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- EMDB-6042: Imaging proteins and cells in liquid solution at nanometer resolu... -

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Entry
Database: EMDB / ID: EMD-6042
TitleImaging proteins and cells in liquid solution at nanometer resolution by electron microscopy
Map dataIMOD 3D reconstruction of bacterium Magnetospirillum magneticum in liquid growth medium
Sample
  • Sample: Magnetospirillum magneticum (strain AMB-1) grown in standard medium
  • Organelle or cellular component: Magnetospirillum magneticum
KeywordsLiquid-cell transmission electron microscopy / protein structure in liquid solution / bacteria structure in growth medium / virus infecting mammalian cell
Biological speciesMagnetospirillum magneticum (bacteria)
Methodelectron tomography / negative staining
AuthorsRen G / Peng B / Zhang L / Lei D / Lu Z / Wong E / Zhang M / Rames MJ / Liu J / De Yoreo JJ / Zhang J
CitationJournal: 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
DepositionAug 19, 2014-
Header (metadata) releaseSep 24, 2014-
Map releaseSep 2, 2015-
UpdateSep 2, 2015-
Current statusSep 2, 2015Processing 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_6042.tif

Downloads & links

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Map

FileDownload / File: emd_6042.map.gz / Format: CCP4 / Size: 781 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationIMOD 3D reconstruction of bacterium Magnetospirillum magneticum in liquid growth medium
Voxel sizeX=Y=Z: 10.72 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)-78.253585819999998 (±71.720863339999994)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin81-599-799
Dimensions27712501121
Spacing27712501121
CellA: 26810.72 Å / B: 29705.121 Å / C: 1297.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z10.7200003998410.72000036088110.72
M x/y/z25012771121
origin x/y/z0.0000.0000.000
length x/y/z26810.72129705.1211297.120
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-59981-799
NC/NR/NS25012771121
D min/max/mean-128.000127.000-78.254

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Supplemental data

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Sample components

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Entire : Magnetospirillum magneticum (strain AMB-1) grown in standard medium

EntireName: Magnetospirillum magneticum (strain AMB-1) grown in standard medium
Components
  • Sample: Magnetospirillum magneticum (strain AMB-1) grown in standard medium
  • Organelle or cellular component: Magnetospirillum magneticum

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Supramolecule #1000: Magnetospirillum magneticum (strain AMB-1) grown in standard medium

SupramoleculeName: 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

SupramoleculeName: Magnetospirillum magneticum / type: organelle_or_cellular_component / ID: 1 / Details: full cell / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Magnetospirillum magneticum (bacteria) / Strain: AMB-1

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron tomography
Aggregation statecell

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Sample preparation

StainingType: NEGATIVE / Details: No stain, in liquid solution, at room temperature
GridDetails: Two 200 mesh copper TEM grids pre-coated with a thin layer of Formvar film, no glow discharge
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeZEISS LIBRA120PLUS
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 13988 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal magnification: 10000
Specialist opticsEnergy filter - Name: Zeiss Libra 120 PLUS / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen 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 °
TemperatureMin: 293 K / Max: 296 K / Average: 295 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 125,000 times magnification
DetailsLow-dose illumination
DateMar 15, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 68 / Average electron dose: 50 e/Å2 / Bits/pixel: 16

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Image processing

CTF correctionDetails: TOMOCTF
Final reconstructionAlgorithm: OTHER / Software - Name: IMOD / Number images used: 68
DetailsMicrographs 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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