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- EMDB-3301: The Structure of the Relaxed Thick Filaments from Lethocerus Flig... -

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Basic information

Entry
Database: EMDB / ID: EMD-3301
TitleThe Structure of the Relaxed Thick Filaments from Lethocerus Flight Muscle
Map dataCryoEM IHRSR of relaxed thick filament from Lethocerus indicus (water bug) flight muscle
Sample
  • Sample: Thick filament from Lethocerus (waterbug) flight muscle
  • Protein or peptide: myosin II
  • Protein or peptide: paramyosin
  • Protein or peptide: flightin
  • Protein or peptide: myofilin
Keywordsthick filament / insect fight muscle / myosin / paramyosin / myofilin / flightin / relaxed state / muscle contraction
Function / homologyMyofilin / Myofilin / Myofilin / Flightin / Myofilin protein
Function and homology information
Biological speciesLethocerus indicus (insect)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 5.5 Å
AuthorsHu Z / Taylor DW / Reedy MK / Edwards RJ / Taylor KA
CitationJournal: Sci Adv / Year: 2016
Title: Structure of myosin filaments from relaxed flight muscle by cryo-EM at 6 Å resolution.
Authors: Zhongjun Hu / Dianne W Taylor / Michael K Reedy / Robert J Edwards / Kenneth A Taylor /
Abstract: We describe a cryo-electron microscopy three-dimensional image reconstruction of relaxed myosin II-containing thick filaments from the flight muscle of the giant water bug . The relaxed thick ...We describe a cryo-electron microscopy three-dimensional image reconstruction of relaxed myosin II-containing thick filaments from the flight muscle of the giant water bug . The relaxed thick filament structure is a key element of muscle physiology because it facilitates the reextension process following contraction. Conversely, the myosin heads must disrupt their relaxed arrangement to drive contraction. Previous models predicted that myosin was unique in having an intermolecular head-head interaction, as opposed to the intramolecular head-head interaction observed in all other species. In contrast to the predicted model, we find an intramolecular head-head interaction, which is similar to that of other thick filaments but oriented in a distinctly different way. The arrangement of myosin's long α-helical coiled-coil rod domain has been hypothesized as either curved layers or helical subfilaments. Our reconstruction is the first report having sufficient resolution to track the rod α helices in their native environment at resolutions ~5.5 Å, and it shows that the layer arrangement is correct for . Threading separate paths through the forest of myosin coiled coils are four nonmyosin peptides. We suggest that the unusual position of the heads and the rod arrangement separated by nonmyosin peptides are adaptations for mechanical signal transduction whereby applied tension disrupts the myosin heads as a component of stretch activation.
History
DepositionJan 18, 2016-
Header (metadata) releaseMar 9, 2016-
Map releaseOct 5, 2016-
UpdateDec 21, 2016-
Current statusDec 21, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.9
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.9
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3301.png

Downloads & links

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Map

FileDownload / File: emd_3301.map.gz / Format: CCP4 / Size: 300.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM IHRSR of relaxed thick filament from Lethocerus indicus (water bug) flight muscle
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.22 Å/pix.
x 432 pix.
= 528.336 Å		1.22 Å/pix.
x 432 pix.
= 528.336 Å		1.22 Å/pix.
x 432 pix.
= 528.336 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.223 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.9 / Movie #1: 2.9
Minimum - Maximum-7.59589577 - 16.792200090000001
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin137137137
Dimensions432432432
Spacing432432432
CellA=B=C: 528.336 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2231.2231.223
M x/y/z432432432
origin x/y/z0.0000.0000.000
length x/y/z528.336528.336528.336
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS137137137
NC/NR/NS432432432
D min/max/mean-7.59616.792-0.000

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Supplemental data

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Sample components

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Entire : Thick filament from Lethocerus (waterbug) flight muscle

EntireName: Thick filament from Lethocerus (waterbug) flight muscle
Components
  • Sample: Thick filament from Lethocerus (waterbug) flight muscle
  • Protein or peptide: myosin II
  • Protein or peptide: paramyosin
  • Protein or peptide: flightin
  • Protein or peptide: myofilin

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Supramolecule #1000: Thick filament from Lethocerus (waterbug) flight muscle

SupramoleculeName: Thick filament from Lethocerus (waterbug) flight muscle
type: sample / ID: 1000
Details: The sample is a bipolar helical structure, with helical repeat 145 Angstrom and helical turn 33.98 degree. The sample has C4 symmetry. The map contains 6 unique features: myosin molecule ...Details: The sample is a bipolar helical structure, with helical repeat 145 Angstrom and helical turn 33.98 degree. The sample has C4 symmetry. The map contains 6 unique features: myosin molecule with completely resolved rods, 4 resolved non-myosin densities among the myosin rods and an annular region inside of annulus occupied by myosin rods that most likely contains paramyosin. The 4 non-myosin densities may contain parts of the proteins myofilin and flightin.
Oligomeric state: bipolar helical structure / Number unique components: 4

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Macromolecule #1: myosin II

MacromoleculeName: myosin II / type: protein_or_peptide / ID: 1
Details: The resolution of LMM and C-terminal of S2 domain is about 5.5 Angstrom; the N-terminal (at first crown) of S2 is about 7 angstrom; free head is about 10 angstrom; block head is about 20 angstrom.
Oligomeric state: Helical assembly / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Lethocerus indicus (insect) / synonym: giant waterbug / Tissue: dorsal longitudinal indirect flight muscle / Cell: myocyte / Organelle: sarcomere / Location in cell: myofibril
Molecular weightTheoretical: 520 KDa

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Macromolecule #2: paramyosin

MacromoleculeName: paramyosin / type: protein_or_peptide / ID: 2
Details: Paramyosin is located in the filament core, and may not have the same symmetry with myosin. The resolution is lower at 10 Angstrom.
Oligomeric state: dimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Lethocerus indicus (insect) / synonym: giant waterbug / Tissue: dorsal longitudinal indirect flight muscle / Cell: myocyte / Organelle: sarcomere / Location in cell: myofibril
Molecular weightTheoretical: 100 KDa

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Macromolecule #3: flightin

MacromoleculeName: flightin / type: protein_or_peptide / ID: 3
Details: We can see some non-myosin densities in the reconstruction. Their identification as flightin is uncertain
Recombinant expression: No / Database: NCBI
Source (natural)Organism: Lethocerus indicus (insect) / synonym: giant waterbug / Tissue: dorsal longitudinal indirect flight muscle / Cell: myocyte / Organelle: sarcomere / Location in cell: myofibril
Molecular weightTheoretical: 19 KDa
SequenceUniProtKB: Flightin

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Macromolecule #4: myofilin

MacromoleculeName: myofilin / type: protein_or_peptide / ID: 4
Details: We can see some non-myosin densities in the reconstruction. Their identification as myofilin is uncertain.
Recombinant expression: No / Database: NCBI
Source (natural)Organism: Lethocerus indicus (insect) / synonym: giant waterbug / Tissue: dorsal longitudinal indirect flight muscle / Cell: myocyte / Organelle: sarcomere / Location in cell: myofibril
Molecular weightTheoretical: 30.3 KDa
SequenceUniProtKB: Myofilin protein / InterPro: Myofilin

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.8
Details: 8 mM MOPS, 10 mM Na acetate, 1 mM Mg acetate, 1 mM ATP, and 1 mM EGTA
StainingType: NEGATIVE
Details: All reconstruction data obtained from unstained, frozen hydrated samples.
GridDetails: R2/1 Quantifoil grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 100 K / Instrument: FEI VITROBOT MARK IV
Details: The Vitrobot environmental chamber was set to 100% relative humidity for freezing and maintained at a temperature of 22 degrees Celsius. The crude filament prep was applied to the grid, ...Details: The Vitrobot environmental chamber was set to 100% relative humidity for freezing and maintained at a temperature of 22 degrees Celsius. The crude filament prep was applied to the grid, incubated for 60 seconds, blotted once and then plunged into liquid ethane to vitrify.
Method: EM samples of relaxed filaments were prepared by applying 4 microliters of the above prep to a R2/1 Quantifoil grid at room temperature for 1 minute, washing with several drops of rinse ...Method: EM samples of relaxed filaments were prepared by applying 4 microliters of the above prep to a R2/1 Quantifoil grid at room temperature for 1 minute, washing with several drops of rinse buffer, identical to the relaxing buffer except NaCl was replaced with Na acetate. Following a 2 minute incubation, the grid was washed with several drops of a low salt rinse consisting of 8 mM MOPS, 10 mM Na Acetate, 1 mM Mg Acetate, 1 mM ATP, and 1 mM EGTA, pH 6.8.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 90 K
Details48 frames were recorded over a 1.5 sec exposure time
DateMar 27, 2015
Image recordingCategory: CCD / Film or detector model: OTHER / Number real images: 4000 / Average electron dose: 65 e/Å2 / Details: The total dose is 65 electrons with 48 frames / Bits/pixel: 32
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsParticles are picked and segmented using Appion system. The defocus was determined and refined using ACE and CTFFIND3. The 3D classification and final reconstruction was performed by RELION.
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Helical parameters - Δz: 145 Å
Applied symmetry - Helical parameters - Δ&Phi: 33.98 °
Applied symmetry - Helical parameters - Axial symmetry: C4 (4 fold cyclic)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 5.5 Å / Resolution method: OTHER / Software - Name: Relion
Details: Damage compensation strategy was used after whole frame alignment. There were 72,000 segments at beginning. After 3D classification, only 24,000 segments were used in the final reconstruction.
Number images used: 24000
Final angle assignmentDetails: The limit of angle tilt is 75 degrees
FSC plot (resolution estimation)

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