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- PDB-5a6u: Native mammalian ribosome-bound Sec61 protein-conducting channel ... -

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Basic information

Entry
Database: PDB / ID: 5a6u
TitleNative mammalian ribosome-bound Sec61 protein-conducting channel in the 'non-inserting' state
Components
  • SEC61A
  • SEC61B
  • SEC61G
KeywordsTRANSLATION / RIBOSOME / SEC61 / TRANSLOCON / ENDOPLASMIC RETICULUM / CRYOELECTRON TOMOGRAPHY / SUBTOMOGRAM ANALYSIS
Function / homology
Function and homology information


Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / membrane docking / endoplasmic reticulum Sec complex / pronephric nephron development / cotranslational protein targeting to membrane / Ssh1 translocon complex / Sec61 translocon complex / protein targeting to ER / protein-transporting ATPase activity / protein insertion into ER membrane ...Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / membrane docking / endoplasmic reticulum Sec complex / pronephric nephron development / cotranslational protein targeting to membrane / Ssh1 translocon complex / Sec61 translocon complex / protein targeting to ER / protein-transporting ATPase activity / protein insertion into ER membrane / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding / post-translational protein targeting to membrane, translocation / protein transmembrane transporter activity / phospholipid binding / ribosome binding / endoplasmic reticulum membrane / endoplasmic reticulum / membrane
Similarity search - Function
Protein transport Sec61-beta/Sbh / Protein transport protein SecG/Sec61-beta/Sbh / Sec61beta family / Protein translocase SEC61 complex, gamma subunit / Protein translocase SecE domain superfamily / Translocon Sec61/SecY, plug domain / Plug domain of Sec61p / Protein secE/sec61-gamma signature. / Protein secY signature 1. / Protein secY signature 2. ...Protein transport Sec61-beta/Sbh / Protein transport protein SecG/Sec61-beta/Sbh / Sec61beta family / Protein translocase SEC61 complex, gamma subunit / Protein translocase SecE domain superfamily / Translocon Sec61/SecY, plug domain / Plug domain of Sec61p / 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
Similarity search - Domain/homology
Protein transport protein Sec61 subunit alpha isoform 1 / Protein transport protein Sec61 subunit gamma / Protein transport protein Sec61 subunit beta
Similarity search - Component
Biological speciesCANIS LUPUS FAMILIARIS (dog)
MethodELECTRON MICROSCOPY / electron tomography / cryo EM / Resolution: 9 Å
AuthorsPfeffer, S. / Burbaum, L. / Unverdorben, P. / Pech, M. / Chen, Y. / Zimmermann, R. / Beckmann, R. / Foerster, F.
CitationJournal: Nat Commun / Year: 2015
Title: Structure of the native Sec61 protein-conducting channel.
Authors: Stefan Pfeffer / Laura Burbaum / Pia Unverdorben / Markus Pech / Yuxiang Chen / Richard Zimmermann / Roland Beckmann / Friedrich Förster /
Abstract: In mammalian cells, secretory and membrane proteins are translocated across or inserted into the endoplasmic reticulum (ER) membrane by the universally conserved protein-conducting channel Sec61, ...In mammalian cells, secretory and membrane proteins are translocated across or inserted into the endoplasmic reticulum (ER) membrane by the universally conserved protein-conducting channel Sec61, which has been structurally studied in isolated, detergent-solubilized states. Here we structurally and functionally characterize native, non-solubilized ribosome-Sec61 complexes on rough ER vesicles using cryo-electron tomography and ribosome profiling. Surprisingly, the 9-Å resolution subtomogram average reveals Sec61 in a laterally open conformation, even though the channel is not in the process of inserting membrane proteins into the lipid bilayer. In contrast to recent mechanistic models for polypeptide translocation and insertion, our results indicate that the laterally open conformation of Sec61 is the only conformation present in the ribosome-bound translocon complex, independent of its functional state. Consistent with earlier functional studies, our structure suggests that the ribosome alone, even without a nascent chain, is sufficient for lateral opening of Sec61 in a lipid environment.
History
DepositionJul 1, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 7, 2015Provider: repository / Type: Initial release
Revision 1.1Oct 28, 2015Group: Database references
Revision 2.0Aug 23, 2017Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations
Category: atom_site / database_PDB_caveat ...atom_site / database_PDB_caveat / em_software / pdbx_struct_sheet_hbond / struct_conf / struct_conn / struct_sheet / struct_sheet_order / struct_sheet_range
Item: _em_software.fitting_id / _em_software.image_processing_id ..._em_software.fitting_id / _em_software.image_processing_id / _em_software.name / _pdbx_struct_sheet_hbond.range_1_auth_comp_id / _pdbx_struct_sheet_hbond.range_1_auth_seq_id / _pdbx_struct_sheet_hbond.range_1_label_comp_id / _pdbx_struct_sheet_hbond.range_1_label_seq_id / _pdbx_struct_sheet_hbond.range_2_auth_comp_id / _pdbx_struct_sheet_hbond.range_2_auth_seq_id / _pdbx_struct_sheet_hbond.range_2_label_comp_id / _pdbx_struct_sheet_hbond.range_2_label_seq_id / _pdbx_struct_sheet_hbond.sheet_id / _struct_sheet.id / _struct_sheet_order.sheet_id / _struct_sheet_range.sheet_id
Revision 2.1Aug 21, 2019Group: Data collection / Category: em_software / Item: _em_software.name

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Assembly

Deposited unit
A: SEC61A
B: SEC61B
G: SEC61G


Theoretical massNumber of molelcules
Total (without water)60,3343
Polymers60,3343
Non-polymers00
Water0
1


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

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Components

#1: Protein SEC61A


Mass: 49321.688 Da / Num. of mol.: 1 / Fragment: UNP RESIDUES 26-476 / Source method: isolated from a natural source / Source: (natural) CANIS LUPUS FAMILIARIS (dog) / Organ: PANCREAS / References: UniProt: P38377
#2: Protein/peptide SEC61B


Mass: 3992.791 Da / Num. of mol.: 1 / Fragment: UNP RESIDUES 61-96 / Source method: isolated from a natural source / Source: (natural) CANIS LUPUS FAMILIARIS (dog) / Organ: PANCREAS / References: UniProt: P60467
#3: Protein SEC61G


Mass: 7019.456 Da / Num. of mol.: 1 / Fragment: UNP RESIDUES 7-68 / Source method: isolated from a natural source / Source: (natural) CANIS LUPUS FAMILIARIS (dog) / Organ: PANCREAS / References: UniProt: P60058

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: electron tomography

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Sample preparation

ComponentName: ER-DERIVED MICROSOMES / Type: ORGANELLE OR CELLULAR COMPONENT
Buffer solutionName: 20MM HEPES, 50MM KCL, 2MM MGCL2 / pH: 7.6 / Details: 20MM HEPES, 50MM KCL, 2MM MGCL2
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE-PROPANE MIXTURE, HUMIDITY- 70, INSTRUMENT- FEI VITROBOT MARK IV, METHOD- BLOT 3 SECONDS BEFORE PLUNGING.,

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Jun 18, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4000 nm / Nominal defocus min: 3000 nm
Specimen holderTilt angle max: 20 ° / Tilt angle min: -20 °
Image recordingElectron dose: 30 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)
Image scansNum. digital images: 2000

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Processing

EM software
IDNameCategory
1MDFFmodel fitting
2AV33D reconstruction
3PyTom3D reconstruction
4TOM Toolbox3D reconstruction
CTF correctionDetails: EACH TILT IMAGE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 9 Å / Num. of particles: 17653 / Nominal pixel size: 2.62 Å / Actual pixel size: 2.62 Å
Magnification calibration: CROSS- -CORRELATION WITH RIBOSOME DENSITIES
Details: WEIGHTED BACKPROJECTION AND CONSTRAINED SUBTOMOGRAM AVERAGING SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3068. (DEPOSITION ID: 13544).
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: PSEUDO-ENERGY / Details: METHOD--FLEXIBLE REFINEMENT PROTOCOL--CRYO-EM
Atomic model buildingPDB-ID: 3J7Q
RefinementHighest resolution: 9 Å
Refinement stepCycle: LAST / Highest resolution: 9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3778 0 0 0 3778

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