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- EMDB-1585: Electron tomography of isometrically contracting insect flight mu... -

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Basic information

Entry
Database: EMDB / ID: 1585
TitleElectron tomography of isometrically contracting insect flight muscle quick frozen after a rapid stretch transient
Keywordsinsect / muscle / myosin / troponin / tropomyosin / actin / light chains / thin filament / thick filament / electron microscopy / image processing / isometric contraction / freezing / freeze substitution / microtomy / multivariate data analysis
SampleIsometrically contracting asynchronous insect flight muscle upon a quick stretch
SourceLethocerus indicus / arthropod / Water bug
Map dataThis is a global average of aligned subvolumes.
Methodsubtomogram averaging
AuthorsWu S / Liu J / Reedy MC / Tregear RT / Winkler H / Franzini-Armstrong C / Sasaki H / Lucaveche C / Goldman YE / Reedy MK / Taylor KA
CitationPLoS ONE, 2012, 7, e39422-e39422

primary. PLoS ONE, 2012, 7, e39422-e39422 StrPapers
Structural changes in isometrically contracting insect flight muscle trapped following a mechanical perturbation.
Shenping Wu / Jun Liu / Mary C Reedy / Robert J Perz-Edwards / Richard T Tregear / Hanspeter Winkler / Clara Franzini-Armstrong / Hiroyuki Sasaki / Carmen Lucaveche / Yale E Goldman / Michael K Reedy / Kenneth A Taylor

1. J. Struct. Biol., 2009, 168, 485-502 StrPapers
Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle.
Shenping Wu / Jun Liu / Mary C Reedy / Hanspeter Winkler / Michael K Reedy / Kenneth A Taylor

DateDeposition: Nov 24, 2008 / Header (metadata) release: Dec 3, 2008 / Map release: Nov 12, 2010 / Last update: Nov 24, 2008

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.118447077
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.118447077
  • Imaged by UCSF CHIMERA
  • Download
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Supplemental images

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Map

Fileemd_1585.map.gz (map file in CCP4 format, 1222 KB)
Projections & slices
Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
88 pix
6.9 A/pix
= 607.2 A
48 pix
6.9 A/pix
= 331.2 A
74 pix
6.9 A/pix
= 510.6 A

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 6.9 A
Density
Contour Level:-0.372 (by emdb), 0.1184471 (movie #1):
Minimum - Maximum-1.94175231 - 1.03447604
Average (Standard dev.)0E-8 (0.45458132)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions487488
Origin-24-37-44
Limit233643
Spacing487488
CellA: 510.6 A / B: 331.2 A / C: 607.2 A
Alpha=beta=gamma: 90 deg.

CCP4 map header:

modeImage stored as Reals
A/pix X/Y/Z6.96.96.9
M x/y/z744888
origin x/y/z0.0000.0000.000
length x/y/z510.600331.200607.200
alpha/beta/gamma90.00090.00090.000
start NX/NY/NZ-100-100-99
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-37-24-44
NC/NR/NS744888
D min/max/mean-1.9421.0340.000

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Supplemental data

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Sample components

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Entire Isometrically contracting asynchronous insect flight muscle upon ...

EntireName: 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

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Component #1: cellular-component, myofibril

Cellular-componentName: 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
SourceSpecies: Lethocerus indicus / arthropod / Water bug
Source (natural)Organelle: myofibril / Location in cell: myoplasm
Organ or tissue: asynchronous dorsal longitudinal flight muscle

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Experimental details

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Sample preparation

Sample solutionBuffer solution: 20 mM MOPS buffer, 5 mM NaN3, and MgCl2, ATP, CaCl2, and EGTA in varying millimolar concentrations
StainingFreeze 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.
VitrificationInstrument: 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

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Electron microscopy imaging

ImagingMicroscope: FEI/PHILIPS CM300FEG/T
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
LensCs: 2 mm / Imaging mode: BRIGHT FIELD
Specimen HolderHolder: side entry, eucentric / Model: OTHER / Tilt Angle: -72 - 72 deg.
CameraDetector: TVIPS TEMCAM-F224 (2k x 2k)

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Image acquisition

Image acquisition
16
Image acquisition
Imagesreco rdedonaTVI PSTemCamF2 242kx2kCCD camera

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Image processing

ProcessingMethod: 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 reconstructionAlgorithm: 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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