|Entry||Database: EMDB / ID: 1585|
|Title||Electron tomography of isometrically contracting insect flight muscle quick frozen after a rapid stretch transient|
|Keywords||insect / muscle / myosin / troponin / tropomyosin / actin / light chains / thin filament / thick filament / electron microscopy / image processing / isometric contraction / freezing / freeze substitution / microtomy / multivariate data analysis|
|Sample||Isometrically contracting asynchronous insect flight muscle upon a quick stretch|
|Source||Lethocerus indicus / arthropod / Water bug|
|Map data||This is a global average of aligned subvolumes.|
|Authors||Wu S / Liu J / Reedy MC / Tregear RT / Winkler H / Franzini-Armstrong C / Sasaki H / Lucaveche C / Goldman YE / Reedy MK / Taylor KA|
|Citation||PLoS ONE, 2012, 7, e39422-e39422|
primary. PLoS ONE, 2012, 7, e39422-e39422 Yorodumi Papers
1. J. Struct. Biol., 2009, 168, 485-502 Yorodumi Papers
|Validation Report||PDB-ID: 2w4g|
About validation report
|Date||Deposition: Nov 24, 2008 / Header (metadata) release: Dec 3, 2008 / Map release: Nov 12, 2010 / Last update: Feb 5, 2014|
Downloads & links
|File||emd_1585.map.gz (map file in CCP4 format, 1222 KB)|
|Projections & slices|
Images are generated by Spider package.
|Voxel size||X=Y=Z: 6.9 Å|
CCP4 map header:
-Entire Isometrically contracting asynchronous insect flight muscle upon ...
|Entire||Name: Isometrically contracting asynchronous insect flight muscle upon a quick stretch|
Details: This specimen is obtained from a quick frozen, isometrically contracting asynchronous insect flight muscle that has been freeze substituted, plastic embedded, and thin sectioned. The fiber was stretched 6 nm per half-sarcomere in 2 ms and length was held constant after the length step. The freezing impact occurred 6-7 ms later.
Number of components: 8 / Oligomeric State: tissue
-Component #1: cellular-component, myofibril
|Cellular-component||Name: myofibril / a.k.a: muscle / Oligomeric Details: muscle fibril|
Details: The sample was enblock stained using uranyl acetate and tannic acid, and post stained with lead citrate and potassium permanganate
Recombinant expression: No
|Source||Species: Lethocerus indicus / arthropod / Water bug|
|Source (natural)||Organelle: myofibril / Location in cell: myoplasm|
Organ or tissue: asynchronous dorsal longitudinal flight muscle
|Sample solution||Buffer solution: 20 mM MOPS buffer, 5 mM NaN3, and MgCl2, ATP, CaCl2, and EGTA in varying millimolar concentrations|
|Staining||Freeze slammed fibers were freeze-substituted in acetone using a tannic aciduranyl acetate sequence, and ultimately embedded in Araldite-506 for thin-section electron microscopy. Ultrathin (25-30 nm) longitudinal sections were stained by permanganate-lead.|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: HELIUM / Temperature: 4.5 K|
Method: smash against a liquid helium cooled Au-coated Cu mirror
Time resolved state: Frozen 6-7 msec after application of the length transient
Details: Vitrification instrument: modified Heuser Cryopress freezing head
-Electron microscopy imaging
|Imaging||Microscope: FEI/PHILIPS CM300FEG/T|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Lens||Cs: 2 mm / Imaging mode: BRIGHT FIELD|
|Specimen Holder||Holder: side entry, eucentric / Model: OTHER / Tilt Angle: -72 - 72 deg.|
|Camera||Detector: TVIPS TEMCAM-F224 (2k x 2k)|
Imagesreco rdedonaTVI PSTemCamF2 242kx2kCCD camera
|Processing||Method: subtomogram averaging|
Details: The fiber was stretched 6 nm per half-sarcomere in 2 ms and length was held constant after the length step. The freezing impact occurred 6-7 ms later. Average number of tilts used in the 3D reconstructions: 70.
|3D reconstruction||Algorithm: Marker free alignment / Software: PROTOMO, IMOD|
Details: Two tilt series at 90 degrees to one another were first independently aligned using marker-free alignment and area matching. The two resulting tomograms were then merged by patch correlation and volume warp using IMOD. Tomogram computed using weighted back projection.
Resolution method: FSC 0.5
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