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- PDB-6z3t: Structure of canine Sec61 inhibited by mycolactone -

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Basic information

Entry
Database: PDB / ID: 6z3t
TitleStructure of canine Sec61 inhibited by mycolactone
Components
  • Protein transport protein Sec61 subunit alpha isoform 1Protein targeting
  • Protein transport protein Sec61 subunit betaProtein targeting
  • Protein transport protein Sec61 subunit gammaProtein targeting
KeywordsMEMBRANE PROTEIN / Sec61 / mycolactone / ribosome-translocon complex / translocation inhibitor
Function / homology
Function and homology information


Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / membrane docking / pronephric nephron development / cotranslational protein targeting to membrane / Ssh1 translocon complex / Sec61 translocon complex / protein targeting to ER / protein insertion into ER membrane / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding ...Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / membrane docking / pronephric nephron development / cotranslational protein targeting to membrane / Ssh1 translocon complex / Sec61 translocon complex / protein targeting to ER / protein insertion into ER membrane / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding / post-translational protein targeting to membrane, translocation / protein transmembrane transporter activity / phospholipid binding / ribosome binding / endoplasmic reticulum membrane
Similarity search - Function
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 ...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
Chem-Q6B / Protein transport protein Sec61 subunit alpha isoform 1 / Protein transport protein Sec61 subunit gamma
Similarity search - Component
Biological speciesCanis lupus familiaris (dog)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.69 Å
AuthorsGerard, S.F. / Higgins, M.K.
Funding support1items
OrganizationGrant numberCountry
Wellcome Trust
CitationJournal: Mol Cell / Year: 2020
Title: Structure of the Inhibited State of the Sec Translocon.
Authors: Samuel F Gérard / Belinda S Hall / Afroditi M Zaki / Katherine A Corfield / Peter U Mayerhofer / Catia Costa / Daniel K Whelligan / Philip C Biggin / Rachel E Simmonds / Matthew K Higgins /
Abstract: Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic ...Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.
History
DepositionMay 21, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 22, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 29, 2020Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Aug 19, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Assembly

Deposited unit
A: Protein transport protein Sec61 subunit alpha isoform 1
B: Protein transport protein Sec61 subunit gamma
C: Protein transport protein Sec61 subunit beta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,4954
Polymers61,7523
Non-polymers7431
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3750 Å2
ΔGint-42 kcal/mol
Surface area28210 Å2
MethodPISA

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Components

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


Mass: 52279.379 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Canis lupus familiaris (dog) / References: UniProt: P38377
#2: 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) Canis lupus familiaris (dog) / References: UniProt: P60058
#3: Protein/peptide Protein transport protein Sec61 subunit beta / Protein targeting


Mass: 1720.111 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Canis lupus familiaris (dog)
#4: Chemical ChemComp-Q6B / [(6~{S},7~{S},9~{Z},12~{R})-12-[(~{Z},2~{S},6~{R},7~{R},9~{R})-4,6-dimethyl-7,9-bis(oxidanyl)dec-4-en-2-yl]-7,9-dimethyl-2-oxidanylidene-1-oxacyclododec-9-en-6-yl] (2~{E},4~{E},6~{E},8~{E},10~{E},12~{S},13~{S},15~{S})-4,6,10-trimethyl-12,13,15-tris(oxidanyl)hexadeca-2,4,6,8,10-pentaenoate


Mass: 743.021 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C44H70O9 / Feature type: SUBJECT OF INVESTIGATION
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: Ribosome-translocon complexes from canine ER microsomes, bound to mycolactone
Type: COMPLEX / Entity ID: #1-#3 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Canis lupus familiaris (dog)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
150 mMHEPES1
20.25 %Digitonin1
3200 mMPotassium acetate1
410 mMMagnesium acetate1
51 mMDTT1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: Blot time 5 seconds Blot force -15 Incubation time 15 seconds

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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 FIELDBright-field microscopy
Image recordingElectron dose: 49 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: BUSTER / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4.1CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 108129
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.69 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 45733 / Symmetry type: POINT
Atomic model building
IDPDB-ID 3D fitting-ID
13JC2

3jc2

1
23J7Q

3j7q

1
Displacement parametersBiso max: 121.46 Å2 / Biso mean: 66.2313 Å2 / Biso min: 5.84 Å2

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