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- EMDB-27587: Cryo-EM structure of the human Sec61 complex inhibited by ipomoea... -

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Basic information

Entry
Database: EMDB / ID: EMD-27587
TitleCryo-EM structure of the human Sec61 complex inhibited by ipomoeassin F
Map dataCryo-EM structure of the human Sec61 complex inhibited by ipomoeassin F
Sample
  • Complex: A human-yeast chimeric Sec complex treated with ipomoeassin F
    • Protein or peptide: Protein transport protein Sec61 subunit alpha isoform 1Protein targeting
    • Protein or peptide: Protein transport protein Sec61 subunit gammaProtein targeting
    • Protein or peptide: Protein transport protein Sec61 subunit betaProtein targeting
  • Ligand: [(1~{S},3~{R},4~{S},5~{R},6~{R},8~{R},10~{S},23~{R},24~{R},25~{R},26~{R})-5-acetyloxy-6-methyl-4,26-bis(oxidanyl)-17,20-bis(oxidanylidene)-10-pentyl-24-[(~{E})-3-phenylprop-2-enoyl]oxy-2,7,9,21,27-pentaoxatricyclo[21.3.1.0^{3,8}]heptacosan-25-yl] (~{E})-2-methylbut-2-enoate
  • Ligand: water
Keywordstranslocon / inhibitor / protein translocation / PROTEIN TRANSPORT / PROTEIN TRANSPORT-INHIBITOR complex
Function / homology
Function and homology information


endoplasmic reticulum Sec complex / endoplasmic reticulum quality control compartment / pronephric nephron development / cotranslational protein targeting to membrane / Ssh1 translocon complex / Sec61 translocon complex / protein targeting to ER / protein insertion into ER membrane / post-translational protein targeting to endoplasmic reticulum membrane / SRP-dependent cotranslational protein targeting to membrane, translocation ...endoplasmic reticulum Sec complex / endoplasmic reticulum quality control compartment / pronephric nephron development / cotranslational protein targeting to membrane / Ssh1 translocon complex / Sec61 translocon complex / protein targeting to ER / protein insertion into ER membrane / post-translational protein targeting to endoplasmic reticulum membrane / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding / SRP-dependent cotranslational protein targeting to membrane / post-translational protein targeting to membrane, translocation / endoplasmic reticulum organization / retrograde protein transport, ER to cytosol / epidermal growth factor binding / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / SRP-dependent cotranslational protein targeting to membrane / protein transmembrane transporter activity / : / calcium channel activity / ribosome binding / ER-Phagosome pathway / endoplasmic reticulum membrane / endoplasmic reticulum / RNA binding / membrane / cytosol
Similarity search - Function
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 / Protein secE/sec61-gamma signature. / Protein secY signature 1. / Protein secY signature 2. ...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 / 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
Similarity search - Domain/homology
Protein transport protein Sec61 subunit gamma / Protein transport protein Sec61 subunit beta / Protein transport protein Sec61 subunit alpha isoform 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsPark E / Itskanov S
Funding support United States, 2 items
OrganizationGrant numberCountry
The Vallee Foundation Inc. United States
The Pew Charitable Trusts United States
CitationJournal: Nat Chem Biol / Year: 2023
Title: A common mechanism of Sec61 translocon inhibition by small molecules.
Authors: Samuel Itskanov / Laurie Wang / Tina Junne / Rumi Sherriff / Li Xiao / Nicolas Blanchard / Wei Q Shi / Craig Forsyth / Dominic Hoepfner / Martin Spiess / Eunyong Park /
Abstract: The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural ...The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit Sec61, generating cellular effects that are useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of human Sec61 inhibited by a comprehensive panel of structurally distinct small molecules-cotransin, decatransin, apratoxin, ipomoeassin, mycolactone, cyclotriazadisulfonamide and eeyarestatin. All inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of Sec61. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain in a closed state, thereby preventing the protein-translocation pore from opening. Our study provides the atomic details of Sec61-inhibitor interactions and the structural framework for further pharmacological studies and drug design.
History
DepositionJul 12, 2022-
Header (metadata) releaseMay 24, 2023-
Map releaseMay 24, 2023-
UpdateSep 6, 2023-
Current statusSep 6, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27587.png

Downloads & links

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Map

FileDownload / File: emd_27587.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the human Sec61 complex inhibited by ipomoeassin F
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.42
Minimum - Maximum-1.69699 - 2.5835009
Average (Standard dev.)0.0033468045 (±0.044249624)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 268.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Cryo-EM structure of the human Sec61 complex inhibited...

Fileemd_27587_half_map_1.map
AnnotationCryo-EM structure of the human Sec61 complex inhibited by ipomoeassin F
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM structure of the human Sec61 complex inhibited...

Fileemd_27587_half_map_2.map
AnnotationCryo-EM structure of the human Sec61 complex inhibited by ipomoeassin F
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : A human-yeast chimeric Sec complex treated with ipomoeassin F

EntireName: A human-yeast chimeric Sec complex treated with ipomoeassin F
Components
  • Complex: A human-yeast chimeric Sec complex treated with ipomoeassin F
    • Protein or peptide: Protein transport protein Sec61 subunit alpha isoform 1Protein targeting
    • Protein or peptide: Protein transport protein Sec61 subunit gammaProtein targeting
    • Protein or peptide: Protein transport protein Sec61 subunit betaProtein targeting
  • Ligand: [(1~{S},3~{R},4~{S},5~{R},6~{R},8~{R},10~{S},23~{R},24~{R},25~{R},26~{R})-5-acetyloxy-6-methyl-4,26-bis(oxidanyl)-17,20-bis(oxidanylidene)-10-pentyl-24-[(~{E})-3-phenylprop-2-enoyl]oxy-2,7,9,21,27-pentaoxatricyclo[21.3.1.0^{3,8}]heptacosan-25-yl] (~{E})-2-methylbut-2-enoate
  • Ligand: water

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Supramolecule #1: A human-yeast chimeric Sec complex treated with ipomoeassin F

SupramoleculeName: A human-yeast chimeric Sec complex treated with ipomoeassin F
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human) / Organelle: endoplasmic reticulum

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Macromolecule #1: Protein transport protein Sec61 subunit alpha isoform 1

MacromoleculeName: Protein transport protein Sec61 subunit alpha isoform 1
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.202438 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAIKFLEVIK PFCVILPEIQ KPERKIQFKE KVLWTAITLF IFLVCCQIPL FGIMSSDSAD PFYWMRVILA SNRGTLMELG ISPIVTSGL IMQLLAGAKI IEVGDTPKDR ALFNGAQKLF GMIITIGQSI VYVMTGMYGD PSEMGAGICL LITIQLFVAG L IVLLLDEL ...String:
MAIKFLEVIK PFCVILPEIQ KPERKIQFKE KVLWTAITLF IFLVCCQIPL FGIMSSDSAD PFYWMRVILA SNRGTLMELG ISPIVTSGL IMQLLAGAKI IEVGDTPKDR ALFNGAQKLF GMIITIGQSI VYVMTGMYGD PSEMGAGICL LITIQLFVAG L IVLLLDEL LQKGYGLGSG ISLFIATNIC ETIVWKAFSP TTVNTGRGME FEGAIIALFH LLATRTDKVR ALREAFYRQN LP NLMNLIA TIFVFAVVIY FQGFRYELPI RSTKVRGQIG IYPIKLFYTS NIPIILQSAL VSNLYVISQM LSARFSGNLL VSL LGTWSD TSSGGPARAY PVGGLCYYLS PPESFGSVLE DPVHAVVYIV FMLGSCAFFS KTWIEVSGSS PRDIAKQFKD QGMV INGKR ETSIYRELKK IIPTAAAFGG LCIGALSVLA DFLGAIGSGT GILLAVTIIY QYFEIFVKEQ SEVGSMGALL F

UniProtKB: Protein transport protein Sec61 subunit alpha isoform 1

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Macromolecule #2: Protein transport protein Sec61 subunit gamma

MacromoleculeName: Protein transport protein Sec61 subunit gamma / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.752325 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MDQVMQFVEP SRQFVKDSIR LVKRCTKPDR KEFQKIAMAT AIGFAIMGFI GFFVKLIHIP INNIIVGG

UniProtKB: Protein transport protein Sec61 subunit gamma

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Macromolecule #3: Protein transport protein Sec61 subunit beta

MacromoleculeName: Protein transport protein Sec61 subunit beta / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 9.987456 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MPGPTPSGTN VGSSGRSPSK AVAARAAGST VRQRKNASCG TRSAGRTTSA GTGGMWRFYT EDSPGLKVGP VPVLVMSLLF IASVFMLHI WGKYTRS

UniProtKB: Protein transport protein Sec61 subunit beta

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Macromolecule #4: [(1~{S},3~{R},4~{S},5~{R},6~{R},8~{R},10~{S},23~{R},24~{R},25~{R}...

MacromoleculeName: [(1~{S},3~{R},4~{S},5~{R},6~{R},8~{R},10~{S},23~{R},24~{R},25~{R},26~{R})-5-acetyloxy-6-methyl-4,26-bis(oxidanyl)-17,20-bis(oxidanylidene)-10-pentyl-24-[(~{E})-3-phenylprop-2-enoyl]oxy- ...Name: [(1~{S},3~{R},4~{S},5~{R},6~{R},8~{R},10~{S},23~{R},24~{R},25~{R},26~{R})-5-acetyloxy-6-methyl-4,26-bis(oxidanyl)-17,20-bis(oxidanylidene)-10-pentyl-24-[(~{E})-3-phenylprop-2-enoyl]oxy-2,7,9,21,27-pentaoxatricyclo[21.3.1.0^{3,8}]heptacosan-25-yl] (~{E})-2-methylbut-2-enoate
type: ligand / ID: 4 / Number of copies: 1 / Formula: SXF
Molecular weightTheoretical: 830.954 Da
Chemical component information

ChemComp-SXF:
[(1~{S},3~{R},4~{S},5~{R},6~{R},8~{R},10~{S},23~{R},24~{R},25~{R},26~{R})-5-acetyloxy-6-methyl-4,26-bis(oxidanyl)-17,20-bis(oxidanylidene)-10-pentyl-24-[(~{E})-3-phenylprop-2-enoyl]oxy-2,7,9,21,27-pentaoxatricyclo[21.3.1.0^{3,8}]heptacosan-25-yl] (~{E})-2-methylbut-2-enoate

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 1 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration10 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mM(HOCH2)3CNH2Tris
100.0 mMNaClSodium chloridesodium chloride
2.0 mMC4H10O2S2dithiothreitol
1.0 mMC10H16N2O8EDTAEthylenediaminetetraacetic acid
3.0 mMC13H17F13NO4PFluorinated Fos-choline-8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 4 seconds before plunging.
DetailsReconsitituted into a peptidisc. Monodisperse peak from a Superose 6 column.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
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 K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 676714
Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 324612
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8do1:
Cryo-EM structure of the human Sec61 complex inhibited by ipomoeassin F

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