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- PDB-7kar: Cryo-EM structure of the Sec complex from S. cerevisiae, Sec63 FN... -

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Basic information

Entry
Database: PDB / ID: 7kar
TitleCryo-EM structure of the Sec complex from S. cerevisiae, Sec63 FN3 mutant, class without Sec62
Components
  • (Protein transport protein ...) x 3
  • (Translocation protein ...) x 2
  • Protein translocation protein SEC63
KeywordsPROTEIN TRANSPORT / Sec61 / translocon / endoplasmic reticulum / protein translocation / Sec62 / Sec63 / channel
Function / homology
Function and homology information


misfolded protein transport / Sec62/Sec63 complex / translocon complex / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / cytosol to endoplasmic reticulum transport / rough endoplasmic reticulum membrane / Ssh1 translocon complex / Sec61 translocon complex / protein-transporting ATPase activity / post-translational protein targeting to endoplasmic reticulum membrane ...misfolded protein transport / Sec62/Sec63 complex / translocon complex / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / cytosol to endoplasmic reticulum transport / rough endoplasmic reticulum membrane / Ssh1 translocon complex / Sec61 translocon complex / protein-transporting ATPase activity / post-translational protein targeting to endoplasmic reticulum membrane / filamentous growth / SRP-dependent cotranslational protein targeting to membrane, translocation / SRP-dependent cotranslational protein targeting to membrane / signal sequence binding / post-translational protein targeting to membrane, translocation / peptide transmembrane transporter activity / nuclear inner membrane / retrograde protein transport, ER to cytosol / protein transmembrane transporter activity / ERAD pathway / guanyl-nucleotide exchange factor activity / cell periphery / ribosome binding / endoplasmic reticulum membrane / structural molecule activity / endoplasmic reticulum / mitochondrion / cytosol
Similarity search - Function
Translocation protein Sec66 / Preprotein translocase subunit Sec66 / 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 / Nt-dnaJ domain signature. ...Translocation protein Sec66 / Preprotein translocase subunit Sec66 / 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 / Nt-dnaJ domain signature. / DnaJ domain, conserved site / 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 / DnaJ domain / Sec63 domain / Sec63 Brl domain / DnaJ molecular chaperone homology domain / dnaJ domain profile. / Chaperone J-domain superfamily / DnaJ domain / Tetratricopeptide repeats / Tetratricopeptide repeat / Tetratricopeptide-like helical domain superfamily / Immunoglobulin E-set
Similarity search - Domain/homology
Protein translocation protein SEC63 / Protein transport protein SEC61 / Translocation protein SEC66 / Protein transport protein SSS1 / Translocation protein SEC72 / Protein transport protein SBH1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae BY4741 (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsItskanov, S. / Park, E.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: Stepwise gating of the Sec61 protein-conducting channel by Sec63 and Sec62.
Authors: Samuel Itskanov / Katie M Kuo / James C Gumbart / Eunyong Park /
Abstract: Many proteins are transported into the endoplasmic reticulum by the universally conserved Sec61 channel. Post-translational transport requires two additional proteins, Sec62 and Sec63, but their ...Many proteins are transported into the endoplasmic reticulum by the universally conserved Sec61 channel. Post-translational transport requires two additional proteins, Sec62 and Sec63, but their functions are poorly defined. In the present study, we determined cryo-electron microscopy (cryo-EM) structures of several variants of Sec61-Sec62-Sec63 complexes from Saccharomyces cerevisiae and Thermomyces lanuginosus and show that Sec62 and Sec63 induce opening of the Sec61 channel. Without Sec62, the translocation pore of Sec61 remains closed by the plug domain, rendering the channel inactive. We further show that the lateral gate of Sec61 must first be partially opened by interactions between Sec61 and Sec63 in cytosolic and luminal domains, a simultaneous disruption of which completely closes the channel. The structures and molecular dynamics simulations suggest that Sec62 may also prevent lipids from invading the channel through the open lateral gate. Our study shows how Sec63 and Sec62 work together in a hierarchical manner to activate Sec61 for post-translational protein translocation.
History
DepositionOct 1, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 6, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 20, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Feb 24, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Protein transport protein SEC61
C: Protein transport protein SSS1
B: Protein transport protein SBH1
D: Protein translocation protein SEC63
E: Translocation protein SEC66
F: Translocation protein SEC72


Theoretical massNumber of molelcules
Total (without water)192,8676
Polymers192,8676
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16950 Å2
ΔGint-146 kcal/mol
Surface area70320 Å2

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Components

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Protein transport protein ... , 3 types, 3 molecules ACB

#1: Protein Protein transport protein SEC61 / Sec61 complex subunit SEC61 / Sec61 complex subunit alpha


Mass: 52978.148 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast)
Plasmid details: SEC63 is tagged with TEV-GFP and recombinantly expressed
Strain: ATCC 204508 / S288c / References: UniProt: P32915
#2: Protein Protein transport protein SSS1 / Sec61 complex subunit SSS1 / Sec61 complex subunit gamma / Ssh1 complex subunit SSS1 / Ssh1 complex ...Sec61 complex subunit SSS1 / Sec61 complex subunit gamma / Ssh1 complex subunit SSS1 / Ssh1 complex subunit gamma


Mass: 8958.641 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast)
Plasmid details: SEC63 is tagged with TEV-GFP and recombinantly expressed
Strain: ATCC 204508 / S288c / References: UniProt: P35179
#3: Protein Protein transport protein SBH1 / Sec61 complex subunit SBH1 / Sec61 complex subunit beta


Mass: 8723.155 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae BY4741 (yeast)
Strain: ATCC 204508 / S288c / Gene: SBH1, SEB1, YER087C-B, YER087BC / Production host: Saccharomyces cerevisiae BY4741 (yeast) / Strain (production host): ATCC 204508 / S288c / References: UniProt: P52870

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Protein , 1 types, 1 molecules D

#4: Protein Protein translocation protein SEC63 / Protein NPL1 / Sec62/63 complex 73 kDa subunit


Mass: 76312.039 Da / Num. of mol.: 1 / Mutation: E440R/F481S/del(441-447)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae BY4741 (yeast)
Strain: ATCC 204508 / S288c / Gene: SEC63, NPL1, PTL1, YOR254C / Production host: Saccharomyces cerevisiae BY4741 (yeast) / Strain (production host): ATCC 204508 / S288c / References: UniProt: P14906

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Translocation protein ... , 2 types, 2 molecules EF

#5: Protein Translocation protein SEC66 / Protein HSS1 / Sec62/63 complex 31.5 kDa subunit


Mass: 24263.939 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast)
Plasmid details: SEC63 is tagged with TEV-GFP and recombinantly expressed
Strain: ATCC 204508 / S288c / References: UniProt: P33754
#6: Protein Translocation protein SEC72 / Sec62/63 complex 23 kDa subunit / p23


Mass: 21631.090 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast)
Plasmid details: SEC63 is tagged with TEV-GFP and recombinantly expressed
Strain: ATCC 204508 / S288c / References: UniProt: P39742

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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: Endoplasmic reticulum protein-transport machinery Sec complex from yeast
Type: COMPLEX
Details: Sec63 has the following mutations: E440R/F481S/del(441-447)
Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae BY4741 (yeast) / Strain: ATCC 204508 / S288c
Buffer solutionpH: 7.5
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 42017 X / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 49.1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
1Warp1.0.7particle selection
2SerialEM3.7image acquisition
4Warp1.0.7CTF correction
5cryoSPARC2.12CTF correction
11cryoSPARC2.12initial Euler assignment
12cryoSPARC2.12final Euler assignment
14cryoSPARC2.123D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1274219 / Details: autopicked particles
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 82671 / Algorithm: FOURIER SPACE / Details: Non-uniform refinement from cryoSPARC / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00310643
ELECTRON MICROSCOPYf_angle_d0.53414426
ELECTRON MICROSCOPYf_dihedral_angle_d11.2166411
ELECTRON MICROSCOPYf_chiral_restr0.0391710
ELECTRON MICROSCOPYf_plane_restr0.0031793

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