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- EMDB-23060: The stress-sensing domain of activated IRE1a forms helical filame... -

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Entry
Database: EMDB / ID: EMD-23060
TitleThe stress-sensing domain of activated IRE1a forms helical filaments in narrow ER membrane tubes
Map dataCryo-CLEM/cryo-ET was used to target IRE1a fused to fluorescent protein mNeonGreen (mNG) in U2OS cells. Helical symmetry applied using sym=H2.5:2:45:5 with --keep=0.9 --maxalt=50.0 --mask=Auto
Sample
  • Organelle or cellular component: Double-helical arrangement of human IRE1a luminal domain in 30 nm ER tubes
    • Protein or peptide: IRE1a lumenal domain
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 30.0 Å
AuthorsCarter SD / Tran NH / De Maziere A / Ashkenazi A / Klumpermann J / Walter P / Jensen GJ
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR)P50 AI150464 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)R35 GM122588 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)R01-GM032384 United States
CitationJournal: Science / Year: 2021
Title: The stress-sensing domain of activated IRE1α forms helical filaments in narrow ER membrane tubes.
Authors: Ngoc-Han Tran / Stephen D Carter / Ann De Mazière / Avi Ashkenazi / Judith Klumperman / Peter Walter / Grant J Jensen /
Abstract: The signaling network of the unfolded protein response (UPR) adjusts the protein-folding capacity of the endoplasmic reticulum (ER) according to need. The most conserved UPR sensor, IRE1α, spans the ...The signaling network of the unfolded protein response (UPR) adjusts the protein-folding capacity of the endoplasmic reticulum (ER) according to need. The most conserved UPR sensor, IRE1α, spans the ER membrane and activates through oligomerization. IRE1α oligomers accumulate in dynamic foci. We determined the in situ structure of IRE1α foci by cryogenic correlated light and electron microscopy combined with electron cryo-tomography and complementary immuno–electron microscopy in mammalian cell lines. IRE1α foci localized to a network of narrow anastomosing ER tubes (diameter, ~28 nm) with complex branching. The lumen of the tubes contained protein filaments, which were likely composed of arrays of IRE1α lumenal domain dimers that were arranged in two intertwined, left-handed helices. This specialized ER subdomain may play a role in modulating IRE1α signaling.
History
DepositionDec 2, 2020-
Header (metadata) releaseNov 10, 2021-
Map releaseNov 10, 2021-
UpdateJun 8, 2022-
Current statusJun 8, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.00542
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.00542
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23060.png

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Map

FileDownload / File: emd_23060.map.gz / Format: CCP4 / Size: 686.5 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-CLEM/cryo-ET was used to target IRE1a fused to fluorescent protein mNeonGreen (mNG) in U2OS cells. Helical symmetry applied using sym=H2.5:2:45:5 with --keep=0.9 --maxalt=50.0 --mask=Auto
Voxel sizeX=Y=Z: 6.52 Å
Density
Contour LevelBy AUTHOR: 0.00542 / Movie #1: 0.00542
Minimum - Maximum-0.017792173 - 0.02924714
Average (Standard dev.)0.0001607504 (±0.0021572465)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions565656
Spacing565656
CellA=B=C: 365.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.526.526.52
M x/y/z565656
origin x/y/z0.0000.0000.000
length x/y/z365.120365.120365.120
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS565656
D min/max/mean-0.0180.0290.000

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Supplemental data

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Mask #1

Fileemd_23060_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Double-helical arrangement of human IRE1a luminal domain in 30 nm...

EntireName: Double-helical arrangement of human IRE1a luminal domain in 30 nm ER tubes
Components
  • Organelle or cellular component: Double-helical arrangement of human IRE1a luminal domain in 30 nm ER tubes
    • Protein or peptide: IRE1a lumenal domain

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Supramolecule #1: Double-helical arrangement of human IRE1a luminal domain in 30 nm...

SupramoleculeName: Double-helical arrangement of human IRE1a luminal domain in 30 nm ER tubes
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Cryo-CLEM was used to target IRE1a fused to fluorescent protein mNeonGreen (mNG) for cryo-ET imaging.
Source (natural)Organism: Homo sapiens (human) / Organelle: ER

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Macromolecule #1: IRE1a lumenal domain

MacromoleculeName: IRE1a lumenal domain / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString: MPARRLLLLL TLLLPGLGIF GSTSTVTLPE TLLFVSTLDG SLHAVSKRTG SIKWTLKEDP VLQVPTHVEE PAFLPDPNDG SLYTLGSKNN EGLTKLPFTI PELVQASPCR SSDGILYMGK KQDIWYVIDL LTGEKQQTLS SAFADSLCPS TSLLYLGRTE YTITMYDTKT ...String:
MPARRLLLLL TLLLPGLGIF GSTSTVTLPE TLLFVSTLDG SLHAVSKRTG SIKWTLKEDP VLQVPTHVEE PAFLPDPNDG SLYTLGSKNN EGLTKLPFTI PELVQASPCR SSDGILYMGK KQDIWYVIDL LTGEKQQTLS SAFADSLCPS TSLLYLGRTE YTITMYDTKT RELRWNATYF DYAASLPEDD VDYKMSHFVS NGDGLVVTVD SESGDVLWIQ NYASPVVAFY VWQREGLRKV MHINVAVETL RYLTFMSGEV GRITKWKYPF PKETEAKSKL TPTLYVGKYS TSLYASPSMV HEGVAVVPRG STLPLLEGPQ TDGVTIGDKG ECVITPSTDV KFDPGLKSKN KLNYLRNYWL LIGHHETPLS ASTKMLERFP NNLPKHRENV IPADSEKKSF EEVINLVDQT SENAPTTVSR DVEEKPAHAP ARPEAPVDSM LKD

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statefilament

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 5.0 nm
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Chamber temperature: 310.15 K / Instrument: FEI VITROBOT MARK III
Details: Manual blotting from the gold side of the grid (opposite side to cells)..
Detailsin situ imaging in a near-native state

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Electron microscopy

MicroscopeFEI POLARA 300
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.5 e/Å2
Details: Images were collected using a bidirectional tilt-scheme.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal magnification: 34000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 32.6 Å
Applied symmetry - Helical parameters - Δ&Phi: 45 °
Applied symmetry - Helical parameters - Axial symmetry: C2 (2 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 (ver. 2.3) / Number subtomograms used: 653
ExtractionNumber tomograms: 3 / Number images used: 653 / Method: Manually every ~85A along the filament / Details: e2boxer.py
CTF correctionSoftware - Name: EMAN2 (ver. 2.3) / Software - details: CTF / Details: CTF correction was performed in EMAN2
Final angle assignmentType: NOT APPLICABLE

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