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Basic information

Entry
Database: PDB / ID: 7aft
TitleCryo-EM structure of the signal sequence-engaged post-translational Sec translocon
Components
  • (Protein transport protein ...Protein targeting) x 3
  • (Translocation protein ...) x 3
  • Mating factor alpha-1,Mating factor alpha-1
  • Protein translocation protein SEC63Protein targeting
KeywordsMEMBRANE PROTEIN / Post-translational translocation / Protein translocation / Sec complex / Signal sequence
Function / homology
Function and homology information


mating pheromone activity / mating / protein transmembrane import into intracellular organelle / misfolded protein transport / pheromone-dependent signal transduction involved in conjugation with cellular fusion / Sec62/Sec63 complex / translocon complex / cytosol to endoplasmic reticulum transport / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / rough endoplasmic reticulum membrane ...mating pheromone activity / mating / protein transmembrane import into intracellular organelle / misfolded protein transport / pheromone-dependent signal transduction involved in conjugation with cellular fusion / Sec62/Sec63 complex / translocon complex / cytosol to endoplasmic reticulum transport / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / 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 / peptide transmembrane transporter activity / signal sequence binding / SRP-dependent cotranslational protein targeting to membrane / post-translational protein targeting to membrane, translocation / cupric ion binding / nuclear inner membrane / retrograde protein transport, ER to cytosol / protein transmembrane transporter activity / : / guanyl-nucleotide exchange factor activity / cell periphery / ribosome binding / endoplasmic reticulum membrane / structural molecule activity / endoplasmic reticulum / mitochondrion / extracellular region / membrane / cytosol
Similarity search - Function
Translocation protein Sec62, ascomycota / Translocation protein Sec62 / Translocation protein Sec62 / Mating factor alpha, C-terminal repeat / Mating factor alpha precursor, N-terminal / Yeast mating factor alpha hormone / Mating factor alpha precursor N-terminus / Translocation protein Sec66 / Preprotein translocase subunit Sec66 / Protein transport Sec61-beta/Sbh ...Translocation protein Sec62, ascomycota / Translocation protein Sec62 / Translocation protein Sec62 / Mating factor alpha, C-terminal repeat / Mating factor alpha precursor, N-terminal / Yeast mating factor alpha hormone / Mating factor alpha precursor N-terminus / 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 / Sec63 domain / Sec63 Brl domain / DnaJ 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
Mating factor alpha-1 / Protein translocation protein SEC63 / Translocation protein SEC62 / Protein transport protein SEC61 / Translocation protein SEC66 / Protein transport protein SSS1 / Translocation protein SEC72 / Protein transport protein SBH1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsWeng, T.-H. / Beatrix, B. / Berninghausen, O. / Becker, T. / Cheng, J. / Beckmann, R.
CitationJournal: EMBO J / Year: 2021
Title: Architecture of the active post-translational Sec translocon.
Authors: Tsai-Hsuan Weng / Wieland Steinchen / Birgitta Beatrix / Otto Berninghausen / Thomas Becker / Gert Bange / Jingdong Cheng / Roland Beckmann /
Abstract: In eukaryotes, most secretory and membrane proteins are targeted by an N-terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein-conducting channel ...In eukaryotes, most secretory and membrane proteins are targeted by an N-terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein-conducting channel (PCC). In the post-translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre-opened state. Here, we present a cryo-EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N-terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C-terminus of Sec63. Together, we obtained a near-complete and integrated model of the active Sec complex.
History
DepositionSep 20, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 2, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 30, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Feb 10, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.year

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Structure visualization

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Assembly

Deposited unit
A: Protein transport protein SEC61
B: Protein transport protein SBH1
C: Protein transport protein SSS1
D: Protein translocation protein SEC63
E: Translocation protein SEC66
F: Translocation protein SEC72
G: Translocation protein SEC62,Translocation protein SEC62
H: Mating factor alpha-1,Mating factor alpha-1


Theoretical massNumber of molelcules
Total (without water)247,4048
Polymers247,4048
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: immunoprecipitation
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

#1: Protein Protein transport protein SEC61 / Protein targeting / 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 (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P32915
#2: Protein Protein transport protein SBH1 / Protein targeting / Sec61 complex subunit SBH1 / Sec61 complex subunit beta


Mass: 8723.155 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P52870
#3: Protein Protein transport protein SSS1 / Protein targeting / 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 (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P35179

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Protein , 2 types, 2 molecules DH

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


Mass: 75432.258 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P14906
#8: Protein Mating factor alpha-1,Mating factor alpha-1 / Alpha-1 mating pheromone


Mass: 19766.402 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: MF(ALPHA)1, MF-ALPHA-1, MFAL1, YPL187W / Plasmid: pET28a / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P01149

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Translocation protein ... , 3 types, 3 molecules EFG

#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 (strain ATCC 204508 / S288c) (yeast)
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 (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P39742
#7: Protein Translocation protein SEC62,Translocation protein SEC62 / Sec62/63 complex 30 kDa subunit


Mass: 35649.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The residue numbering in chain G is based on the transmembrane helix prediction of Sec62.
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: SEC62, YPL094C, LPG14C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P21825

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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

Component
IDNameTypeEntity IDParent-IDSource
1Signal sequence-engaged post-translational Sec transloconCOMPLEXall0MULTIPLE SOURCES
2Signal sequence-engaged post-translational Sec transloconCOMPLEX#1-#61NATURAL
3Translocation protein SEC62COMPLEX#71RECOMBINANT
4Mating factor alpha-1COMPLEX#81RECOMBINANT
Molecular weightValue: 0.255 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
23Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
34Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
13Saccharomyces cerevisiae (brewer's yeast)4932
24Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2100 mMKOAcCH3CO2K1
32.5 mMMg(OAc)2Mg(CH3COO)21
41 mMDTTC4H10O2S21
50.02 %GDNC56H92O251
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R2/2
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 FIELDBright-field microscopy
Image recording
IDImaging-IDElectron dose (e/Å2)Detector modeFilm or detector modelNum. of grids imaged
1136COUNTINGGATAN K2 QUANTUM (4k x 4k)1
2148COUNTINGGATAN K2 QUANTUM (4k x 4k)1
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategoryImage processing-ID
4GctfCTF correction1
7Cootmodel fitting
9RELION3initial Euler assignment1
10RELION3.1final Euler assignment1
11RELION3.1classification1
12RELION3.13D reconstruction1
13RELION3.1particle selection2
14GctfCTF correction2
15RELION3.1initial Euler assignment2
16RELION3.1final Euler assignment2
17RELION3.1classification2
18RELION3.13D reconstruction2
Image processing
IDImage recording-ID
11
22
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstruction

Algorithm: FOURIER SPACE / Entry-ID: 7AFT / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Symmetry type: POINT

IDNum. of particlesImage processing-ID
1745581
2157252
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 6N3Q

6n3q

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00610431
ELECTRON MICROSCOPYf_angle_d0.75714156
ELECTRON MICROSCOPYf_dihedral_angle_d22.1521421
ELECTRON MICROSCOPYf_chiral_restr0.0421704
ELECTRON MICROSCOPYf_plane_restr0.0061761

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