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Yorodumi- EMDB-18194: Cryo-electron tomogram of GEM2-labelled Mito-EGFP in HeLa cells -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-18194 | |||||||||||||||||||||
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Title | Cryo-electron tomogram of GEM2-labelled Mito-EGFP in HeLa cells | |||||||||||||||||||||
Map data | Unfiltered tomogram corresponding to tilt series TS_010.mrc of Dataset 2 (220330) of EMPIAR-11561 | |||||||||||||||||||||
Sample |
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Keywords | Encapsulin / Protein Engineering / Synechococcus elongatus Srp1 / intracellular labeling of mitochondrial-targeted EGFP / CYTOSOLIC PROTEIN | |||||||||||||||||||||
Biological species | Synechococcus elongatus PCC 7942 = FACHB-805 (bacteria) | |||||||||||||||||||||
Method | electron tomography / cryo EM / Resolution: 13.7 Å | |||||||||||||||||||||
Authors | Fung HKH / Hayashi Y / Salo VT / Babenko A / Zagoriy I / Brunner A / Ellenberg J / Mueller CW / Cuylen-Haering S / Mahamid J | |||||||||||||||||||||
Funding support | Germany, European Union, France, Finland, 6 items
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Citation | Journal: Nat Methods / Year: 2023 Title: Genetically encoded multimeric tags for subcellular protein localization in cryo-EM. Authors: Herman K H Fung / Yuki Hayashi / Veijo T Salo / Anastasiia Babenko / Ievgeniia Zagoriy / Andreas Brunner / Jan Ellenberg / Christoph W Müller / Sara Cuylen-Haering / Julia Mahamid / Abstract: Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest ...Cryo-electron tomography (cryo-ET) allows for label-free high-resolution imaging of macromolecular assemblies in their native cellular context. However, the localization of macromolecules of interest in tomographic volumes can be challenging. Here we present a ligand-inducible labeling strategy for intracellular proteins based on fluorescent, 25-nm-sized, genetically encoded multimeric particles (GEMs). The particles exhibit recognizable structural signatures, enabling their automated detection in cryo-ET data by convolutional neural networks. The coupling of GEMs to green fluorescent protein-tagged macromolecules of interest is triggered by addition of a small-molecule ligand, allowing for time-controlled labeling to minimize disturbance to native protein function. We demonstrate the applicability of GEMs for subcellular-level localization of endogenous and overexpressed proteins across different organelles in human cells using cryo-correlative fluorescence and cryo-ET imaging. We describe means for quantifying labeling specificity and efficiency, and for systematic optimization for rare and abundant protein targets, with emphasis on assessing the potential effects of labeling on protein function. | |||||||||||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_18194.map.gz | 2.5 GB | EMDB map data format | |
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Header (meta data) | emd-18194-v30.xml emd-18194.xml | 11.4 KB 11.4 KB | Display Display | EMDB header |
Images | emd_18194.png | 337.4 KB | ||
Filedesc metadata | emd-18194.cif.gz | 4.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-18194 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-18194 | HTTPS FTP |
-Validation report
Summary document | emd_18194_validation.pdf.gz | 557 KB | Display | EMDB validaton report |
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Full document | emd_18194_full_validation.pdf.gz | 556.5 KB | Display | |
Data in XML | emd_18194_validation.xml.gz | 5 KB | Display | |
Data in CIF | emd_18194_validation.cif.gz | 5.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-18194 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-18194 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_18194.map.gz / Format: CCP4 / Size: 2.7 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||
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Annotation | Unfiltered tomogram corresponding to tilt series TS_010.mrc of Dataset 2 (220330) of EMPIAR-11561 | ||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. generated in cubic-lattice coordinate | ||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 13.7 Å | ||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Sample components
-Entire : Cryo-focused ion beam lamella of a HeLa cell expressing an engine...
Entire | Name: Cryo-focused ion beam lamella of a HeLa cell expressing an engineered genetically encoded multimeric tag GEM2 coupled to stably expressed Mito-EGFP upon treatment with rapalog |
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Components |
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-Supramolecule #1: Cryo-focused ion beam lamella of a HeLa cell expressing an engine...
Supramolecule | Name: Cryo-focused ion beam lamella of a HeLa cell expressing an engineered genetically encoded multimeric tag GEM2 coupled to stably expressed Mito-EGFP upon treatment with rapalog type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Synechococcus elongatus PCC 7942 = FACHB-805 (bacteria) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | electron tomography |
Aggregation state | cell |
-Sample preparation
Buffer | pH: 7.4 |
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Vitrification | Cryogen name: ETHANE / Instrument: LEICA EM GP |
Sectioning | Focused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.05 / Focused ion beam - Duration: 480 / Focused ion beam - Temperature: 87 K / Focused ion beam - Initial thickness: 1000 / Focused ion beam - Final thickness: 200 Focused ion beam - Details: The value given for _em_focused_ion_beam.instrument is TFS Aquilos. 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 K3 BIOQUANTUM (6k x 4k) / Average electron dose: 2.0 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: 4.0 µm / Nominal defocus min: 1.5 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 13.7 Å / Software - Version: 1.3.1 Details: Tilt-series alignment and tomogram reconstruction performed with AreTomo version 1.3.1, doi: 10.1016/j.yjsbx.2022.100068. Number images used: 53 |
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