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- EMDB-22779: Cryo-EM structure of the Sec complex from S. cerevisiae, Sec61 po... -

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Basic information

Entry
Database: EMDB / ID: EMD-22779
TitleCryo-EM structure of the Sec complex from S. cerevisiae, Sec61 pore mutant, class with Sec62, conformation 1 (C1)
Map dataUnsharpened, lowpass-filtered map
Sample
  • Complex: Endoplasmic reticulum protein-transport machinery Sec complex from yeast with Sec61 pore ring mutated.
    • Protein or peptide: Protein transport protein SEC61Protein targeting
    • Protein or peptide: Protein transport protein SSS1Protein targeting
    • Protein or peptide: Protein transport protein SBH1Protein targeting
    • Protein or peptide: Protein translocation protein SEC63Protein targeting
    • Protein or peptide: Translocation protein SEC66
    • Protein or peptide: Translocation protein SEC72
    • Protein or peptide: Protein transport protein Sec62Protein targeting
KeywordsSec61 / translocon / endoplasmic reticulum / protein translocation / Sec62 / Sec63 / channel / PROTEIN TRANSPORT
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 BY4741 (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsItskanov S / Park E
Funding support United States, 1 items
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, 2020-
Header (metadata) releaseJan 6, 2021-
Map releaseJan 6, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kap
  • Surface level: 0.45
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22779.png

Downloads & links

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Map

FileDownload / File: emd_22779.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened, lowpass-filtered map
Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.45 / Movie #1: 0.45
Minimum - Maximum-0.48002195 - 1.3358262
Average (Standard dev.)0.008672541 (±0.06334379)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 294.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z294.400294.400294.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.4801.3360.009

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Supplemental data

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Mask #1

Fileemd_22779_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Additional map: Sharpened map

Fileemd_22779_additional_1.map
AnnotationSharpened map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_22779_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_22779_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Endoplasmic reticulum protein-transport machinery Sec complex fro...

EntireName: Endoplasmic reticulum protein-transport machinery Sec complex from yeast with Sec61 pore ring mutated.
Components
  • Complex: Endoplasmic reticulum protein-transport machinery Sec complex from yeast with Sec61 pore ring mutated.
    • Protein or peptide: Protein transport protein SEC61Protein targeting
    • Protein or peptide: Protein transport protein SSS1Protein targeting
    • Protein or peptide: Protein transport protein SBH1Protein targeting
    • Protein or peptide: Protein translocation protein SEC63Protein targeting
    • Protein or peptide: Translocation protein SEC66
    • Protein or peptide: Translocation protein SEC72
    • Protein or peptide: Protein transport protein Sec62Protein targeting

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Supramolecule #1: Endoplasmic reticulum protein-transport machinery Sec complex fro...

SupramoleculeName: Endoplasmic reticulum protein-transport machinery Sec complex from yeast with Sec61 pore ring mutated.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Sec61 has the following mutations: M90L/T185I/M294I/M450L
Source (natural)Organism: Saccharomyces cerevisiae BY4741 (yeast) / Strain: ATCC 204508 / S288c

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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 BY4741 (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 52.936086 KDa
SequenceString: MSSNRVLDLF KPFESFLPEV IAPERKVPYN QKLIWTGVSL LIFLILGQIP LYGIVSSETS DPLYWLRAML ASNRGTLLEL GVSPIITSS LIFQFLQGTQ LLQIRPESKQ DRELFQIAQK VCAIILILGQ ALVVVMTGNY GAPSDLGLPI CLLLIFQLMF A SLIVMLLD ...String:
MSSNRVLDLF KPFESFLPEV IAPERKVPYN QKLIWTGVSL LIFLILGQIP LYGIVSSETS DPLYWLRAML ASNRGTLLEL GVSPIITSS LIFQFLQGTQ LLQIRPESKQ DRELFQIAQK VCAIILILGQ ALVVVMTGNY GAPSDLGLPI CLLLIFQLMF A SLIVMLLD ELLSKGYGLG SGISLFIATN IAEQIFWRAF APTTVNSGRG KEFEGAVIAF FHLLAVRKDK KRALVEAFYR TN LPNMFQV LMTVAIFLFV LYLQGFRYEL PIRSTKVRGQ IGIYPIKLFY TSNTPIILQS ALTSNIFLIS QILFQKYPTN PLI RLIGVW GIRPGTQGPQ MALSGLAYYI QPLMSLSEAL LDPIKTIVYI TFVLGSCAVF SKTWIEISGT SPRDIAKQFK DQGM VINGK RETSIYRELK KIIPTAAAFG GATIGALSVG SDLLGTLGSG ASILLATTTI YGYYEAAAKE GGFTKNLVPG FSDLM

UniProtKB: Protein transport protein SEC61

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Macromolecule #2: Protein transport protein SSS1

MacromoleculeName: Protein transport protein SSS1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae BY4741 (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 8.958641 KDa
SequenceString:
MARASEKGEE KKQSNNQVEK LVEAPVEFVR EGTQFLAKCK KPDLKEYTKI VKAVGIGFIA VGIIGYAIKL IHIPIRYVIV

UniProtKB: Protein transport protein SSS1

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Macromolecule #3: Protein transport protein SBH1

MacromoleculeName: Protein transport protein SBH1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae BY4741 (yeast) / Strain: ATCC 204508 / S288c
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 #4: Protein translocation protein SEC63

MacromoleculeName: Protein translocation protein SEC63 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae BY4741 (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 77.93368 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae BY4741 (yeast)
SequenceString: GGSGGSGGSG GSGGSPTNYE YDEASETWPS FILTGLLMVV GPMTLLQIYQ IFFGANAEDG NSGKSKEFNE EVFKNLNEEY TSDEIKQFR RKFDKNSNKK SKIWSRRNII IIVGWILVAI LLQRINSNDA IKDAATKLFD PYEILGISTS ASDRDIKSAY R KLSVKFHP ...String:
GGSGGSGGSG GSGGSPTNYE YDEASETWPS FILTGLLMVV GPMTLLQIYQ IFFGANAEDG NSGKSKEFNE EVFKNLNEEY TSDEIKQFR RKFDKNSNKK SKIWSRRNII IIVGWILVAI LLQRINSNDA IKDAATKLFD PYEILGISTS ASDRDIKSAY R KLSVKFHP DKLAKGLTPD EKSVMEETYV QITKAYESLT DELVRQNYLK YGHPDGPQST SHGIALPRFL VDGSASPLLV VC YVALLGL ILPYFVSRWW ARTQSYTKKG IHNVTASNFV SNLVNYKPSE IVTTDLILHW LSFAHEFKQF FPDLQPTDFE KLL QDHINR RDSGKLNNAK FRIVAKCHSL LHGLLDIACG FRNLDIALGA INTFKCIVQA VPLTPNCQIL QLPNVDKEHF ITKT GDIHT LGKLFTLEDA KIGEVLGIKD QAKLNETLRV ASHIPNLKII KADFLVPGEN QVTPSSTPYI SLKVLVRSAK QPLIP TSLI PEENLTEPQD FESQRDPFAM MSKQPLVPYS FAPFFPTKRR GSWCCLVSSQ KDGKILQTPI IIEKLSYKNL NDDKDF FDK RIKMDLTKHE KFDINDWEIG TIKIPLGQPA PETVGDFFFR VIVKSTDYFT TDLDITMNMK VRDSPAVEQV EVYSEED DE YSTDDDETES DDESDASDYT DIDTDTEAED DESPEAGGAT TASGTGENLY FQ

UniProtKB: Protein translocation protein SEC63

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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 BY4741 (yeast) / Strain: ATCC 204508 / S288c
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 BY4741 (yeast) / Strain: ATCC 204508 / S288c
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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Macromolecule #7: Protein transport protein Sec62

MacromoleculeName: Protein transport protein Sec62 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae BY4741 (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 4.783888 KDa
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)

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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

Concentration5 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 35 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 43478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 48.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 195915 / Details: autopicked particles
Startup modelType of model: OTHER / Details: Ab initio reconstruction from cryoSPARC
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2.12)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 2.12) / Details: Non-uniform refinement from cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.12) / Details: Non-uniform refinement from cryoSPARC / Number images used: 17341
FSC plot (resolution estimation)

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