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- EMDB-1561: Electron tomography of isometrically contracting insect flight muscle -

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Basic information

Entry
Database: EMDB / ID: EMD-1561
TitleElectron tomography of isometrically contracting insect flight muscle
Map dataThis is a dual axis tomogram of quick frozen, freeze This is a dual axis tomogram of quick frozen, freeze substituted, plastic embedded, thin sectioned insect flight muscle from Lethocerus sp.
Sample
  • Sample: Isometrically contracting asynchronous insect flight muscle
  • 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)
Methodelectron tomography / 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
DepositionSep 26, 2008-
Header (metadata) releaseSep 29, 2008-
Map releaseApr 12, 2010-
UpdateAug 12, 2015-
Current statusAug 12, 2015Processing site: PDBe / Status: Released

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Structure visualization

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Structure viewerEM map:
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Supplemental images

1561.gif

Downloads & links

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Map

FileDownload / File: emd_1561.map.gz / Format: CCP4 / Size: 560.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a dual axis tomogram of quick frozen, freeze This is a dual axis tomogram of quick frozen, freeze substituted, plastic embedded, thin sectioned insect flight muscle from Lethocerus sp.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.9 Å/pix.
x 70 pix.
= 483. Å		6.9 Å/pix.
x 1344 pix.
= 11040. Å		6.9 Å/pix.
x 1600 pix.
= 9273.601 Å

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)
Minimum - Maximum-3296.960000000000036 - 4848.859999999999673
Average (Standard dev.)5.83993 (±532.039999999999964)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions1344160070
Spacing1344160070
CellA: 9273.6 Å / B: 11040 Å / C: 483 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.90000074404766.96.9
M x/y/z1344160070
origin x/y/z0.0000.0000.000
length x/y/z9273.60111040.000483.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS1600134470
D min/max/mean-3296.9594848.8645.840

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Supplemental data

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Sample components

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Entire : Isometrically contracting asynchronous insect flight muscle

EntireName: Isometrically contracting asynchronous insect flight muscle
Components
  • Sample: Isometrically contracting asynchronous insect flight muscle
  • Organelle or cellular component: myofibril

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Supramolecule #1000: Isometrically contracting asynchronous insect flight muscle

SupramoleculeName: Isometrically contracting asynchronous insect flight muscle
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
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: myooplasm

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingelectron tomography

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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
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: Gatan model 670 Ultrahigh tilt analytical holder
Specimen holder model: OTHER / Tilt series - Axis1 - Min angle: -72 ° / Tilt series - Axis1 - Max angle: 72 °

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Image processing

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.
Number images used: 70

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