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- EMDB-21706: Cryogenic Single-Molecule Fluorescence Annotations for Electron T... -

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Entry
Database: EMDB / ID: EMD-21706
TitleCryogenic Single-Molecule Fluorescence Annotations for Electron Tomography Reveal In Situ Organization of Key Proteins in Caulobacter
Map dataTomographic Reconstruction (bin by 4) of C. Crescentus that underwent no correlative imaging.
Sample
  • Cell: Caulobacter crescentus cells
KeywordsBacteria / Tomography / Regulatory proteins / CELL CYCLE
Biological speciesCaulobacter vibrioides (bacteria)
Methodelectron tomography / cryo EM
AuthorsDahlberg PD / Saurabh S / Sartor AM / Wang J / Mitchell P / Chiu W / Shapiro L / Moerner WE
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118067 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118071 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P01NS092525 United States
Department of Energy (DOE, United States)FWP100463 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Cryogenic single-molecule fluorescence annotations for electron tomography reveal in situ organization of key proteins in .
Authors: Peter D Dahlberg / Saumya Saurabh / Annina M Sartor / Jiarui Wang / Patrick G Mitchell / Wah Chiu / Lucy Shapiro / W E Moerner /
Abstract: Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence ...Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of ∼30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium : McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.
History
DepositionApr 16, 2020-
Header (metadata) releaseMay 27, 2020-
Map releaseMay 27, 2020-
UpdateAug 30, 2023-
Current statusAug 30, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Supplemental images

emd_21706.png

Downloads & links

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Map

FileDownload / File: emd_21706.map.gz / Format: CCP4 / Size: 590.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomographic Reconstruction (bin by 4) of C. Crescentus that underwent no correlative imaging.
Voxel sizeX=Y=Z: 29.16 Å
Density
Minimum - Maximum-76.492324999999994 - 84.078789999999998
Average (Standard dev.)0.00000000841601 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-321677
Dimensions960960168
Spacing960960168
CellA: 27993.6 Å / B: 27993.6 Å / C: 4898.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z29.1629.1629.16
M x/y/z960960168
origin x/y/z0.0000.0000.000
length x/y/z27993.60027993.6004898.880
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS16-3277
NC/NR/NS960960168
D min/max/mean-76.49284.0790.000

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Supplemental data

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Additional map: Tomographic Reconstruction (bin 4) of C. Crescentus that...

Fileemd_21706_additional_1.map
AnnotationTomographic Reconstruction (bin 4) of C. Crescentus that underwent correlative imaging. See corresponding file for single-molecule fluorescence localizations annotating the positions of PAmKate-PopZ.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Single-molecule fluorescence localizations annotating the positions of PAmKate-PopZ....

Fileemd_21706_additional_2.map
AnnotationSingle-molecule fluorescence localizations annotating the positions of PAmKate-PopZ.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Tomographic Reconstruction (bin 4) of C. Crescentus that...

Fileemd_21706_additional_3.map
AnnotationTomographic Reconstruction (bin 4) of C. Crescentus that underwent correlative imaging. See corresponding file for single-molecule fluorescence localizations annotating the positions of PAmKate-McpA.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Single-molecule fluorescence localizations annotating the positions of PAmKate-McpA....

Fileemd_21706_additional_4.map
AnnotationSingle-molecule fluorescence localizations annotating the positions of PAmKate-McpA.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Tomographic Reconstruction (bin by 4) of C. Crescentus...

Fileemd_21706_additional_5.map
AnnotationTomographic Reconstruction (bin by 4) of C. Crescentus that underwent correlative imaging. Fluorescence imaging was performed using an excitation beam with an intensity of ~80W/cm^2 for ~3 hours.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Caulobacter crescentus cells

EntireName: Caulobacter crescentus cells
Components
  • Cell: Caulobacter crescentus cells

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Supramolecule #1: Caulobacter crescentus cells

SupramoleculeName: Caulobacter crescentus cells / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Caulobacter vibrioides (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: 6.8 / Details: Cells cultured and vitrified in M2G growth media
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: GATAN CRYOPLUNGE 3 / Details: double sided blot for 3.5 seconds.
Cryo protectant20% ethylene glycol
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: EMS / Diameter: 15 nm

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus min: 10.0 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Average electron dose: 1.1 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 91

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