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

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Basic information

Entry
Database: EMDB / ID: EMD-1585
TitleElectron tomography of isometrically contracting insect flight muscle quick frozen after a rapid stretch transient
Map dataThis is a global average of aligned subvolumes.
Sample
  • Sample: Isometrically contracting asynchronous insect flight muscle upon a quick stretch
  • Organelle or cellular component: myofibril
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
Function / homology
Function and homology information


troponin C binding / contractile muscle fiber / troponin complex / Striated Muscle Contraction / regulation of muscle contraction / muscle myosin complex / myosin filament / myosin II complex / myosin complex / cytoskeletal motor activator activity ...troponin C binding / contractile muscle fiber / troponin complex / Striated Muscle Contraction / regulation of muscle contraction / muscle myosin complex / myosin filament / myosin II complex / myosin complex / cytoskeletal motor activator activity / sarcomere organization / microfilament motor activity / myofibril / tropomyosin binding / myosin heavy chain binding / mesenchyme migration / troponin I binding / filamentous actin / actin filament bundle / skeletal muscle thin filament assembly / actin filament bundle assembly / striated muscle thin filament / skeletal muscle contraction / skeletal muscle myofibril / actin monomer binding / cardiac muscle contraction / skeletal muscle fiber development / stress fiber / titin binding / skeletal muscle tissue development / muscle contraction / actin filament polymerization / filopodium / actin filament organization / actin filament / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / calcium-dependent protein binding / actin filament binding / actin cytoskeleton / lamellipodium / actin binding / cell body / calmodulin binding / hydrolase activity / protein heterodimerization activity / protein domain specific binding / calcium ion binding / positive regulation of gene expression / magnesium ion binding / protein homodimerization activity / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
Troponin T / : / Tropomyosins signature. / Troponin / Troponin domain superfamily / Troponin / : / Tropomyosin / Tropomyosin / EF-hand domain ...Troponin T / : / Tropomyosins signature. / Troponin / Troponin domain superfamily / Troponin / : / Tropomyosin / Tropomyosin / EF-hand domain / DNA repair protein XRCC4-like, C-terminal / Myosin tail / Myosin tail / Myosin N-terminal SH3-like domain / Myosin S1 fragment, N-terminal / : / Myosin, N-terminal, SH3-like / Myosin N-terminal SH3-like domain profile. / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif, EF-hand binding site / Myosin head, motor domain / Myosin head (motor domain) / Myosin motor domain profile. / Myosin. Large ATPases. / IQ motif profile. / Kinesin motor domain superfamily / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / EF-hand domain pair / EF-hand, calcium binding motif / ATPase, nucleotide binding domain / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Troponin C, skeletal muscle / Myosin light chain 3, skeletal muscle isoform / Myosin regulatory light chain 11 / Myosin essential light chain, striated adductor muscle / Troponin T, fast skeletal muscle isoforms / Myosin heavy chain, skeletal muscle, adult / Myosin regulatory light chain, striated adductor muscle / Myosin heavy chain, striated muscle / Tropomyosin alpha-1 chain / Actin, alpha skeletal muscle / Troponin I, fast skeletal muscle
Similarity search - Component
Biological speciesLethocerus indicus (insect)
Methodsubtomogram averaging / cryo EM / negative staining
AuthorsWu S / Liu J / Reedy MC / Tregear RT / Winkler H / Franzini-Armstrong C / Sasaki H / Lucaveche C / Goldman YE / Reedy MK / Taylor KA
CitationJournal: J Struct Biol / Year: 2009
Title: Methods for identifying and averaging variable molecular conformations in tomograms of actively contracting insect flight muscle.
Authors: Shenping Wu / Jun Liu / Mary C Reedy / Hanspeter Winkler / Michael K Reedy / Kenneth A Taylor /
Abstract: During active muscle contraction, tension is generated through many simultaneous, independent interactions between the molecular motor myosin and the actin filaments. The ensemble of myosin motors ...During active muscle contraction, tension is generated through many simultaneous, independent interactions between the molecular motor myosin and the actin filaments. The ensemble of myosin motors displays heterogeneous conformations reflecting different mechanochemical steps of the ATPase pathway. We used electron tomography of actively contracting insect flight muscle fast-frozen, freeze substituted, Araldite embedded, thin-sectioned and stained, to obtain 3D snapshots of the multiplicity of actin-attached myosin structures. We describe procedures for alignment of the repeating lattice of sub-volumes (38.7 nm cross-bridge repeats bounded by troponin) and multivariate data analysis to identify self-similar repeats for computing class averages. Improvements in alignment and classification of repeat sub-volumes reveals (for the first time in active muscle images) the helix of actin subunits in the thin filament and the troponin density with sufficient clarity that a quasiatomic model of the thin filament can be built into the class averages independent of the myosin cross-bridges. We show how quasiatomic model building can identify both strong and weak myosin attachments to actin. We evaluate the accuracy of image classification to enumerate the different types of actin-myosin attachments.
History
DepositionNov 24, 2008-
Header (metadata) releaseDec 3, 2008-
Map releaseNov 12, 2010-
UpdateFeb 5, 2014-
Current statusFeb 5, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.118447077
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  • Surface view colored by cylindrical radius
  • Surface level: 0.118447077
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2w4u, PDB-2w4v
  • Surface level: 0.118447077
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2w4u, PDB-2w4w
  • Surface level: 0.118447077
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2w4g
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2w4h
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2w4u
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2w4v
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2w4w
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1585.png

Downloads & links

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Map

FileDownload / File: emd_1585.map.gz / Format: CCP4 / Size: 1.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a global average of aligned subvolumes.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.9 Å/pix.
x 88 pix.
= 607.2 Å		6.9 Å/pix.
x 48 pix.
= 331.2 Å		6.9 Å/pix.
x 74 pix.
= 510.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 6.9 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: -0.372 / Movie #1: 0.1184471
Minimum - Maximum-1.94175231 - 1.03447604
Average (Standard dev.)0.0 (±0.45458132)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-24-37-44
Dimensions487488
Spacing487488
CellA: 510.6 Å / B: 331.2 Å / C: 607.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/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
α/β/γ90.00090.00090.000
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
Components
  • Sample: Isometrically contracting asynchronous insect flight muscle upon a quick stretch
  • Organelle or cellular component: myofibril

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Supramolecule #1000: Isometrically contracting asynchronous insect flight muscle upon ...

SupramoleculeName: Isometrically contracting asynchronous insect flight muscle upon a quick stretch
type: sample / ID: 1000
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 ...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.
Oligomeric state: tissue / Number unique components: 8

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Supramolecule #1: myofibril

SupramoleculeName: myofibril / type: organelle_or_cellular_component / ID: 1 / Name.synonym: muscle
Details: The sample was enblock stained using uranyl acetate and tannic acid, and post stained with lead citrate and potassium permanganate
Oligomeric state: muscle fibril / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Lethocerus indicus (insect) / synonym: Water bug / Tissue: asynchronous dorsal longitudinal flight muscle / Organelle: myofibril / Location in cell: myoplasm

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsubtomogram averaging

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Sample preparation

BufferDetails: 20 mM MOPS buffer, 5 mM NaN3, and MgCl2, ATP, CaCl2, and EGTA in varying millimolar concentrations
StainingType: NEGATIVE
Details: 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 ...Details: 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.
VitrificationCryogen name: HELIUM / Chamber temperature: 4.5 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: modified Heuser Cryopress freezing head
Timed resolved state: Frozen 6-7 msec after application of the length transient
Method: smash against a liquid helium cooled Au-coated Cu mirror

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Electron microscopy

MicroscopeFEI/PHILIPS CM300FEG/T
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F224 (2k x 2k)
Details: Images recorded on a TVIPS Tem-Cam F224 2k x 2k CCD camera
Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm
Sample stageSpecimen holder: side entry, eucentric / Specimen holder model: OTHER / Tilt series - Axis1 - Min angle: -72 ° / Tilt series - Axis1 - Max angle: 72 °

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Image processing

DetailsThe 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.
Final reconstructionAlgorithm: OTHER / Resolution method: FSC 0.5 CUT-OFF / Software - Name: 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 ...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.

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