translation elongation factor EFTu-like, domain 2 / Translation elongation factor EF1A, eukaryotic/archaeal / Small GTP-binding protein domain / Ribosomal protein S12/S23 / L13a-mediated translational silencing of Ceruloplasmin expression ...Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Ribosomal protein S23, eukaryotic/archaeal / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Transcription factor, GTP-binding domain / Translation elongation factor EFTu/EF1A, C-terminal / Translation elongation factor EFTu-like, domain 2 / Translation elongation factor EF1A, eukaryotic/archaeal / Small GTP-binding protein domain / Ribosomal protein S12/S23 / L13a-mediated translational silencing of Ceruloplasmin expression / Translation protein, beta-barrel domain superfamily / Ribosomal protein S12 signature. / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase / Tr-type G domain, conserved site / Elongation factor Tu GTP binding domain / Ribosomal protein S12/S23 / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Elongation factor Tu C-terminal domain / Elongation factor Tu domain 2 / Formation of the ternary complex, and subsequently, the 43S complex / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Regulation of expression of SLITs and ROBOs / Eukaryotic Translation Termination / GTP hydrolysis and joining of the 60S ribosomal subunit / Ribosomal scanning and start codon recognition / Formation of a pool of free 40S subunits / Peptide chain elongation / Translation initiation complex formation / Major pathway of rRNA processing in the nucleolus and cytosol / Selenocysteine synthesis / Viral mRNA Translation / SRP-dependent cotranslational protein targeting to membrane / stress granule assembly / SRP-dependent cotranslational protein targeting to membrane / translational initiation / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / polysomal ribosome / translation elongation factor activity / maintenance of translational fidelity / rough endoplasmic reticulum / cytoplasmic translation / cytosolic small ribosomal subunit / ribosome / structural constituent of ribosome / translation / GTPase activity / synapse / GTP binding / endoplasmic reticulum / RNA binding / membrane / nucleoplasm / cytosol / cytoplasm / 40S ribosomal protein S23 / Elongation factor 1-alpha / gb:4338: / gb:36162: / gb:337381:
Function and homology information
Specimen sourceESCHERICHIA COLI (E. coli)
ORYCTOLAGUS CUNICULUS (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 8.9 Å resolution
AuthorsBudkevich, T.V. / Giesebrecht, J. / Behrmann, E. / Loerke, J. / Ramrath, D.J.F. / Mielke, T. / Ismer, J. / Hildebrand, P. / Tung, C.-S. / Nierhaus, K.H. / Sanbonmatsu, K.Y. / Spahn, C.M.T.
CitationJournal: Cell / Year: 2014
Title: Regulation of the mammalian elongation cycle by subunit rolling: a eukaryotic-specific ribosome rearrangement.
Authors: Tatyana V Budkevich / Jan Giesebrecht / Elmar Behrmann / Justus Loerke / David J F Ramrath / Thorsten Mielke / Jochen Ismer / Peter W Hildebrand / Chang-Shung Tung / Knud H Nierhaus / Karissa Y Sanbonmatsu / Christian M T Spahn
Abstract: The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present subnanometer resolution ...The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present subnanometer resolution cryoelectron microscopy maps of the mammalian 80S ribosome in the posttranslocational state and in complex with the eukaryotic eEF1A⋅Val-tRNA⋅GMPPNP ternary complex, revealing significant differences in the elongation mechanism between bacteria and mammals. Surprisingly, and in contrast to bacterial ribosomes, a rotation of the small subunit around its long axis and orthogonal to the well-known intersubunit rotation distinguishes the posttranslocational state from the classical pretranslocational state ribosome. We term this motion "subunit rolling." Correspondingly, a mammalian decoding complex visualized in substates before and after codon recognition reveals structural distinctions from the bacterial system. These findings suggest how codon recognition leads to GTPase activation in the mammalian system and demonstrate that in mammalia subunit rolling occurs during tRNA selection.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Apr 7, 2014 / Release: Jul 16, 2014
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jul 16, 2014Structure modelrepositoryInitial release
2.0Aug 23, 2017Structure modelAdvisory / Atomic model / Data collection / Derived calculations / Refinement description / Structure summaryatom_site / database_PDB_caveat / em_3d_fitting / em_software / entity / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_close_contact / struct_conn_atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_seq_id / _atom_site.occupancy / _atom_site.type_symbol / _em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id / _entity.src_method / _pdbx_validate_close_contact.auth_atom_id_2
Remark 700 SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "XA" IN EACH CHAIN ON SHEET RECORDS BELOW ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "XA" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 5-STRANDED BARREL THIS IS REPRESENTED BY A 6-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL.

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Structure visualization

Movie
  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-2624
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Structure viewerMolecule:
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Downloads & links

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Assembly

Deposited unit
1: 18S RRNA - H44
2: 28S RRNA - H89
A: ELONGATION FACTOR 1A
X: 40S RIBOSOMAL PROTEIN US12
Y: TRANSFER RNA
a: 18S RRNA - H5-H14
b: 18S RRNA - H8
c: 28S RRNA - H95
x: MESSENGER RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)184,58110
Polyers184,4169
Non-polymers1651
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPQS

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Components

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RNA chain , 7 types, 7 molecules 12Yabcx

#1: RNA chain 18S RRNA - H44


Mass: 43635.969 Da / Num. of mol.: 1 / Source: (natural) ORYCTOLAGUS CUNICULUS (rabbit) / References: GenBank: 36162
#2: RNA chain 28S RRNA - H89


Mass: 15974.376 Da / Num. of mol.: 1 / Source: (natural) ORYCTOLAGUS CUNICULUS (rabbit) / References: GenBank: 337381
#5: RNA chain TRANSFER RNA /


Mass: 24688.023 Da / Num. of mol.: 1 / Source: (natural) ORYCTOLAGUS CUNICULUS (rabbit) / References: GenBank: 4338
#6: RNA chain 18S RRNA - H5-H14


Mass: 15424.328 Da / Num. of mol.: 1 / Source: (natural) ORYCTOLAGUS CUNICULUS (rabbit) / References: GenBank: 36162
#7: RNA chain 18S RRNA - H8


Mass: 5445.253 Da / Num. of mol.: 1 / Source: (natural) ORYCTOLAGUS CUNICULUS (rabbit) / References: GenBank: 36162
#8: RNA chain 28S RRNA - H95


Mass: 6140.738 Da / Num. of mol.: 1 / Source: (natural) ORYCTOLAGUS CUNICULUS (rabbit) / References: GenBank: 337381
#9: RNA chain MESSENGER RNA /


Mass: 8527.686 Da / Num. of mol.: 1 / Source: (gene. exp.) ESCHERICHIA COLI (E. coli)
Description: FOR MODELING MRNA (CHAIN 1, PDB ID CODE 3I8G) WAS USED
Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): K-12

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Protein/peptide , 2 types, 2 molecules AX

#3: Protein/peptide ELONGATION FACTOR 1A / EF-1-ALPHA / ELONGATION FACTOR TU / EF-TU / ELONGATION FACTOR 1A


Mass: 48734.527 Da / Num. of mol.: 1 / Source: (natural) ORYCTOLAGUS CUNICULUS (rabbit) / References: UniProt: Q9YAV0
#4: Protein/peptide 40S RIBOSOMAL PROTEIN US12 / / 40S RIBOSOMAL PROTEIN S23


Mass: 15844.666 Da / Num. of mol.: 1 / Source: (natural) ORYCTOLAGUS CUNICULUS (rabbit) / References: UniProt: P62266

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Non-polymers , 1 types, 1 molecules

#10: Chemical ChemComp-PHE / PHENYLALANINE


Mass: 165.189 Da / Num. of mol.: 1 / Formula: C9H11NO2 / Phenylalanine

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: CODON RECOGNITION - GTPASE ACTIVATION STATE OF MAMMALIAN 80S RIBOSOME
Type: RIBOSOME
Buffer solutionName: 20 MM HEPES, 5 MM MGCL2, 100 MM NH4CL, 6 MM BETA- MERCAPTOETHANOL, 0.8 MM SPERMIDINE, 0.6 MM SPERMINE
Details: 20 MM HEPES, 5 MM MGCL2, 100 MM NH4CL, 6 MM BETA- MERCAPTOETHANOL, 0.8 MM SPERMIDINE, 0.6 MM SPERMINE
pH: 7.5
SpecimenConc.: 1.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 80, INSTRUMENT - FEI VITROBOT MARK II, METHOD- BLOT FOR 2 SEC BEFORE PLUNGING,

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TECNAI F30 / Date: Sep 22, 2008 / Details: MINIMAL DOSE SYSTEM
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 39000 / Calibrated magnification: 65520 / Nominal defocus max: 4000 nm / Nominal defocus min: 2000 nm / Cs: 2 mm
Image recordingElectron dose: 20 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNumber digital images: 230
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1Cootmodel fitting
2UCSF Chimeramodel fitting
3SPARX3D reconstruction
4SPIDER3D reconstruction
CTF correctionDetails: DEFOCUS GROUP
SymmetryPoint symmetry: C1
3D reconstructionMethod: MULTI-REFERENCE TEMPLATE MATCHING / Resolution: 8.9 Å / Number of particles: 52686 / Nominal pixel size: 1.26 / Actual pixel size: 1.26
Magnification calibration: CROSS- -CORRELATION WITH KNOWN STRUCTURE
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD -2624. (DEPOSITION ID: 12412).
Symmetry type: POINT
Atomic model buildingDetails: METHOD--RIGID BODY / Ref protocol: RIGID BODY FIT / Ref space: REAL / Target criteria: Cross-correlation coefficient
Least-squares processHighest resolution: 8.9 Å
Refine hist #LASTHighest resolution: 8.9 Å
Number of atoms included #LASTProtein: 4319 / Nucleic acid: 7940 / Ligand: 11 / Solvent: 0 / Total: 12270

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