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- EMDB-18194: Cryo-electron tomogram of GEM2-labelled Mito-EGFP in HeLa cells -

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Basic information

Entry
Database: EMDB / ID: EMD-18194
TitleCryo-electron tomogram of GEM2-labelled Mito-EGFP in HeLa cells
Map dataUnfiltered tomogram corresponding to tilt series TS_010.mrc of Dataset 2 (220330) of EMPIAR-11561
Sample
  • Cell: 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
KeywordsEncapsulin / Protein Engineering / Synechococcus elongatus Srp1 / intracellular labeling of mitochondrial-targeted EGFP / CYTOSOLIC PROTEIN
Biological speciesSynechococcus elongatus PCC 7942 = FACHB-805 (bacteria)
Methodelectron tomography / cryo EM / Resolution: 13.7 Å
AuthorsFung 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
OrganizationGrant numberCountry
German Research Foundation (DFG)419120233 Germany
EIPOD fellowship under Marie Sklodowska-Curie Actions COFUND664726 Germany
H2020 Marie Curie Actions of the European Commission101028297European Union
Human Frontier Science Program (HFSP)CDA00045/2019 France
Other privateBiomedicum Helsinki Foundation Finland
Other privateOrion Foundation Finland
CitationJournal: 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
DepositionAug 14, 2023-
Header (metadata) releaseAug 30, 2023-
Map releaseAug 30, 2023-
UpdateDec 20, 2023-
Current statusDec 20, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18194.png

Downloads & links

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Map

FileDownload / File: emd_18194.map.gz / Format: CCP4 / Size: 2.7 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnfiltered tomogram corresponding to tilt series TS_010.mrc of Dataset 2 (220330) of EMPIAR-11561
Voxel sizeX=Y=Z: 13.7 Å
Density
Minimum - Maximum-198735.89000000001397 - 89517.059999999997672
Average (Standard dev.)-4.772659 (±9734.845999999999549)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions14401022500
Spacing10221440500
CellA: 14001.399 Å / B: 19728.0 Å / C: 6850.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Cryo-focused ion beam lamella of a HeLa cell expressing an engine...

EntireName: 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
Components
  • Cell: 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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Supramolecule #1: Cryo-focused ion beam lamella of a HeLa cell expressing an engine...

SupramoleculeName: 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
Source (natural)Organism: Synechococcus elongatus PCC 7942 = FACHB-805 (bacteria)

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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: 7.4
VitrificationCryogen name: ETHANE / Instrument: LEICA EM GP
SectioningFocused 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.

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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: 4.0 µm / Nominal defocus min: 1.5 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 2.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: 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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