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- EMDB-0440: CryoEM structure of the post-translational protein translocation ... -

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Basic information

Entry
Database: EMDB / ID: EMD-0440
TitleCryoEM structure of the post-translational protein translocation machinery of the ER membrane
Map dataEM map after focused refinement
Sample
  • Complex: The yeast Sec Complex involved in post-translational protein translocation
    • Protein or peptide: Protein transport protein SEC61Protein targeting
    • Protein or peptide: Protein translocation protein SEC63Protein targeting
    • Protein or peptide: Protein transport protein SSS1Protein targeting
    • Protein or peptide: Protein transport protein SBH1Protein targeting
    • Protein or peptide: Translocation protein SEC66
    • Protein or peptide: Translocation protein SEC72
KeywordsSec61 / post-translational translocation / yeast / Sec63 / PROTEIN TRANSPORT / TRANSPORT PROTEIN
Function / homology
Function and homology information


protein transmembrane import into intracellular organelle / misfolded protein transport / 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 ...protein transmembrane import into intracellular organelle / misfolded protein transport / 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 / nuclear inner membrane / retrograde protein transport, ER to cytosol / protein transmembrane transporter activity / : / guanyl-nucleotide exchange factor activity / cell periphery / ribosome binding / structural molecule activity / endoplasmic reticulum membrane / endoplasmic reticulum / mitochondrion / membrane / 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 / 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
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 (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsWu X / Cabanos C
Funding support United States, 1 items
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.
History
DepositionDec 13, 2018-
Header (metadata) releaseJan 9, 2019-
Map releaseJan 9, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nd1
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0440.png

Downloads & links

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Map

FileDownload / File: emd_0440.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM map after focused refinement
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.028 / Movie #1: 0.035
Minimum - Maximum-0.13241692 - 0.19763649
Average (Standard dev.)0.000037265527 (±0.008323493)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 216.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z216.000216.000216.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.1320.1980.000

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Supplemental data

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Additional map: EM map before focused refinement, without sharpening

Fileemd_0440_additional.map
AnnotationEM map before focused refinement, without sharpening
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The yeast Sec Complex involved in post-translational protein tran...

EntireName: The yeast Sec Complex involved in post-translational protein translocation
Components
  • Complex: The yeast Sec Complex involved in post-translational protein translocation
    • Protein or peptide: Protein transport protein SEC61Protein targeting
    • Protein or peptide: Protein translocation protein SEC63Protein targeting
    • Protein or peptide: Protein transport protein SSS1Protein targeting
    • Protein or peptide: Protein transport protein SBH1Protein targeting
    • Protein or peptide: Translocation protein SEC66
    • Protein or peptide: Translocation protein SEC72

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Supramolecule #1: The yeast Sec Complex involved in post-translational protein tran...

SupramoleculeName: The yeast Sec Complex involved in post-translational protein translocation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Protein transport protein SEC61

MacromoleculeName: Protein transport protein SEC61 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 52.978148 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSSNRVLDLF KPFESFLPEV IAPERKVPYN QKLIWTGVSL LIFLILGQIP LYGIVSSETS DPLYWLRAML ASNRGTLLEL GVSPIITSS MIFQFLQGTQ LLQIRPESKQ DRELFQIAQK VCAIILILGQ ALVVVMTGNY GAPSDLGLPI CLLLIFQLMF A SLIVMLLD ...String:
MSSNRVLDLF KPFESFLPEV IAPERKVPYN QKLIWTGVSL LIFLILGQIP LYGIVSSETS DPLYWLRAML ASNRGTLLEL GVSPIITSS MIFQFLQGTQ LLQIRPESKQ DRELFQIAQK VCAIILILGQ ALVVVMTGNY GAPSDLGLPI CLLLIFQLMF A SLIVMLLD ELLSKGYGLG SGISLFTATN IAEQIFWRAF APTTVNSGRG KEFEGAVIAF FHLLAVRKDK KRALVEAFYR TN LPNMFQV LMTVAIFLFV LYLQGFRYEL PIRSTKVRGQ IGIYPIKLFY TSNTPIMLQS ALTSNIFLIS QILFQKYPTN PLI RLIGVW GIRPGTQGPQ MALSGLAYYI QPLMSLSEAL LDPIKTIVYI TFVLGSCAVF SKTWIEISGT SPRDIAKQFK DQGM VINGK RETSIYRELK KIIPTAAAFG GATIGALSVG SDLLGTLGSG ASILMATTTI YGYYEAAAKE GGFTKNLVPG FSDLM

UniProtKB: Protein transport protein SEC61

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Macromolecule #2: Protein translocation protein SEC63

MacromoleculeName: Protein translocation protein SEC63 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 76.831602 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MPTNYEYDEA SETWPSFILT GLLMVVGPMT LLQIYQIFFG ANAEDGNSGK SKEFNEEVFK NLNEEYTSDE IKQFRRKFDK NSNKKSKIW SRRNIIIIVG WILVAILLQR INSNDAIKDA ATKLFDPYEI LGISTSASDR DIKSAYRKLS VKFHPDKLAK G LTPDEKSV ...String:
MPTNYEYDEA SETWPSFILT GLLMVVGPMT LLQIYQIFFG ANAEDGNSGK SKEFNEEVFK NLNEEYTSDE IKQFRRKFDK NSNKKSKIW SRRNIIIIVG WILVAILLQR INSNDAIKDA ATKLFDPYEI LGISTSASDR DIKSAYRKLS VKFHPDKLAK G LTPDEKSV MEETYVQITK AYESLTDELV RQNYLKYGHP DGPQSTSHGI ALPRFLVDGS ASPLLVVCYV ALLGLILPYF VS RWWARTQ SYTKKGIHNV TASNFVSNLV NYKPSEIVTT DLILHWLSFA HEFKQFFPDL QPTDFEKLLQ DHINRRDSGK LNN AKFRIV AKCHSLLHGL LDIACGFRNL DIALGAINTF KCIVQAVPLT PNCQILQLPN VDKEHFITKT GDIHTLGKLF TLED AKIGE VLGIKDQAKL NETLRVASHI PNLKIIKADF LVPGENQVTP SSTPYISLKV LVRSAKQPLI PTSLIPEENL TEPQD FESQ RDPFAMMSKQ PLVPYSFAPF FPTKRRGSWC CLVSSQKDGK ILQTPIIIEK LSYKNLNDDK DFFDKRIKMD LTKHEK FDI NDWEIGTIKI PLGQPAPETV GDFFFRVIVK STDYFTTDLD ITMNMKVRDS PAVEQVEVYS EEDDEYSTDD DETESDD ES DASDYTDIDT DTEAEDDESP EGSGGSGDYK DDDDK

UniProtKB: Protein translocation protein SEC63

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Macromolecule #3: Protein transport protein SSS1

MacromoleculeName: Protein transport protein SSS1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 8.958641 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MARASEKGEE KKQSNNQVEK LVEAPVEFVR EGTQFLAKCK KPDLKEYTKI VKAVGIGFIA VGIIGYAIKL IHIPIRYVIV

UniProtKB: Protein transport protein SSS1

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Macromolecule #4: Protein transport protein SBH1

MacromoleculeName: Protein transport protein SBH1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 8.723155 KDa
SequenceString:
MSSPTPPGGQ RTLQKRKQGS SQKVAASAPK KNTNSNNSIL KIYSDEATGL RVDPLVVLFL AVGFIFSVVA LHVISKVAGK LF

UniProtKB: Protein transport protein SBH1

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Macromolecule #5: Translocation protein SEC66

MacromoleculeName: Translocation protein SEC66 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 24.263939 KDa
SequenceString: MSEFNETKFS NNGTFFETEE PIVETKSISV YTPLIYVFIL VVSLVMFASS YRKKQAKKIS EQPSIFDEND AHDLYFQIKE MSENEKIHE KVLKAALLNR GAESVRRSLK LKELAPQINL LYKNGSIGED YWKRFETEVK LIELEFKDTL QEAERLQPGW V QLFVMVCK ...String:
MSEFNETKFS NNGTFFETEE PIVETKSISV YTPLIYVFIL VVSLVMFASS YRKKQAKKIS EQPSIFDEND AHDLYFQIKE MSENEKIHE KVLKAALLNR GAESVRRSLK LKELAPQINL LYKNGSIGED YWKRFETEVK LIELEFKDTL QEAERLQPGW V QLFVMVCK EICFNQALSR RYQSILKRKE VCIKEWELKI NNDGRLVN

UniProtKB: Translocation protein SEC66

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Macromolecule #6: Translocation protein SEC72

MacromoleculeName: Translocation protein SEC72 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 21.63109 KDa
SequenceString: MVTLEYNANS KLITASDAVV ALSTETNIDQ INVLTTSLIG ETNPNFTPQP NEALSKMIKG LFESGMKNLQ QKKLNEALKN VSLAIEMAQ RKRAPWEAFA IQLPELHFML RSKIDLCLIL GKHLEALQDL DFLLGTGLIQ PDVFVRKADC LLKLRQWEEA R ATCERGLA ...String:
MVTLEYNANS KLITASDAVV ALSTETNIDQ INVLTTSLIG ETNPNFTPQP NEALSKMIKG LFESGMKNLQ QKKLNEALKN VSLAIEMAQ RKRAPWEAFA IQLPELHFML RSKIDLCLIL GKHLEALQDL DFLLGTGLIQ PDVFVRKADC LLKLRQWEEA R ATCERGLA LAPEDMKLRA LLIETARNLA EYNGE

UniProtKB: Translocation protein SEC72

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
25.0 mMC8H18N2O4SHEPES
0.05 %C56H92O29Digitonin
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: GATAN CRYOPLUNGE 3 / Details: blot for 2.5 seconds before plunging.
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 54.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 91218

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

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

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