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- PDB-8b6l: Subtomogram average of the human Sec61-TRAP-OSTA-translocon -

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Entry
Database: PDB / ID: 8b6l
TitleSubtomogram average of the human Sec61-TRAP-OSTA-translocon
Components
  • (Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ...) x 5
  • (Protein transport protein Sec61 subunit ...Protein targeting) x 3
  • (Translocon-associated protein subunit ...) x 4
  • Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit
  • Oligosaccharyltransferase complex subunit OSTCOligosaccharyltransferase
  • Signal peptide mix
  • Transmembrane protein 258Transmembrane protein
KeywordsMEMBRANE PROTEIN / membrane protein complex / protein translocation / N-glycosylation / signal peptide insertion
Function / homology
Function and homology information


oligosaccharyltransferase complex binding / oligosaccharyltransferase I complex / oligosaccharyltransferase III complex / Asparagine N-linked glycosylation / membrane-bounded organelle / endoplasmic reticulum Sec complex / co-translational protein modification / endoplasmic reticulum quality control compartment / pronephric nephron development / oligosaccharyltransferase complex ...oligosaccharyltransferase complex binding / oligosaccharyltransferase I complex / oligosaccharyltransferase III complex / Asparagine N-linked glycosylation / membrane-bounded organelle / endoplasmic reticulum Sec complex / co-translational protein modification / endoplasmic reticulum quality control compartment / pronephric nephron development / oligosaccharyltransferase complex / dolichyl-diphosphooligosaccharide-protein glycotransferase / dolichyl-diphosphooligosaccharide-protein glycotransferase activity / cotranslational protein targeting to membrane / Ssh1 translocon complex / Sec61 translocon complex / protein targeting to ER / protein insertion into ER membrane / protein N-linked glycosylation via asparagine / XBP1(S) activates chaperone genes / 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 / protein N-linked glycosylation / epidermal growth factor binding / epithelial cell apoptotic process / retrograde protein transport, ER to cytosol / azurophil granule membrane / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / : / protein glycosylation / blastocyst development / Advanced glycosylation endproduct receptor signaling / SRP-dependent cotranslational protein targeting to membrane / protein transmembrane transporter activity / endomembrane system / enzyme activator activity / rough endoplasmic reticulum / T cell activation / response to endoplasmic reticulum stress / post-translational protein modification / response to cytokine / calcium channel activity / protein modification process / regulation of protein stability / melanosome / ribosome binding / ER-Phagosome pathway / Maturation of spike protein / membrane => GO:0016020 / nuclear body / inflammatory response / intracellular membrane-bounded organelle / apoptotic process / Neutrophil degranulation / positive regulation of cell population proliferation / endoplasmic reticulum membrane / negative regulation of apoptotic process / endoplasmic reticulum / protein-containing complex / RNA binding / extracellular exosome / membrane / metal ion binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Translocon-associated protein (TRAP), alpha subunit / Translocon-associated protein (TRAP), alpha subunit / Translocon-associated / Translocon-associated protein subunit beta / Translocon-associated protein subunit gamma / Translocon-associated protein, delta subunit precursor (TRAP-delta) / Translocon-associated protein, gamma subunit (TRAP-gamma) / DAD/Ost2 / Oligosaccharyltransferase complex subunit / DAD family ...Translocon-associated protein (TRAP), alpha subunit / Translocon-associated protein (TRAP), alpha subunit / Translocon-associated / Translocon-associated protein subunit beta / Translocon-associated protein subunit gamma / Translocon-associated protein, delta subunit precursor (TRAP-delta) / Translocon-associated protein, gamma subunit (TRAP-gamma) / DAD/Ost2 / Oligosaccharyltransferase complex subunit / DAD family / Oligosaccharyltransferase subunit 5 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48kDa subunit / Oligosaccharyltransferase complex subunit OSTC / Oligosaccharyltransferase 48 kDa subunit beta / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit Swp1 / Oligosaccharyltransferase subunit Ribophorin II / Oligosaccaryltransferase / Oligosaccharyltransferase subunit ost4p superfamily / Oligosaccaryltransferase / Ribophorin I / Ribophorin I / Oligosaccharyl transferase complex, subunit OST3/OST6 / OST3 / OST6 family, transporter family / : / STT3/PglB/AglB core domain / : / Oligosaccharyl transferase, STT3 subunit / Oligosaccharyl transferase STT3, N-terminal / 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
Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 4 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit / Translocon-associated protein subunit alpha / Translocon-associated protein subunit beta / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A / Translocon-associated protein subunit delta / Protein transport protein Sec61 subunit gamma / Protein transport protein Sec61 subunit beta ...Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 4 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit / Translocon-associated protein subunit alpha / Translocon-associated protein subunit beta / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A / Translocon-associated protein subunit delta / Protein transport protein Sec61 subunit gamma / Protein transport protein Sec61 subunit beta / Transmembrane protein 258 / Protein transport protein Sec61 subunit alpha isoform 1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit DAD1 / Oligosaccharyltransferase complex subunit OSTC / Translocon-associated protein subunit gamma
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 7.6 Å
AuthorsGemmer, M. / Fedry, J.M.M. / Forster, F.G.
Funding supportEuropean Union, Netherlands, 3items
OrganizationGrant numberCountry
European Research Council (ERC)724425European Union
Netherlands Organisation for Scientific Research (NWO)724.016.001 Netherlands
Netherlands Organisation for Scientific Research (NWO)212.152 Netherlands
CitationJournal: Nature / Year: 2023
Title: Visualization of translation and protein biogenesis at the ER membrane.
Authors: Max Gemmer / Marten L Chaillet / Joyce van Loenhout / Rodrigo Cuevas Arenas / Dimitrios Vismpas / Mariska Gröllers-Mulderij / Fujiet A Koh / Pascal Albanese / Richard A Scheltema / Stuart C ...Authors: Max Gemmer / Marten L Chaillet / Joyce van Loenhout / Rodrigo Cuevas Arenas / Dimitrios Vismpas / Mariska Gröllers-Mulderij / Fujiet A Koh / Pascal Albanese / Richard A Scheltema / Stuart C Howes / Abhay Kotecha / Juliette Fedry / Friedrich Förster /
Abstract: The dynamic ribosome-translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome. It governs the synthesis, translocation, membrane ...The dynamic ribosome-translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome. It governs the synthesis, translocation, membrane insertion, N-glycosylation, folding and disulfide-bond formation of nascent proteins. Although individual components of this machinery have been studied at high resolution in isolation, insights into their interplay in the native membrane remain limited. Here we use cryo-electron tomography, extensive classification and molecular modelling to capture snapshots of mRNA translation and protein maturation at the ER membrane at molecular resolution. We identify a highly abundant classical pre-translocation intermediate with eukaryotic elongation factor 1a (eEF1a) in an extended conformation, suggesting that eEF1a may remain associated with the ribosome after GTP hydrolysis during proofreading. At the ER membrane, distinct polysomes bind to different ER translocons specialized in the synthesis of proteins with signal peptides or multipass transmembrane proteins with the translocon-associated protein complex (TRAP) present in both. The near-complete atomic model of the most abundant ER translocon variant comprising the protein-conducting channel SEC61, TRAP and the oligosaccharyltransferase complex A (OSTA) reveals specific interactions of TRAP with other translocon components. We observe stoichiometric and sub-stoichiometric cofactors associated with OSTA, which are likely to include protein isomerases. In sum, we visualize ER-bound polysomes with their coordinated downstream machinery.
History
DepositionSep 27, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 7, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 25, 2023Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
Revision 1.2Feb 8, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein transport protein Sec61 subunit alpha isoform 1
B: Protein transport protein Sec61 subunit beta
C: Protein transport protein Sec61 subunit gamma
D: Signal peptide mix
E: Translocon-associated protein subunit alpha
F: Translocon-associated protein subunit beta
G: Translocon-associated protein subunit gamma
H: Translocon-associated protein subunit delta
I: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A
J: Oligosaccharyltransferase complex subunit OSTC
K: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 4
L: Transmembrane protein 258
M: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit DAD1
N: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit
O: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1
P: Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2


Theoretical massNumber of molelcules
Total (without water)476,56816
Polymers476,56816
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area43340 Å2
ΔGint-388 kcal/mol
Surface area188330 Å2

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Components

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Protein transport protein Sec61 subunit ... , 3 types, 3 molecules ABC

#1: Protein Protein transport protein Sec61 subunit alpha isoform 1 / Protein targeting / Sec61 alpha-1


Mass: 52304.406 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: UniProt: P61619
#2: Protein Protein transport protein Sec61 subunit beta / Protein targeting


Mass: 9987.456 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: UniProt: P60468
#3: Protein Protein transport protein Sec61 subunit gamma / Protein targeting


Mass: 7752.325 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: UniProt: P60059

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Protein/peptide , 1 types, 1 molecules D

#4: Protein/peptide Signal peptide mix


Mass: 1890.321 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)

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Translocon-associated protein subunit ... , 4 types, 4 molecules EFGH

#5: Protein Translocon-associated protein subunit alpha / TRAP-alpha / Signal sequence receptor subunit alpha / SSR-alpha


Mass: 32263.219 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P43307
#6: Protein Translocon-associated protein subunit beta / TRAP-beta / Signal sequence receptor subunit beta / SSR-beta


Mass: 20154.986 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P43308
#7: Protein Translocon-associated protein subunit gamma / TRAP-gamma / Signal sequence receptor subunit gamma / SSR-gamma


Mass: 21106.465 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9UNL2
#8: Protein Translocon-associated protein subunit delta / TRAP-delta / Signal sequence receptor subunit delta / SSR-delta


Mass: 19015.547 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P51571

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Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit ... , 5 types, 5 molecules IKMOP

#9: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A / STT3-A / B5 / Integral membrane protein 1 / Transmembrane protein TMC


Mass: 80607.320 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
References: UniProt: P46977, dolichyl-diphosphooligosaccharide-protein glycotransferase
#11: Protein/peptide Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 4


Mass: 4196.004 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P0C6T2
#13: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit DAD1 / Oligosaccharyl transferase subunit DAD1 / Defender against cell death 1 / DAD-1


Mass: 12503.631 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P61803
#15: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 1 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 67 kDa subunit / Ribophorin I / RPN- ...Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 67 kDa subunit / Ribophorin I / RPN-I / Ribophorin-1


Mass: 68656.156 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P04843
#16: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit 2 / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 63 kDa subunit / RIBIIR / Ribophorin ...Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 63 kDa subunit / RIBIIR / Ribophorin II / RPN-II / Ribophorin-2


Mass: 69347.508 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P04844

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Protein , 3 types, 3 molecules JLN

#10: Protein Oligosaccharyltransferase complex subunit OSTC / Oligosaccharyltransferase / Hydrophobic protein HSF-28


Mass: 16844.215 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9NRP0
#12: Protein Transmembrane protein 258 / Transmembrane protein / Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit TMEM258 / Oligosaccharyl ...Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit TMEM258 / Oligosaccharyl transferase subunit TMEM258


Mass: 9083.804 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P61165
#14: Protein Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit / DDOST 48 kDa subunit / Oligosaccharyl transferase 48 kDa subunit


Mass: 50854.578 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P39656

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: Sec61-TRAP-OSTA translocon complex / Type: ORGANELLE OR CELLULAR COMPONENT
Details: Translocon complex embedded in its native membrane and bound to cytosolic ribosomes
Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: ER-derived vesicles decorated with cytosolic ribosomes.
Specimen supportGrid material: COPPER / Grid type: Quantifoil
VitrificationCryogen name: ETHANE-PROPANE / Details: Manual plunger

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 79000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 0.7 sec. / Electron dose: 2.3 e/Å2 / Avg electron dose per subtomogram: 80 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 8
Image scansMovie frames/image: 7 / Used frames/image: 7-7

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Processing

EM software
IDNameVersionCategory
1PyTom0.994volume selection
2SerialEM3.8image acquisition
3DigitalMicrograph3.3.2image acquisition
5WarpCTF correction
8UCSF Chimeramodel fitting
10PHENIXmodel refinement
11ISOLDEmodel refinement
12Cootmodel refinement
14RELION3.1.1final Euler assignment
15RELION3.1.1classification
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 600 Å / B: 600 Å / C: 600 Å / Space group name: C1 / Space group num: 1
CTF correctionDetails: Estimation and correction in WARP/M / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 7.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 42215 / Algorithm: BACK PROJECTION / Symmetry type: 3D CRYSTAL
EM volume selectionMethod: Template matching / Num. of tomograms: 869 / Num. of volumes extracted: 134350
Reference model: Subtomogram average of ER membrane-associated ribosome
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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