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Yorodumi- EMDB-6042: Imaging proteins and cells in liquid solution at nanometer resolu... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6042 | |||||||||
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Title | Imaging proteins and cells in liquid solution at nanometer resolution by electron microscopy | |||||||||
Map data | IMOD 3D reconstruction of bacterium Magnetospirillum magneticum in liquid growth medium | |||||||||
Sample |
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Keywords | Liquid-cell transmission electron microscopy / protein structure in liquid solution / bacteria structure in growth medium / virus infecting mammalian cell | |||||||||
Biological species | Magnetospirillum magneticum (bacteria) | |||||||||
Method | electron tomography / negative staining | |||||||||
Authors | Ren G / Peng B / Zhang L / Lei D / Lu Z / Wong E / Zhang M / Rames MJ / Liu J / De Yoreo JJ / Zhang J | |||||||||
Citation | Journal: PLoS One / Year: 2012 Title: IPET and FETR: experimental approach for studying molecular structure dynamics by cryo-electron tomography of a single-molecule structure. Authors: Lei Zhang / Gang Ren / Abstract: The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional ...The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a "focused electron tomography reconstruction" (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single "snapshot" molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6042.map.gz | 626.4 MB | EMDB map data format | |
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Header (meta data) | emd-6042-v30.xml emd-6042.xml | 10.7 KB 10.7 KB | Display Display | EMDB header |
Images | emd_6042.tif | 69.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6042 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6042 | HTTPS FTP |
-Validation report
Summary document | emd_6042_validation.pdf.gz | 79.2 KB | Display | EMDB validaton report |
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Full document | emd_6042_full_validation.pdf.gz | 78.3 KB | Display | |
Data in XML | emd_6042_validation.xml.gz | 499 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6042 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6042 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_6042.map.gz / Format: CCP4 / Size: 781 MB / Type: IMAGE STORED AS SIGNED BYTE | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | IMOD 3D reconstruction of bacterium Magnetospirillum magneticum in liquid growth medium | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 10.72 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Magnetospirillum magneticum (strain AMB-1) grown in standard medium
Entire | Name: Magnetospirillum magneticum (strain AMB-1) grown in standard medium |
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Components |
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-Supramolecule #1000: Magnetospirillum magneticum (strain AMB-1) grown in standard medium
Supramolecule | Name: Magnetospirillum magneticum (strain AMB-1) grown in standard medium type: sample / ID: 1000 / Details: label-free, live bacterium in growth medium / Number unique components: 1 |
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-Supramolecule #1: Magnetospirillum magneticum
Supramolecule | Name: Magnetospirillum magneticum / type: organelle_or_cellular_component / ID: 1 / Details: full cell / Number of copies: 1 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Magnetospirillum magneticum (bacteria) / Strain: AMB-1 |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | electron tomography |
Aggregation state | cell |
-Sample preparation
Staining | Type: NEGATIVE / Details: No stain, in liquid solution, at room temperature |
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Grid | Details: Two 200 mesh copper TEM grids pre-coated with a thin layer of Formvar film, no glow discharge |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | ZEISS LIBRA120PLUS |
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Temperature | Min: 293 K / Max: 296 K / Average: 295 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 125,000 times magnification |
Specialist optics | Energy filter - Name: Zeiss Libra 120 PLUS / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV |
Details | Low-dose illumination |
Date | Mar 15, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 68 / Average electron dose: 50 e/Å2 / Bits/pixel: 16 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Calibrated magnification: 13988 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal magnification: 10000 |
Sample stage | Specimen holder: Normal room-temperature specimen holder / Specimen holder model: OTHER / Tilt series - Axis1 - Min angle: -66 ° / Tilt series - Axis1 - Max angle: 68 ° / Tilt series - Axis1 - Angle increment: 1 ° |
-Image processing
Details | Micrographs were initially aligned using the IMOD software package. CTF was then corrected using TOMOCTF. The tilt series of full micrographs was then reconstructed using IMOD. |
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Final reconstruction | Algorithm: OTHER / Software - Name: IMOD / Number images used: 68 |
CTF correction | Details: TOMOCTF |