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- EMDB-11064: Structure of canine Sec61 inhibited by mycolactone -

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Basic information

Entry
Database: EMDB / ID: EMD-11064
TitleStructure of canine Sec61 inhibited by mycolactone
Map dataExperimental map used for model building
Sample
  • Complex: Ribosome-translocon complexes from canine ER microsomes, bound to mycolactone
    • 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: [(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
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
Protein transport protein Sec61 subunit alpha isoform 1 / Protein transport protein Sec61 subunit gamma
Similarity search - Component
Biological speciesCanis lupus familiaris (dog) / Dog (dog)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.69 Å
AuthorsGerard SF / Higgins MK
Funding support1 items
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, 2020-
Header (metadata) releaseJul 22, 2020-
Map releaseJul 22, 2020-
UpdateAug 19, 2020-
Current statusAug 19, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0087
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0087
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6z3t
  • Surface level: 0.0087
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6z3t
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11064.png

Downloads & links

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Map

FileDownload / File: emd_11064.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationExperimental map used for model building
Voxel sizeX=Y=Z: 1.033 Å
Density
Contour LevelBy AUTHOR: 0.0087 / Movie #1: 0.0087
Minimum - Maximum-0.06044175 - 0.10117586
Average (Standard dev.)0.00059515 (±0.0042546308)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 433.86 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0331.0331.033
M x/y/z420420420
origin x/y/z0.0000.0000.000
length x/y/z433.860433.860433.860
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ280280280
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS420420420
D min/max/mean-0.0600.1010.001

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Supplemental data

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Mask #1

Fileemd_11064_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Map for mycolactone-free control sample

Fileemd_11064_additional_1.map
AnnotationMap for mycolactone-free control sample
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Map for mycolactone-bound sample filtered to 5A resolution,...

Fileemd_11064_additional_2.map
AnnotationMap for mycolactone-bound sample filtered to 5A resolution, using for Figure 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 1

Fileemd_11064_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_11064_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ribosome-translocon complexes from canine ER microsomes, bound to...

EntireName: Ribosome-translocon complexes from canine ER microsomes, bound to mycolactone
Components
  • Complex: Ribosome-translocon complexes from canine ER microsomes, bound to mycolactone
    • 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: [(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

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Supramolecule #1: Ribosome-translocon complexes from canine ER microsomes, bound to...

SupramoleculeName: Ribosome-translocon complexes from canine ER microsomes, bound to mycolactone
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Canis lupus familiaris (dog)

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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: Dog (dog)
Molecular weightTheoretical: 52.279379 KDa
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 FQGFRVDLPI KSARYRGQYN TYPIKLFYTS NIPIILQSAL VSNLYVISQM LSARFSGNLL VSL LGTWSD TSSGGPARAY PVGGLCHYLS PPESFGSVLE DPVHAVVYIV FMLGSCAFFS KTWIEVSGSS AKDVAKQLKE QQMV MRGHR ETSMVHELNR YIPTAAAFGG LCIGALSVLA DFLGAIGSGT GILLAVTIIY QYFEIFVKEQ SEVGSMGALL F

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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: Dog (dog)
Molecular weightTheoretical: 7.752325 KDa
SequenceString:
MDQVMQFVEP SRQFVKDSIR LVKRCTKPDR KEFQKIAMAT AIGFAIMGFI GFFVKLIHIP INNIIVGG

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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: Dog (dog)
Molecular weightTheoretical: 1.720111 KDa
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)

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Macromolecule #4: [(6~{S},7~{S},9~{Z},12~{R})-12-[(~{Z},2~{S},6~{R},7~{R},9~{R})-4,...

MacromoleculeName: [(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] ...Name: [(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
type: ligand / ID: 4 / Number of copies: 1 / Formula: Q6B
Molecular weightTheoretical: 743.021 Da
Chemical component information

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

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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

BufferpH: 7.5
Component:
ConcentrationName
50.0 mMHEPES
0.25 %Digitonin
200.0 mMPotassium acetate
10.0 mMMagnesium acetate
1.0 mMDTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: GRAPHENE OXIDE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: Blot time 5 seconds Blot force -15 Incubation time 15 seconds.

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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
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 49.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 108129
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.69 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 45733
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

3jc2

,

3j7q

)
Output model

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

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