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- EMDB-22776: Cryo-EM structure of the Sec complex from T. lanuginosus lacking Sec62 -

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Basic information

Entry
Database: EMDB / ID: EMD-22776
TitleCryo-EM structure of the Sec complex from T. lanuginosus lacking Sec62
Map dataUnsharpened, lowpass-filtered map
Sample
  • Complex: Endoplasmic reticulum protein-transport machinery Sec complex from T. lanuginosus without Sec62
    • Protein or peptide: Protein transport channel Sec61 complex, alpha subunit (Sec61)
    • Protein or peptide: Protein transport channel Sec61 complex, gamma subunit (Sss1)
    • Protein or peptide: Protein transport channel Sec61 complex, beta subunit (Sbh1)
    • Protein or peptide: Protein transport protein Sec63
    • Protein or peptide: Protein transport protein Sec66/Sec71
    • Protein or peptide: Protein transport protein Sec72
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
KeywordsSec61 / translocon / endoplasmic reticulum / protein translocation / Sec62 / Sec63 / channel / PROTEIN TRANSPORT
Function / homology
Function and homology information


misfolded protein transport / Sec62/Sec63 complex / translocon complex / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / cytosol to endoplasmic reticulum transport / rough endoplasmic reticulum membrane / Ssh1 translocon complex / Sec61 translocon complex / protein-transporting ATPase activity / post-translational protein targeting to endoplasmic reticulum membrane ...misfolded protein transport / Sec62/Sec63 complex / translocon complex / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / cytosol to endoplasmic reticulum transport / 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 / SRP-dependent cotranslational protein targeting to membrane / signal sequence binding / post-translational protein targeting to membrane, translocation / peptide transmembrane transporter activity / nuclear inner membrane / retrograde protein transport, ER to cytosol / protein transmembrane transporter activity / ERAD pathway / guanyl-nucleotide exchange factor activity / cell periphery / ribosome binding / endoplasmic reticulum membrane / structural molecule activity / endoplasmic reticulum / mitochondrion / 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 / DnaJ domain / Sec63 domain / Sec63 Brl 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 speciesThermomyces lanuginosus (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 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) releaseFeb 3, 2021-
Map releaseFeb 3, 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: 0.46
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.46
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kan
  • Surface level: 0.46
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22776.png

Downloads & links

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Map

FileDownload / File: emd_22776.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened, lowpass-filtered map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.14 Å/pix.
x 256 pix.
= 291.84 Å		1.14 Å/pix.
x 256 pix.
= 291.84 Å		1.14 Å/pix.
x 256 pix.
= 291.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.14 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.46 / Movie #1: 0.46
Minimum - Maximum-0.5402787 - 1.5601128
Average (Standard dev.)0.0036001448 (±0.05827471)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 291.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.141.141.14
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z291.840291.840291.840
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.5401.5600.004

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Supplemental data

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Mask #1

Fileemd_22776_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Sharpened map

Fileemd_22776_additional_1.map
AnnotationSharpened map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-volume 1

Fileemd_22776_half_map_1.map
Annotationhalf-volume 1
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-volume 2

Fileemd_22776_half_map_2.map
Annotationhalf-volume 2
Projections & Slices
AxesZYX

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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 T. lanuginosus without Sec62
Components
  • Complex: Endoplasmic reticulum protein-transport machinery Sec complex from T. lanuginosus without Sec62
    • Protein or peptide: Protein transport channel Sec61 complex, alpha subunit (Sec61)
    • Protein or peptide: Protein transport channel Sec61 complex, gamma subunit (Sss1)
    • Protein or peptide: Protein transport channel Sec61 complex, beta subunit (Sbh1)
    • Protein or peptide: Protein transport protein Sec63
    • Protein or peptide: Protein transport protein Sec66/Sec71
    • Protein or peptide: Protein transport protein Sec72
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

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

SupramoleculeName: Endoplasmic reticulum protein-transport machinery Sec complex from T. lanuginosus without Sec62
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Thermomyces lanuginosus (fungus)

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

MacromoleculeName: Protein transport channel Sec61 complex, alpha subunit (Sec61)
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermomyces lanuginosus (fungus)
Molecular weightTheoretical: 52.42743 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae BY4741 (yeast)
SequenceString: MSGLRFLDLI KPFTPLLPEV AAPETKVPFN QKLMWTGLTL LIFLVMSQMP LYGIVSSDTS DPLYWLRMML ASNRGTLMEL GITPIISSG MVFQLLAGTH LIDVNLDLKT DRELYQTAQK LFAIILSFGQ ACVHVLTGLY GQPSDLGAGI CVLLIVQLVV A GLVVILLD ...String:
MSGLRFLDLI KPFTPLLPEV AAPETKVPFN QKLMWTGLTL LIFLVMSQMP LYGIVSSDTS DPLYWLRMML ASNRGTLMEL GITPIISSG MVFQLLAGTH LIDVNLDLKT DRELYQTAQK LFAIILSFGQ ACVHVLTGLY GQPSDLGAGI CVLLIVQLVV A GLVVILLD ELLQKGYGLG SGISLFIATN ICESIVWKAF SPTTINTGRG PEFEGAIIAL FHLLLTWPDK QRALREAFYR QS LPNIMNL LATLLVFAAV IYLQGFRVEI PVKSARQRGV RGSYPVRLFY TSNMPIMLQS ALCSNVFLIS QMLYSRFSDN LLV RLLGVW EPREGSAQLH AASGIAYYMS PPLNFKEALL DPVHTVVYIT FMLVACALFS KTWIEVSGSS PRDVAKQLKD QGLV MAGHR EQSMYKELKR VIPTAAAFGG ACIGALSVAS DLLGALGSGT GILLAVTIIY GYFEMAAREG DFGQGLRGLV PGNGS

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Macromolecule #2: Protein transport channel Sec61 complex, gamma subunit (Sss1)

MacromoleculeName: Protein transport channel Sec61 complex, gamma subunit (Sss1)
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermomyces lanuginosus (fungus)
Molecular weightTheoretical: 7.852352 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae BY4741 (yeast)
SequenceString:
MSEQVQELLD IPRDFLKDGM QFIHKCQKPD RKEFKKVCQA VAIGFVAMGA IGYIVKLVHI PINNILVAGS

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Macromolecule #3: Protein transport channel Sec61 complex, beta subunit (Sbh1)

MacromoleculeName: Protein transport channel Sec61 complex, beta subunit (Sbh1)
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermomyces lanuginosus (fungus)
Molecular weightTheoretical: 12.491097 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae BY4741 (yeast)
SequenceString:
MASSGAESGS ESKSPNPGAG SGPGSASGSS AGVIRPSSPT PPGGPRAAIR RRAAADHKES LRNARPSSTR AAGAGGSSGT MLKLYTDES PGLRVDPVVV LVLSLCFIFS VVGLHVIAKI TRKFSS

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Macromolecule #4: Protein transport protein Sec63

MacromoleculeName: Protein transport protein Sec63 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermomyces lanuginosus (fungus)
Molecular weightTheoretical: 79.976703 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae BY4741 (yeast)
SequenceString: GGSGGSGGSG GSGGSMSSRE YNYDENGQFF PFFVLTLTGL VTLPLTYSLL KPPKKVESTA PRIKSDFKPQ HDDIIQNQKR KRLRKERRV KRAIAVVVGW AIIGYMVYLI IVTRRTAPKI WDPYEILGIS RSADERAIAR RYKRLSLLYH PDKVRPDPSK N ETMEMLNQ ...String:
GGSGGSGGSG GSGGSMSSRE YNYDENGQFF PFFVLTLTGL VTLPLTYSLL KPPKKVESTA PRIKSDFKPQ HDDIIQNQKR KRLRKERRV KRAIAVVVGW AIIGYMVYLI IVTRRTAPKI WDPYEILGIS RSADERAIAR RYKRLSLLYH PDKVRPDPSK N ETMEMLNQ RFVELTKAYK ALTDEEIRNN YLQYGHPDGK QSYSIGIALP KLIIEEGSGK YVLMLYASLL GILLPYIVGR WW YGSQRYT REKVLAASAG NMFREYEGTM IGGPIVNALS TGEEYKEMLS GPKAEEGLAK VEKKVLALDE KILSAKDREV LRK IDNPVR RKALALLWAY LNRIDLEDPV LNEEKYEAGS IALSLTESFT AIALAFGNLI PIIGAYRISQ CIVQAISPGS SPLL QLPYF TPKVVESVEG ADVKTHLSVQ KYLDMPEERR RSLTVGPGLL TEDQYNSAIA VAKQLPLFAI SKAFFKVAGE RVVTP SSLV QLVIKGRIIP PGSTGVPDVT EKDLEDIDPD EADVNAIIGR KGATKPSGKS GDENDGDRVQ PPLAHAPYLP RDHPPR WHI FLADAKQGKI AVPPFTFTTF DKPIFDEQGK PTFNMQTLRM QFQAPPQVGN FSFVLHMISD SYMGFDVKQE ITLQVED PS KAAVLQEEDD ISEPDEDSIA GQMQALKTGV PPKKKKVVES DDDESDTEGD EEDTSETDTE TDTDEEGSGT GENLYFQ

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Macromolecule #5: Protein transport protein Sec66/Sec71

MacromoleculeName: Protein transport protein Sec66/Sec71 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermomyces lanuginosus (fungus)
Molecular weightTheoretical: 27.555285 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae BY4741 (yeast)
SequenceString: MDWLTLVVPF AYLGVLIGCL ATFSSLYRRR KAAKAASLEP WFPPHLQRDI YHSLLHLDQQ QQNEKKTRVP ETVLKAALLR RAAEDIKRV MAIREQKQAL ALLLQRGSVG DELWQRFLRA EKEMEDEVRD VVAEANSYAP NWGQVIFQSA REMDANATYR A RMEEYQAT ...String:
MDWLTLVVPF AYLGVLIGCL ATFSSLYRRR KAAKAASLEP WFPPHLQRDI YHSLLHLDQQ QQNEKKTRVP ETVLKAALLR RAAEDIKRV MAIREQKQAL ALLLQRGSVG DELWQRFLRA EKEMEDEVRD VVAEANSYAP NWGQVIFQSA REMDANATYR A RMEEYQAT VAEERAWWDK KRASIQEGFM KELDAEKERP ATAASTATNT TSTTSDDDAV LVEAEKEGTS SPAPGKKKKK GK KGS

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Macromolecule #6: Protein transport protein Sec72

MacromoleculeName: Protein transport protein Sec72 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermomyces lanuginosus (fungus)
Molecular weightTheoretical: 23.382234 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae BY4741 (yeast)
SequenceString: MSSDLDTYTH YPLHLDPSSK AVSLATTEGQ TPAQTEAVEA ELQQLNALHR SLISLDPPNV PPPPLPINPK RSAQITKLKE TANTAYKRG NHGEAVRLYS YAIEMAAGRP GWEPVNLARE ELSGLYANRA QAHMAQQMWP EGWVDAKCSV ESKPVGNAKG W WRGGKCLV ...String:
MSSDLDTYTH YPLHLDPSSK AVSLATTEGQ TPAQTEAVEA ELQQLNALHR SLISLDPPNV PPPPLPINPK RSAQITKLKE TANTAYKRG NHGEAVRLYS YAIEMAAGRP GWEPVNLARE ELSGLYANRA QAHMAQQMWP EGWVDAKCSV ESKPVGNAKG W WRGGKCLV EMGRYDEARA WIEQALGIEG PASDGGKELA ALLEEIKAGS QRRQGS

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Macromolecule #7: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 7 / Number of copies: 2 / Formula: PC1
Molecular weightTheoretical: 790.145 Da
Chemical component information

ChemComp-PC1:
1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / phospholipid*YM

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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 TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 43860 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 546712 / Details: autopicked particles
Startup modelType of model: OTHER / Details: Ab initio reconstruction from cryoSPARC
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.12) / Number images used: 222047
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
FSC plot (resolution estimation)

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