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- EMDB-43978: Subtomogram average of extracellular intermediate filaments from ... -

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Basic information

Entry
Database: EMDB / ID: EMD-43978
TitleSubtomogram average of extracellular intermediate filaments from Jurkat cells
Map dataCryoSparc generated subtomogram average of extracellular intermediate filaments from Jurkat cells.
Sample
  • Organelle or cellular component: Extracellular intermediate filament
KeywordsFilament Intermediate Vimentin Jurkat cell / STRUCTURAL PROTEIN
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 10.8 Å
AuthorsOliver SL
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI20459 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R21AI159375 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM130332 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM139166 United States
CitationJournal: Nat Commun / Year: 2025
Title: Extracellular filaments revealed by affinity capture cryogenic-electron tomography.
Authors: Leeya Engel / Magda Zaoralová / Momei Zhou / Alexander R Dunn / Stefan L Oliver /
Abstract: Cryogenic-electron tomography (cryo-ET) has provided an unprecedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron ...Cryogenic-electron tomography (cryo-ET) has provided an unprecedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron tomography. Micropatterning of extracellular matrix proteins is increasingly used as a method to prepare adherent cell types for cryo-ET as it promotes optimal positioning of cells and subcellular regions of interest for vitrification, cryo-focused ion beam (cryo-FIB) milling, and data acquisition. Here we demonstrate a micropatterning workflow for capturing minimally adherent cell types, human T cells and Jurkat cells, for cryo-FIB and cryo-ET. Our affinity capture system facilitated the nanoscale imaging of Jurkat cells, revealing extracellular filamentous structures. It improved workflow efficiency by consistently producing grids with a sufficient number of well-positioned cells for an entire cryo-FIB session. Affinity capture can be extended to facilitate high-resolution imaging of other adherent and non-adherent cell types with cryo-ET.
History
DepositionMar 7, 2024-
Header (metadata) releaseSep 10, 2025-
Map releaseSep 10, 2025-
UpdateNov 19, 2025-
Current statusNov 19, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43978.png

Downloads & links

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Map

FileDownload / File: emd_43978.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoSparc generated subtomogram average of extracellular intermediate filaments from Jurkat cells.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.47 Å/pix.
x 240 pix.
= 831.6 Å		3.47 Å/pix.
x 240 pix.
= 831.6 Å		3.47 Å/pix.
x 240 pix.
= 831.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.465 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.9
Minimum - Maximum-0.7009581 - 3.6417184
Average (Standard dev.)-0.0018256338 (±0.12203227)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 831.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43978_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CryoSparc generated subtomogram average of extracellular intermediate filaments...

Fileemd_43978_half_map_1.map
AnnotationCryoSparc generated subtomogram average of extracellular intermediate filaments from Jurkat cells; half map A.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: CryoSparc generated subtomogram average of extracellular intermediate filaments...

Fileemd_43978_half_map_2.map
AnnotationCryoSparc generated subtomogram average of extracellular intermediate filaments from Jurkat cells; half map B.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Extracellular intermediate filament

EntireName: Extracellular intermediate filament
Components
  • Organelle or cellular component: Extracellular intermediate filament

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Supramolecule #1: Extracellular intermediate filament

SupramoleculeName: Extracellular intermediate filament / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statefilament

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil R2/4 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 99 % / Chamber temperature: 298 K / Instrument: LEICA EM GP
DetailsExtracellular filaments were revealed by cryo-FIB/SEM of Jurkat cells.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.755 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 26000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsCryoSPARC was used to generate a reconstruction of the intermediate mediate filaments using the helical processing pipeline. CryoSPARC does not currently have a dedicated cryo-ET processing pipeline. However, due to the limited number of tiltseries (three) for intermediate filaments at the periphery of the Jurkat cells, individual movie stacks from each tilt of the cryo-ET data acquisition were imported (Import Movies) into a CryoSPARC project with the accumulated dose information for each movie stack. Movies where the edges of the lamella wall entered the field of view were removed, leaving a total of 110 movies. CryoSPARC patch motion correction and CTF estimation was performed on the 110 movies (micrographs). To generate a 2D class average for template picking, 238 particles were manually picked from 12 micrographs. These particles were subjected to 2D classification into 4 classes with the align filament classes vertically option checked; three classes contained the majority of particles (237) and were used for filament tracing with the following parameters; Filament diameter (100 Angstrom), Separation distance between segments (25 Angstrom), Minimum filament length to consider (400 Angstrom), Angular sampling (5 Degrees). The 250,941 particles generated were extracted from the 110 micrographs with a box size of 120 pixels, which were used for a further round of 2D classification into 50 classes. Of these, 3 classes (46,827) were used for an additional round of filament tracing, extraction of particles from the micrographs with a box size of 240 pixels to increase the length of the filaments captured, and 2D classification of the newly extracted particles (382,315) into 50 classes. Of these, 85,475 particles from 5 classes were used for de novo reconstruction using Helix refine then subjected to 3D classification. One class containing 83,607 particles was further processed to remove duplicate particles. Helix refine was performed on the remaining 35,789 particles yielding a 13.0 Angstrom cryo-ET map as determined by FSC estimation with loose masking. To quantitatively assess directional resolution anisotropy of the cryo-ET map, 3DFSC was used with the half maps and the refinement mask from Helix refine used as input.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 10.8 Å / Resolution method: FSC 0.143 CUT-OFF
Details: The 35789 'subtomograms' were individual particles generated from template picking from individual tilts from the 3 tomograms.
Number subtomograms used: 35789
ExtractionNumber tomograms: 3 / Number images used: 250941
Details: The 250941 'subtomograms' were individual particles generated from template picking from individual tilts from the 3 tomograms.
CTF correctionType: NONE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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