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- PDB-6nd1: CryoEM structure of the Sec Complex from yeast -

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Basic information

Entry
Database: PDB / ID: 6nd1
TitleCryoEM structure of the Sec Complex from yeast
Components
  • (Protein transport protein ...Protein targeting) x 3
  • (Translocation protein ...) x 2
  • Protein translocation protein SEC63Protein targeting
KeywordsPROTEIN TRANSPORT / Sec61 / post-translational translocation / yeast / Sec63 / TRANSPORT PROTEIN
Function / homology
Function and homology information


Sec62/Sec63 complex / Ssh1 translocon complex / misfolded protein transport / protein transmembrane import into intracellular organelle / cytosol to endoplasmic reticulum transport / endoplasmic reticulum Sec complex / Sec61 translocon complex / posttranslational protein targeting to membrane, translocation / posttranslational protein targeting to endoplasmic reticulum membrane / filamentous growth ...Sec62/Sec63 complex / Ssh1 translocon complex / misfolded protein transport / protein transmembrane import into intracellular organelle / cytosol to endoplasmic reticulum transport / endoplasmic reticulum Sec complex / Sec61 translocon complex / posttranslational protein targeting to membrane, translocation / posttranslational protein targeting to endoplasmic reticulum membrane / filamentous growth / SRP-dependent cotranslational protein targeting to membrane, translocation / ARF guanyl-nucleotide exchange factor activity / P-P-bond-hydrolysis-driven protein transmembrane transporter activity / signal sequence binding / retrograde protein transport, ER to cytosol / nuclear inner membrane / peptide transmembrane transporter activity / protein transmembrane transporter activity / SRP-dependent cotranslational protein targeting to membrane / ubiquitin-dependent ERAD pathway / cell periphery / endoplasmic reticulum membrane / structural molecule activity / endoplasmic reticulum / mitochondrion / integral component of membrane
Preprotein translocase subunit Sec66 / SecY domain superfamily / Sec63 domain / Protein translocase SEC61 complex, gamma subunit / Tetratricopeptide-like helical domain superfamily / Immunoglobulin E-set / Protein transport protein SecG/Sec61-beta/Sbh / DnaJ domain, conserved site / Translocation protein Sec66 / Translocon Sec61/SecY, plug domain ...Preprotein translocase subunit Sec66 / SecY domain superfamily / Sec63 domain / Protein translocase SEC61 complex, gamma subunit / Tetratricopeptide-like helical domain superfamily / Immunoglobulin E-set / Protein transport protein SecG/Sec61-beta/Sbh / DnaJ domain, conserved site / Translocation protein Sec66 / Translocon Sec61/SecY, plug domain / Tetratricopeptide repeat / Protein translocase SecE domain superfamily / DnaJ domain / Translocation protein Sec63 / SecY conserved site / Protein transport Sec61-beta/Sbh / Chaperone J-domain superfamily / DnaJ domain / SecY translocase / Plug domain of Sec61p / SecE/Sec61-gamma subunits of protein translocation complex / Sec61beta family / Protein translocase complex, SecE/Sec61-gamma subunit / SecY/SEC61-alpha family
Protein transport protein SBH1 / Translocation protein SEC72 / Translocation protein SEC66 / Protein transport protein SEC61 / Protein translocation protein SEC63 / Protein transport protein SSS1
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsWu, X. / Cabanos, C. / Rapoport, T.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM052586 United States
CitationJournal: Nature / Year: 2019
Title: Structure of the post-translational protein translocation machinery of the ER membrane.
Authors: Xudong Wu / Cerrone Cabanos / Tom A Rapoport /
Abstract: Many proteins must translocate through the protein-conducting Sec61 channel in the eukaryotic endoplasmic reticulum membrane or the SecY channel in the prokaryotic plasma membrane. Proteins with ...Many proteins must translocate through the protein-conducting Sec61 channel in the eukaryotic endoplasmic reticulum membrane or the SecY channel in the prokaryotic plasma membrane. Proteins with highly hydrophobic signal sequences are first recognized by the signal recognition particle (SRP) and then moved co-translationally through the Sec61 or SecY channel by the associated translating ribosome. Substrates with less hydrophobic signal sequences bypass the SRP and are moved through the channel post-translationally. In eukaryotic cells, post-translational translocation is mediated by the association of the Sec61 channel with another membrane protein complex, the Sec62-Sec63 complex, and substrates are moved through the channel by the luminal BiP ATPase. How the Sec62-Sec63 complex activates the Sec61 channel for post-translational translocation is not known. Here we report the electron cryo-microscopy structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins. Sec63 causes wide opening of the lateral gate of the Sec61 channel, priming it for the passage of low-hydrophobicity signal sequences into the lipid phase, without displacing the channel's plug domain. Lateral channel opening is triggered by Sec63 interacting both with cytosolic loops in the C-terminal half of Sec61 and transmembrane segments in the N-terminal half of the Sec61 channel. The cytosolic Brl domain of Sec63 blocks ribosome binding to the channel and recruits Sec71 and Sec72, positioning them for the capture of polypeptides associated with cytosolic Hsp70. Our structure shows how the Sec61 channel is activated for post-translational protein translocation.
Validation Report
SummaryFull reportAbout validation report
History
DepositionDec 13, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 9, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 30, 2019Group: Data collection / Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Feb 20, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year
Revision 1.3Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)193,3876
Polymers193,3876
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, All components were purified by affinity purification and subjected to gel filtration, the bands were confirmed by mass-spec
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TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

#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 genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P32915
#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 genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P35179
#4: 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 (baker's yeast) / References: UniProt: P52870

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Protein , 1 types, 1 molecules A

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


Mass: 76831.602 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Production host: Saccharomyces cerevisiae (baker's yeast) / 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 (baker's yeast) / 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 (baker's yeast) / 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: The yeast Sec Complex involved in post-translational protein translocation
Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaClSodium chloride1
225 mMHEPESC8H18N2O4S1
30.05 %DigitoninC56H92O291
SpecimenConc.: 6 mg/ml / Details: This sample was monodisperse / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 298 K / Details: blot for 2.5 seconds before plunging

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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: OTHER
Electron lensMode: OTHER
Image recordingElectron dose: 54.8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
4GctfCTF correction
9RELION3initial Euler assignment
10RELION3final Euler assignment
13PHENIXmodel refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91218 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
RefinementHighest resolution: 4.1 Å

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