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- EMDB-14503: Cryo-tomogram of FIB-sectioned non-depleted Brl1 control cells -

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Basic information

Entry
Database: EMDB / ID: EMD-14503
TitleCryo-tomogram of FIB-sectioned non-depleted Brl1 control cells
Map dataCryo-tomogram of FIB-sectioned non-depleted Brl1 control cells
Sample
  • Cell: KWY165, BRL1::BRL1-V5-IAA7-KanMX trp1::pADH1-dsRED-HDEL-TRP1
KeywordsSaccharomyces cerevisiae / nuclear envelope / NUCLEAR PROTEIN
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodelectron tomography / cryo EM
AuthorsKralt A / Wojtynek M / Fischer JS / Agote-Aran A / Mancini R / Dultz E / Noor E / Uliana F / Tatarek-Nossol M / Antonin W ...Kralt A / Wojtynek M / Fischer JS / Agote-Aran A / Mancini R / Dultz E / Noor E / Uliana F / Tatarek-Nossol M / Antonin W / Onischenko E / Medalia O / Weis K
Funding support Switzerland, Norway, 3 items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_179275 Switzerland
Swiss National Science Foundation31003A_179418 Switzerland
Research Council of Norway315615 Norway
CitationJournal: Elife / Year: 2022
Title: An amphipathic helix in Brl1 is required for nuclear pore complex biogenesis in .
Authors: Annemarie Kralt / Matthias Wojtynek / Jonas S Fischer / Arantxa Agote-Aran / Roberta Mancini / Elisa Dultz / Elad Noor / Federico Uliana / Marianna Tatarek-Nossol / Wolfram Antonin / Evgeny ...Authors: Annemarie Kralt / Matthias Wojtynek / Jonas S Fischer / Arantxa Agote-Aran / Roberta Mancini / Elisa Dultz / Elad Noor / Federico Uliana / Marianna Tatarek-Nossol / Wolfram Antonin / Evgeny Onischenko / Ohad Medalia / Karsten Weis /
Abstract: The nuclear pore complex (NPC) is the central portal for macromolecular exchange between the nucleus and cytoplasm. In all eukaryotes, NPCs assemble into an intact nuclear envelope (NE) during ...The nuclear pore complex (NPC) is the central portal for macromolecular exchange between the nucleus and cytoplasm. In all eukaryotes, NPCs assemble into an intact nuclear envelope (NE) during interphase, but the process of NPC biogenesis remains poorly characterized. Furthermore, little is known about how NPC assembly leads to the fusion of the outer and inner NE, and no factors have been identified that could trigger this event. Here, we characterize the transmembrane protein Brl1 as an NPC assembly factor required for NE fusion in budding yeast. Brl1 preferentially associates with NPC assembly intermediates and its depletion halts NPC biogenesis, leading to NE herniations that contain inner and outer ring nucleoporins but lack the cytoplasmic export platform. Furthermore, we identify an essential amphipathic helix in the luminal domain of Brl1 that mediates interactions with lipid bilayers. Mutations in this amphipathic helix lead to NPC assembly defects, and cryo-electron tomography analyses reveal multilayered herniations of the inner nuclear membrane with NPC-like structures at the neck, indicating a failure in NE fusion. Taken together, our results identify a role for Brl1 in NPC assembly and suggest a function of its amphipathic helix in mediating the fusion of the inner and outer nuclear membranes.
History
DepositionMar 4, 2022-
Header (metadata) releaseMar 23, 2022-
Map releaseMar 23, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14503.png

Downloads & links

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Map

FileDownload / File: emd_14503.map.gz / Format: CCP4 / Size: 110.4 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationCryo-tomogram of FIB-sectioned non-depleted Brl1 control cells
Voxel sizeX=Y=Z: 13.8 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)12.5727005 (±18.763023)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin16-17-65
Dimensions928960130
Spacing960928130
CellA: 13248.0 Å / B: 12806.4 Å / C: 1794.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : KWY165, BRL1::BRL1-V5-IAA7-KanMX trp1::pADH1-dsRED-HDEL-TRP1

EntireName: KWY165, BRL1::BRL1-V5-IAA7-KanMX trp1::pADH1-dsRED-HDEL-TRP1
Components
  • Cell: KWY165, BRL1::BRL1-V5-IAA7-KanMX trp1::pADH1-dsRED-HDEL-TRP1

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Supramolecule #1: KWY165, BRL1::BRL1-V5-IAA7-KanMX trp1::pADH1-dsRED-HDEL-TRP1

SupramoleculeName: KWY165, BRL1::BRL1-V5-IAA7-KanMX trp1::pADH1-dsRED-HDEL-TRP1
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 6
Details: synthetic complete medium (SCD, 6.7 g/L yeast nitrogen base without amino acids, 2% dextrose)
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Blotted from backside.
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.24 / Focused ion beam - Duration: 60 / Focused ion beam - Temperature: 113 K / Focused ion beam - Initial thickness: 1000 / Focused ion beam - Final thickness: 250
Focused ion beam - Details: The value given for _em_focused_ion_beam.instrument is Zeiss Auriga 40 crossbeam. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.

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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 / Nominal defocus max: 7.0 µm / Nominal defocus min: 4.0 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 41 / Average exposure time: 4.0 sec. / Average electron dose: 3.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.11) / Number images used: 41

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