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- PDB-8dnx: Cryo-EM structure of the human Sec61 complex inhibited by cotransin -

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Basic information

Entry
Database: PDB / ID: 8dnx
TitleCryo-EM structure of the human Sec61 complex inhibited by cotransin
Components
  • Cotransin analogue peptide inhibitor
  • Protein transport protein Sec61 subunit alpha isoform 1
  • Protein transport protein Sec61 subunit beta
  • Protein transport protein Sec61 subunit gamma
KeywordsPROTEIN TRANSPORT/INHIBITOR / translocon / inhibitor / protein translocation / PROTEIN TRANSPORT / PROTEIN TRANSPORT-INHIBITOR complex
Function / homology
Function and homology information


endoplasmic reticulum Sec complex / pronephric nephron development / endoplasmic reticulum quality control compartment / 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 / pronephric nephron development / endoplasmic reticulum quality control compartment / 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 / endoplasmic reticulum organization / post-translational protein targeting to membrane, translocation / epidermal growth factor binding / retrograde protein transport, ER to cytosol / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / SRP-dependent cotranslational protein targeting to membrane / protein transmembrane transporter activity / ERAD pathway / guanyl-nucleotide exchange factor 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)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsPark, E. / Itskanov, S.
Funding support United States, 2items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 24, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 6, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 2.0Nov 15, 2023Group: Atomic model / Data collection / Derived calculations
Category: atom_site / chem_comp_atom ...atom_site / chem_comp_atom / chem_comp_bond / pdbx_validate_rmsd_bond / pdbx_validate_torsion / struct_conn
Item: _atom_site.auth_atom_id / _atom_site.label_atom_id ..._atom_site.auth_atom_id / _atom_site.label_atom_id / _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_1 / _chem_comp_bond.atom_id_2 / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr2_label_atom_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Protein transport protein Sec61 subunit gamma
C: Protein transport protein Sec61 subunit beta
A: Protein transport protein Sec61 subunit alpha isoform 1
D: Cotransin analogue peptide inhibitor


Theoretical massNumber of molelcules
Total (without water)70,8954
Polymers70,8954
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Protein transport protein Sec61 subunit gamma


Mass: 7752.325 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SEC61G / Plasmid: pFastBac / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P60059
#2: Protein Protein transport protein Sec61 subunit beta


Mass: 9987.456 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SEC61B / Plasmid: pFastBac / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P60468
#3: Protein Protein transport protein Sec61 subunit alpha isoform 1 / Sec61 alpha-1


Mass: 52202.438 Da / Num. of mol.: 1
Mutation: V263L, D264E, K268R, A270T, R271K, Y272V, Y276I, N277G, T278I, L394F, E346D, Q348G, M401I, R402N, H404K, M409I, V410Y, H411R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SEC61A1, SEC61A / Plasmid: pFastBac / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P61619
#4: Protein/peptide Cotransin analogue peptide inhibitor


Mass: 953.219 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
Has ligand of interestY

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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: A human-yeast chimeric Sec complex treated with cotransin
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human) / Organelle: endoplasmic reticulum
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf9 / Plasmid: pFastBac
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris(HOCH2)3CNH21
2100 mMsodium chlorideNaCl1
32 mMdithiothreitolC4H10O2S21
41 mMEDTAC10H16N2O81
53 mMFluorinated Fos-choline-8C13H17F13NO4P1
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Reconsitituted into a peptidisc. Monodisperse peak from a Superose 6 column.
Specimen supportDetails: Used a PELCO easiGlow glow discharge cleaner / Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 4 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4Warp1.0.9CTF correction
5cryoSPARC3.3CTF correction
8Coot0.9.8model fitting
10cryoSPARC3.3initial Euler assignment
11cryoSPARC3.3final Euler assignment
13cryoSPARC3.33D reconstruction
14PHENIX1.19model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 274970
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 137441 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 71.29 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00364363
ELECTRON MICROSCOPYf_angle_d0.57955911
ELECTRON MICROSCOPYf_chiral_restr0.0404698
ELECTRON MICROSCOPYf_plane_restr0.0041719
ELECTRON MICROSCOPYf_dihedral_angle_d5.7854585

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