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- PDB-7kal: Cryo-EM structure of the Sec complex from T. lanuginosus, wild-ty... -

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Basic information

Entry
Database: PDB / ID: 7kal
TitleCryo-EM structure of the Sec complex from T. lanuginosus, wild-type, class with Sec62, plug-open conformation
Components
  • (Protein transport channel Sec61 complex, ...) x 3
  • (Protein transport protein ...Protein targeting) x 4
KeywordsPROTEIN TRANSPORT / Sec61 / translocon / endoplasmic reticulum / protein translocation / Sec62 / Sec63 / channel
Biological speciesThermomyces lanuginosus (fungus)
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 channel Sec61 complex, alpha subunit (Sec61)
C: Protein transport channel Sec61 complex, gamma subunit (Sss1)
B: Protein transport channel Sec61 complex, beta subunit (Sbh1)
D: Protein transport protein Sec63
E: Protein transport protein Sec66/Sec71
F: Protein transport protein Sec72
G: Protein transport protein Sec62


Theoretical massNumber of molelcules
Total (without water)248,1457
Polymers248,1457
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17480 Å2
ΔGint-177 kcal/mol
Surface area75910 Å2

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Components

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Protein transport channel Sec61 complex, ... , 3 types, 3 molecules ACB

#1: Protein Protein transport channel Sec61 complex, alpha subunit (Sec61)


Mass: 52427.430 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermomyces lanuginosus (fungus) / Gene: Sec61a / Production host: Saccharomyces cerevisiae BY4741 (yeast) / Strain (production host): ATCC 204508 / S288c
#2: Protein Protein transport channel Sec61 complex, gamma subunit (Sss1)


Mass: 7852.352 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermomyces lanuginosus (fungus) / Gene: Sec61G / Production host: Saccharomyces cerevisiae BY4741 (yeast) / Strain (production host): ATCC 204508 / S288c
#3: Protein Protein transport channel Sec61 complex, beta subunit (Sbh1)


Mass: 12491.097 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermomyces lanuginosus (fungus) / Gene: Sec61b / Production host: Saccharomyces cerevisiae BY4741 (yeast) / Strain (production host): ATCC 204508 / S288c

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Protein transport protein ... , 4 types, 4 molecules DEFG

#4: Protein Protein transport protein Sec63 / Protein targeting


Mass: 79976.703 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermomyces lanuginosus (fungus) / Gene: Sec63 / Production host: Saccharomyces cerevisiae BY4741 (yeast) / Strain (production host): ATCC 204508 / S288c
#5: Protein Protein transport protein Sec66/Sec71 / Protein targeting


Mass: 27555.285 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermomyces lanuginosus (fungus) / Gene: SEC66 / Production host: Saccharomyces cerevisiae BY4741 (yeast) / Strain (production host): ATCC 204508 / S288c
#6: Protein Protein transport protein Sec72 / Protein targeting


Mass: 23382.234 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermomyces lanuginosus (fungus) / Gene: Sec72 / Production host: Saccharomyces cerevisiae BY4741 (yeast) / Strain (production host): ATCC 204508 / S288c
#7: Protein Protein transport protein Sec62 / Protein targeting


Mass: 44460.074 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermomyces lanuginosus (fungus) / Gene: SEC62 / Production host: Saccharomyces cerevisiae BY4741 (yeast) / Strain (production host): ATCC 204508 / S288c

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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 T. lanuginosus
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Thermomyces lanuginosus (fungus)
Source (recombinant)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: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 43860 X / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / 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: 1632659 / Details: autopicked particles
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 114704 / Algorithm: FOURIER SPACE / Details: cryoSPARC non-uniform refinement / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211029
ELECTRON MICROSCOPYf_angle_d0.48114998
ELECTRON MICROSCOPYf_dihedral_angle_d22.7731530
ELECTRON MICROSCOPYf_chiral_restr0.0361757
ELECTRON MICROSCOPYf_plane_restr0.0031881

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