- EMDB-18194: Cryo-electron tomogram of GEM2-labelled Mito-EGFP in HeLa cells -
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IDまたはキーワード:
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基本情報
登録情報
データベース: EMDB / ID: EMD-18194
タイトル
Cryo-electron tomogram of GEM2-labelled Mito-EGFP in HeLa cells
マップデータ
Unfiltered tomogram corresponding to tilt series TS_010.mrc of Dataset 2 (220330) of EMPIAR-11561
試料
細胞: 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
キーワード
Encapsulin / Protein Engineering / Synechococcus elongatus Srp1 / intracellular labeling of mitochondrial-targeted EGFP / CYTOSOLIC PROTEIN
EIPOD fellowship under Marie Sklodowska-Curie Actions COFUND
664726
ドイツ
H2020 Marie Curie Actions of the European Commission
101028297
European Union
Human Frontier Science Program (HFSP)
CDA00045/2019
フランス
Other private
Biomedicum Helsinki Foundation
フィンランド
Other private
Orion Foundation
フィンランド
引用
ジャーナル: Nat Methods / 年: 2023 タイトル: Genetically encoded multimeric tags for subcellular protein localization in cryo-EM. 著者: 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 / 要旨: 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.
ダウンロード / ファイル: emd_18194.map.gz / 形式: CCP4 / 大きさ: 2.7 GB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
Unfiltered tomogram corresponding to tilt series TS_010.mrc of Dataset 2 (220330) of EMPIAR-11561
ボクセルのサイズ
X=Y=Z: 13.7 Å
密度
最小 - 最大
-198735.89000000001397 - 89517.059999999997672
平均 (標準偏差)
-4.772659 (±9734.845999999999549)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
0
0
0
サイズ
1440
1022
500
Spacing
1022
1440
500
セル
A: 14001.399 Å / B: 19728.0 Å / C: 6850.0 Å α=β=γ: 90.0 °
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添付データ
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試料の構成要素
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全体 : Cryo-focused ion beam lamella of a HeLa cell expressing an engine...
全体
名称: 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
要素
細胞: 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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超分子 #1: Cryo-focused ion beam lamella of a HeLa cell expressing an engine...
超分子
名称: 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 タイプ: cell / ID: 1 / 親要素: 0 / 含まれる分子: #1
集束イオンビーム - 装置: OTHER / 集束イオンビーム - イオン: OTHER / 集束イオンビーム - 電圧: 30 / 集束イオンビーム - 電流: 0.05 / 集束イオンビーム - 時間: 480 / 集束イオンビーム - 温度: 87 K / 集束イオンビーム - Initial thickness: 1000 / 集束イオンビーム - 最終 厚さ: 200 集束イオンビーム - 詳細: 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.
アルゴリズム: BACK PROJECTION / 解像度のタイプ: BY AUTHOR / 解像度: 13.7 Å / ソフトウェア - バージョン: 1.3.1 詳細: Tilt-series alignment and tomogram reconstruction performed with AreTomo version 1.3.1, doi: 10.1016/j.yjsbx.2022.100068. 使用した粒子像数: 53