+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-14924 | |||||||||
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Title | Cryo-STEM tomography of fibroblast nuclear periphery | |||||||||
Map data | Cryo-STEM tomography (CSTET) of an intact fibroblast showing nuclear periphery, processed by 3D deconvolution. | |||||||||
Sample |
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Keywords | nucleus / chromatin / nuclear envelope / mitochondria / STEM / CSTET / DNA | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | electron tomography / cryo EM | |||||||||
Authors | Wolf SG / Fass D / Elbaum M | |||||||||
Funding support | Israel, 1 items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2020 Title: Three-dimensional deconvolution processing for STEM cryotomography. Authors: Barnali Waugh / Sharon G Wolf / Deborah Fass / Eric Branlund / Zvi Kam / John W Sedat / Michael Elbaum / Abstract: The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end, one of the primary tools in ...The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryoscanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles and then in representative cryotomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin. #1: Journal: To Be Published Title: A Proposed Unified Interphase Nucleus Chromosome Structure: Preliminary Preponderance of Evidence Authors: Sedat J / McDonald A / Cang H / Lucas J / Arigovindan M / Kam Z / Murre C / Elbaum M | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_14924.map.gz | 1.7 GB | EMDB map data format | |
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Header (meta data) | emd-14924-v30.xml emd-14924.xml | 11.8 KB 11.8 KB | Display Display | EMDB header |
Images | emd_14924.png | 234.4 KB | ||
Filedesc metadata | emd-14924.cif.gz | 4.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-14924 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-14924 | HTTPS FTP |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_14924.map.gz / Format: CCP4 / Size: 2 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Cryo-STEM tomography (CSTET) of an intact fibroblast showing nuclear periphery, processed by 3D deconvolution. | ||||||||||||||||||||
Voxel size | X=Y=Z: 33.51 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Sample components
-Entire : WI-38 fibroblast cell
Entire | Name: WI-38 fibroblast cell |
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Components |
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-Supramolecule #1: WI-38 fibroblast cell
Supramolecule | Name: WI-38 fibroblast cell / type: cell / ID: 1 / Parent: 0 / Details: cells cultured directly on gold EM grid |
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Source (natural) | Organism: Homo sapiens (human) / Organ: lung / Tissue: cell culture |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | electron tomography |
Aggregation state | cell |
-Sample preparation
Buffer | pH: 7.4 Details: Minimal Essential Medium (Gibco) supplemented with 15% fetal calf serum, L-glutamine, and penicillin/streptomycin. |
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Grid | Model: Quantifoil R3.5/1 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. |
Vitrification | Cryogen name: ETHANE / Instrument: LEICA EM GP |
Details | WI-38 embryonic lung fibroblast cells (obtained from Coriell Institute) were cultured directly on gold EM grids and imaged intact by cryo-STEM tomography (CSTET). |
Sectioning | Other: NO SECTIONING |
Fiducial marker | Manufacturer: obtained from L Duchesne (https://doi.org/10.1021/la802876u ) Diameter: 10 nm |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 20.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm |
Specialist optics | Details: STEM data collection in BF mode. |
Sample stage | Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER Cooling holder cryogen: NITROGEN |
Details | NanoProbe STEM acquisition. |
Image recording | Film or detector model: OTHER / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number grids imaged: 1 / Number real images: 61 / Average exposure time: 12.6 sec. / Average electron dose: 3.8 e/Å2 / Details: Cryo-STEM tilt series |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Algorithm: BACK PROJECTION / Software: (Name: TOMO3D, PRIISM/IVE) Details: The original alignment was done using etomo (IMOD), and then optimized using tomoalign as a "thin" specimen. A new aligned stack (.ali) was produced using tomowarpalign (tomoalign) and ...Details: The original alignment was done using etomo (IMOD), and then optimized using tomoalign as a "thin" specimen. A new aligned stack (.ali) was produced using tomowarpalign (tomoalign) and reconstructed by SIRT using tomo3d. The reconstruction was then binned (by 2) and processed by 3D deconvolution as described in Waugh et al (doi:10.1073/pnas.2000700117) with smoothing parameter 0.1. Number images used: 56 |
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Details | Gatan STEM BF/ADF detector |