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Yorodumi- EMDB-14505: Cryo-tomogram of FIB-sectioned Brl1(I395D) overexpressing cells -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-14505 | ||||||||||||
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Title | Cryo-tomogram of FIB-sectioned Brl1(I395D) overexpressing cells | ||||||||||||
Map data | Cryo-tomogram of FIB-sectioned Brl1(I395D) overexpressing cells | ||||||||||||
Sample |
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Keywords | Saccharomyces cerevisiae / nuclear envelope / NUCLEAR PROTEIN | ||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||
Method | electron tomography / cryo EM | ||||||||||||
Authors | Kralt 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
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Citation | Journal: 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 |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_14505.map.gz | 74 MB | EMDB map data format | |
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Header (meta data) | emd-14505-v30.xml emd-14505.xml | 10.8 KB 10.8 KB | Display Display | EMDB header |
Images | emd_14505.png | 233.7 KB | ||
Filedesc metadata | emd-14505.cif.gz | 4.1 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-14505 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-14505 | HTTPS FTP |
-Validation report
Summary document | emd_14505_validation.pdf.gz | 578.8 KB | Display | EMDB validaton report |
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Full document | emd_14505_full_validation.pdf.gz | 578.3 KB | Display | |
Data in XML | emd_14505_validation.xml.gz | 3.7 KB | Display | |
Data in CIF | emd_14505_validation.cif.gz | 4.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-14505 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-14505 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_14505.map.gz / Format: CCP4 / Size: 110.4 MB / Type: IMAGE STORED AS SIGNED BYTE | ||||||||||||||||||||
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Annotation | Cryo-tomogram of FIB-sectioned Brl1(I395D) overexpressing cells | ||||||||||||||||||||
Voxel size | X=Y=Z: 13.8 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Sample components
-Entire : KWY165, ura3::pGAL1-BRL1(I395D)-CaURA3
Entire | Name: KWY165, ura3::pGAL1-BRL1(I395D)-CaURA3 |
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Components |
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-Supramolecule #1: KWY165, ura3::pGAL1-BRL1(I395D)-CaURA3
Supramolecule | Name: KWY165, ura3::pGAL1-BRL1(I395D)-CaURA3 / type: cell / ID: 1 / Parent: 0 |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | electron tomography |
Aggregation state | cell |
-Sample preparation
Buffer | pH: 6 Details: synthetic complete medium (SCD, 6.7 g/L yeast nitrogen base without amino acids, 2% dextrose) |
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Vitrification | Cryogen name: ETHANE / Instrument: HOMEMADE PLUNGER / Details: Blotted from backside. |
Sectioning | Focused 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. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film 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 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 7.0 µm / Nominal defocus min: 4.0 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Algorithm: BACK PROJECTION / Software - Name: IMOD (ver. 4.11) / Number images used: 37 |
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