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- PDB-3j45: Structure of a non-translocating SecY protein channel with the 70... -

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Basic information

Entry
Database: PDB / ID: 3j45
TitleStructure of a non-translocating SecY protein channel with the 70S ribosome
Components
  • (23S ribosomal ...) x 5
  • (50S ribosomal protein ...) x 3
  • Preprotein translocase subunit SecE
  • Protein translocase subunit SecY
  • Protein-export membrane protein SecG
KeywordsRIBOSOME/PROTEIN TRANSPORT / 70S / SECYEG / protein translocation channel / RIBOSOME-PROTEIN TRANSPORT complex
Function / homology
Function and homology information


protein insertion into membrane from inner side / cell envelope Sec protein transport complex / protein transport by the Sec complex / intracellular protein transmembrane transport / protein-transporting ATPase activity / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding / protein transmembrane transporter activity / protein secretion / intracellular protein transport ...protein insertion into membrane from inner side / cell envelope Sec protein transport complex / protein transport by the Sec complex / intracellular protein transmembrane transport / protein-transporting ATPase activity / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding / protein transmembrane transporter activity / protein secretion / intracellular protein transport / ribosomal large subunit assembly / large ribosomal subunit rRNA binding / cytoplasmic translation / cytosolic large ribosomal subunit / rRNA binding / structural constituent of ribosome / translation / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Preprotein translocase SecG subunit / Preprotein translocase SecG subunit / SecE subunit of protein translocation complex, bacterial-like / SecE superfamily / Protein translocase subunit SecY / Protein secE/sec61-gamma signature. / Protein secY signature 1. / Protein secY signature 2. / SecE/Sec61-gamma subunits of protein translocation complex / Protein translocase complex, SecE/Sec61-gamma subunit ...Preprotein translocase SecG subunit / Preprotein translocase SecG subunit / SecE subunit of protein translocation complex, bacterial-like / SecE superfamily / Protein translocase subunit SecY / Protein secE/sec61-gamma signature. / Protein secY signature 1. / Protein secY signature 2. / SecE/Sec61-gamma subunits of protein translocation complex / Protein translocase complex, SecE/Sec61-gamma subunit / SecY/SEC61-alpha family / SecY domain superfamily / SecY conserved site / SecY / Ribosomal proteins 50S L24/mitochondrial 39S L24 / Ribosomal protein L24 / Ribosomal protein L23/L25, conserved site / Ribosomal protein L23 signature. / Ribosomal protein L29, conserved site / Ribosomal protein L29 signature. / Ribosomal protein L29/L35 / Ribosomal protein L29/L35 superfamily / Ribosomal L29 protein / Ribosomal protein L24/L26, conserved site / KOW (Kyprides, Ouzounis, Woese) motif. / Ribosomal protein L26/L24, KOW domain / Ribosomal protein L24 signature. / Ribosomal protein L23 / Ribosomal protein L25/L23 / Translation protein SH3-like domain superfamily / Ribosomal protein L23/L15e core domain superfamily / KOW motif / KOW / Ribosomal protein L2, domain 2 / Nucleotide-binding alpha-beta plait domain superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein uL23 / Protein translocase subunit SecE / Protein-export membrane protein SecG / Protein translocase subunit SecY / Large ribosomal subunit protein uL24
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.5 Å
AuthorsMenetret, J.F. / Park, E. / Gumbart, J.C. / Ludtke, S.J. / Li, W. / Whynot, A. / Rapoport, T.A. / Akey, C.W.
CitationJournal: Nature / Year: 2014
Title: Structure of the SecY channel during initiation of protein translocation.
Authors: Eunyong Park / Jean-François Ménétret / James C Gumbart / Steven J Ludtke / Weikai Li / Andrew Whynot / Tom A Rapoport / Christopher W Akey /
Abstract: Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY or eukaryotic Sec61 complexes, and are translocated across the membrane during ...Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY or eukaryotic Sec61 complexes, and are translocated across the membrane during their synthesis. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome-nascent chain complex binds to the SecY (or Sec61) complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here we have addressed this question by determining structures of inactive and active ribosome-channel complexes with cryo-electron microscopy. Non-translating ribosome-SecY channel complexes derived from Methanocaldococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome-channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the amino- and carboxy-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than entering the channel directly.
History
DepositionJun 18, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 23, 2013Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2013Group: Database references
Revision 1.2Feb 5, 2014Group: Database references
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id
Revision 1.4Feb 21, 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 / struct_ref_seq_dif
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_leaving_atom_flag / _struct_ref_seq_dif.details

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
y: Protein translocase subunit SecY
E: Preprotein translocase subunit SecE
G: Protein-export membrane protein SecG
T: 50S ribosomal protein L23
U: 50S ribosomal protein L24
Y: 50S ribosomal protein L29
1: 23S ribosomal RNA
2: 23S ribosomal RNA
3: 23S ribosomal RNA
4: 23S ribosomal RNA
5: 23S ribosomal RNA


Theoretical massNumber of molelcules
Total (without water)182,43011
Polymers182,43011
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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Protein , 3 types, 3 molecules yEG

#1: Protein Protein translocase subunit SecY


Mass: 47643.305 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: secY / Plasmid: pBAD-EhisYG / Production host: Escherichia coli (E. coli) / Strain (production host): C43(DE3) / References: UniProt: P0AGA2
#2: Protein Preprotein translocase subunit SecE


Mass: 6123.306 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: secE / Plasmid: pBAD-EhisYG / Production host: Escherichia coli (E. coli) / Strain (production host): C43(DE3) / References: UniProt: P0AG96
#3: Protein Protein-export membrane protein SecG


Mass: 6542.755 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: secG / Plasmid: pBAD-EhisYG / Production host: Escherichia coli (E. coli) / Strain (production host): C43(DE3) / References: UniProt: P0AG99

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50S ribosomal protein ... , 3 types, 3 molecules TUY

#4: Protein 50S ribosomal protein L23 /


Mass: 11222.160 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: P0ADZ0
#5: Protein 50S ribosomal protein L24 /


Mass: 11208.054 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: P60624
#6: Protein 50S ribosomal protein L29 /


Mass: 7286.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: P0A7M6

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23S ribosomal ... , 5 types, 5 molecules 12345

#7: RNA chain 23S ribosomal RNA /


Mass: 20350.104 Da / Num. of mol.: 1 / Fragment: helix 6 - helix 7 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600
#8: RNA chain 23S ribosomal RNA /


Mass: 11661.961 Da / Num. of mol.: 1 / Fragment: helix 50 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600
#9: RNA chain 23S ribosomal RNA /


Mass: 5803.507 Da / Num. of mol.: 1 / Fragment: helix 59 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600
#10: RNA chain 23S ribosomal RNA /


Mass: 19776.762 Da / Num. of mol.: 1 / Fragment: helix 68 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600
#11: RNA chain 23S ribosomal RNA /


Mass: 34811.531 Da / Num. of mol.: 1 / Fragment: helix 76 - helix 78 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY / Number of used crystals: 1
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeParent-ID
1non-translating E coli ribosome-SECYEG channel complexCOMPLEX0
2non-translating 70S ribosome1
3SecYEbetaG1
Molecular weightValue: 2.5 MDa / Experimental value: NO
Buffer solutionName: 50 mM HEPES-KOH, 100 mM KOAc, 10 mM Mg(OAc)2, 0.05% DDM
pH: 7.5
Details: 50 mM HEPES-KOH, 100 mM KOAc, 10 mM Mg(OAc)2, 0.05% DDM
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 400 mesh Cu grids with continuous or holey carbon films
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temp: 90 K / Humidity: 95 %
Details: Blot 1 second before plunging into liquid ethane (HOMEMADE PLUNGER).

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Apr 10, 2006 / Details: low dose imaging with manual data collection
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Calibrated magnification: 51000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / Specimen holder type: 626 single tilt / Temperature: 93 K / Tilt angle max: 30 ° / Tilt angle min: 0 °
Image recordingElectron dose: 20 e/Å2 / Film or detector model: KODAK SO-163 FILM / Details: Kodak SO163 film
Image scansNum. digital images: 360
Radiation wavelengthRelative weight: 1

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Processing

Software
NameVersionClassificationNB
REFMACrefinement
PDB_EXTRACT3.11data extraction
EM software
IDNameVersionCategory
1MDFFmodel fitting
2UCSF Chimeramodel fitting
3EMAN13D reconstruction
CTF correctionDetails: per micrograph
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: projection matching / Resolution: 9.5 Å / Resolution method: OTHER / Num. of particles: 39000 / Nominal pixel size: 2.73 Å / Actual pixel size: 2.73 Å
Details: CTF correction was done on untilted and 30 degree tilted images. Resolution method was comparison of 3D map with calculated map of docked ribosomal components, with the second map made with ...Details: CTF correction was done on untilted and 30 degree tilted images. Resolution method was comparison of 3D map with calculated map of docked ribosomal components, with the second map made with EMAN at 7 Angstrom resolution.
Num. of class averages: 1900 / Symmetry type: POINT
Atomic model building
IDProtocolSpaceDetails
1FLEXIBLE FITREALREFINEMENT PROTOCOL--FLEXIBLE
2FLEXIBLE FITREALREFINEMENT PROTOCOL--FLEXIBLE
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
12I2P

2i2p
PDB Unreleased entry

12I2P1PDBexperimental model
23J01

3j01
PDB Unreleased entry

23J012PDBexperimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms6336 6129 0 0 12465

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