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- EMDB-16303: In situ subtomogram average of GEM2 particles in human cells -

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Basic information

Entry
Database: EMDB / ID: EMD-16303
TitleIn situ subtomogram average of GEM2 particles in human cells
Map dataFSC-weighted non-sharpened map
Sample
  • Cell: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159) expressing the engineered genetically encoded multimeric tag GEM2
    • Protein or peptide: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159) expressing the engineered genetically encoded multimeric tag GEM2
KeywordsEncapsulin / Protein Engineering / CYTOSOLIC PROTEIN
Function / homologyType 2A encapsulin shell protein SrpI-like / : / Type 2A encapsulin shell protein SrpI-like / encapsulin nanocompartment / Type 2A encapsulin shell protein SrpI
Function and homology information
Biological speciesSynechococcus elongatus PCC 7942 = FACHB-805 (bacteria) / Homo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 31.3 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, 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
Other privateOrion Foundation
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
DepositionDec 7, 2022-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateDec 20, 2023-
Current statusDec 20, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16303.png

Downloads & links

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Map

FileDownload / File: emd_16303.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFSC-weighted non-sharpened map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.85 Å/pix.
x 128 pix.
= 876.8 Å		6.85 Å/pix.
x 128 pix.
= 876.8 Å		6.85 Å/pix.
x 128 pix.
= 876.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 6.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.22
Minimum - Maximum-0.23084995 - 0.5300017
Average (Standard dev.)0.0020575528 (±0.037265513)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 876.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map

Fileemd_16303_half_map_1.map
AnnotationHalf map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map

Fileemd_16303_half_map_2.map
AnnotationHalf map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159...

EntireName: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159) expressing the engineered genetically encoded multimeric tag GEM2
Components
  • Cell: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159) expressing the engineered genetically encoded multimeric tag GEM2
    • Protein or peptide: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159) expressing the engineered genetically encoded multimeric tag GEM2

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Supramolecule #1: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159...

SupramoleculeName: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159) expressing the engineered genetically encoded multimeric tag GEM2
type: cell / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Synechococcus elongatus PCC 7942 = FACHB-805 (bacteria)

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Macromolecule #1: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159...

MacromoleculeName: Cryo-focused ion beam lamellae of human cells (HeLa, U2OS, Sum159) expressing the engineered genetically encoded multimeric tag GEM2
type: protein_or_peptide / ID: 1
Details: Fusion of Synechococcus elongatus Srp1 with Halo-tag and the FKBP-rapamycin-binding (FRB) domain of mTOR
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: MGSTDNAPQL ALRDVAARQL ANATKTVPQL RTITPRWLVR LLHWTPVEAG IYRVNQVKDA SQITVACSER DESELPETFV DYIDNPREYL LSAVNTVVDV HTRISDLYSN PHDQIREQLR LTIEIMKERQ ESELINSREY GLLNNVAPGQ LVHTRNGAPT PDDLDELLIR ...String:
MGSTDNAPQL ALRDVAARQL ANATKTVPQL RTITPRWLVR LLHWTPVEAG IYRVNQVKDA SQITVACSER DESELPETFV DYIDNPREYL LSAVNTVVDV HTRISDLYSN PHDQIREQLR LTIEIMKERQ ESELINSREY GLLNNVAPGQ LVHTRNGAPT PDDLDELLIR VWKEPAFFLA HPQAIAAFGR ECTRRGVPPA TVSLFGSSFI TWRGVPLIPS DKVPLENGKT KILLLRVGES RQGVVGLYQP NLPGEQGMGL SVRFMGINRK ALASYLVSLY CSLAVLTDDA LAVLDNVDVT QYHTYRYNGT GGGGSGGGSG GGSAEIGTGF PFDPHYVEVL GERMHYVDVG PRDGTPVLFL HGNPTSSYVW RNIIPHVAPT HRCIAPDLIG MGKSDKPDLG YFFDDHVRFM DAFIEALGLE EVVLVIHDWG SALGFHWAKR NPERVKGIAF MEFIRPIPTW DEWPEFARET FQAFRTTDVG RKLIIDQNVF IEGTLPMGVV RPLTEVEMDH YREPFLNPVD REPLWRFPNE LPIAGEPANI VALVEEYMDW LHQSPVPKLL FWGTPGVLIP PAEAARLAKS LPNCKAVDIG PGLNLLQEDN PDLIGSEIAR WLSTLEISGK LGSAGSAAGS GEGILWHEMW HEGLEEASRL YFGERNVKGM FEVLEPLHAM MERGPQTLKE TSFNQAYGRD LMEAQEWCRK YMKSGNVKDL LQAWDLYYHV FRRISK

UniProtKB: Type 2A encapsulin shell protein SrpI

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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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
Image recording#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / #0 - Average electron dose: 2.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #1 - Average electron dose: 2.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 71 / Number images used: 1284 / Software - Name: Warp (ver. 1.0.9)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0.0-beta-2)
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 31.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0.0-beta-2) / Number subtomograms used: 1284
DetailsAll data acquired (with/without Volta phase plate, K2/K3) were pooled for subtomogram averaging.
Image recording ID1
FSC plot (resolution estimation)

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