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- EMDB-43428: Tomogram 6 - Thickness measurement -

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Basic information

Entry
Database: EMDB / ID: EMD-43428
TitleTomogram 6 - Thickness measurement
Map data
Sample
  • Cell: ER-HOXB8 sample prepared by FIB_SEM milling
KeywordsER-HOXB8 cells / neutrophils / cytoplasm / RIBOSOME
Biological speciesMus musculus (house mouse)
Methodelectron tomography / cryo EM
AuthorsElferich J / Kong L / Zottig X / Grigorieff N
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Citation
Journal: Elife / Year: 2024
Title: CTFFIND5 provides improved insight into quality, tilt, and thickness of TEM samples.
Authors: Johannes Elferich / Lingli Kong / Ximena Zottig / Nikolaus Grigorieff /
Abstract: Images taken by transmission electron microscopes are usually affected by lens aberrations and image defocus, among other factors. These distortions can be modeled in reciprocal space using the ...Images taken by transmission electron microscopes are usually affected by lens aberrations and image defocus, among other factors. These distortions can be modeled in reciprocal space using the contrast transfer function (CTF). Accurate estimation and correction of the CTF is essential for restoring the high-resolution signal in cryogenic electron microscopy (cryoEM). Previously, we described the implementation of algorithms for this task in the TEM software package (Grant et al., 2018). Here we show that taking sample characteristics, such as thickness and tilt, into account can improve CTF estimation. This is particularly important when imaging cellular samples, where measurement of sample thickness and geometry derived from accurate modeling of the Thon ring pattern helps judging the quality of the sample. This improved CTF estimation has been implemented in CTFFIND5, a new version of the TEM program CTFFIND. We evaluated the accuracy of these estimates using images of tilted aquaporin crystals and eukaryotic cells thinned by focused ion beam milling. We estimate that with micrographs of sufficient quality CTFFIND5 can measure sample tilt with an accuracy of 3° and sample thickness with an accuracy of 5 nm.
#1: Journal: Elife / Year: 2024
Title: CTFFIND5 provides improved insight into quality, tilt and thickness of TEM sample
Authors: Elferich J / Kong L / Zottig X / Grigorieff N
History
DepositionJan 17, 2024-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateJan 1, 2025-
Current statusJan 1, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43428.png

Downloads & links

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Map

FileDownload / File: emd_43428.map.gz / Format: CCP4 / Size: 1.7 GB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
8.35 Å/pix.
x 1440 pix.
= 12022.56 Å		8.35 Å/pix.
x 625 pix.
= 5218.125 Å		8.35 Å/pix.
x 1024 pix.
= 8549.376 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 8.349 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-85.0 - 92.0
Average (Standard dev.)6.0640793 (±11.324092)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-312208-209
Dimensions62510241440
Spacing10246251440
CellA: 8549.376 Å / B: 5218.125 Å / C: 12022.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : ER-HOXB8 sample prepared by FIB_SEM milling

EntireName: ER-HOXB8 sample prepared by FIB_SEM milling
Components
  • Cell: ER-HOXB8 sample prepared by FIB_SEM milling

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Supramolecule #1: ER-HOXB8 sample prepared by FIB_SEM milling

SupramoleculeName: ER-HOXB8 sample prepared by FIB_SEM milling / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse)

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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: 7.5
VitrificationCryogen name: ETHANE / Instrument: LEICA PLUNGER
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.03 / Focused ion beam - Duration: 60 / Focused ion beam - Temperature: 273 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 Aquilos 2. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 3.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 6.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT) / Resolution method: OTHER / Number images used: 33

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