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- EMDB-6514: 3D reconstruction of tarantula thick filaments -

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Basic information

Entry
Database: EMDB / ID: EMD-6514
Title3D reconstruction of tarantula thick filaments
Map data3D reconstruction of Tarantula thick filament at 30 Angstrom resolution
Sample
  • Sample: tarantula thick filament
  • Protein or peptide: thick filament
Keywordssingle particle analysis / thick filament / muscle contraction
Biological speciesGrammostola rosea (Chilean rose tarantula)
Methodsingle particle reconstruction / cryo EM / Resolution: 30.0 Å
AuthorsYang S / Woodhead JL / Zhao F / Sulbaran G / Craig R
CitationJournal: J Struct Biol / Year: 2016
Title: An approach to improve the resolution of helical filaments with a large axial rise and flexible subunits.
Authors: Shixin Yang / John L Woodhead / Fa-Qing Zhao / Guidenn Sulbarán / Roger Craig /
Abstract: Single particle analysis is widely used for three-dimensional reconstruction of helical filaments. Near-atomic resolution has been obtained for several well-ordered filaments. However, it is still a ...Single particle analysis is widely used for three-dimensional reconstruction of helical filaments. Near-atomic resolution has been obtained for several well-ordered filaments. However, it is still a challenge to achieve high resolution for filaments with flexible subunits and a large axial rise per subunit relative to pixel size. Here, we describe an approach that improves the resolution in such cases. In filaments with a large axial rise, many segments must be shifted a long distance along the filament axis to match with a reference projection, potentially causing loss of alignment accuracy and hence resolution. In our study of myosin filaments, we overcame this problem by pre-determining the axial positions of myosin head crowns within segments to decrease the alignment error. In addition, homogeneous, well-ordered segments were selected from the raw data set by checking the assigned azimuthal rotation angle of segments in each filament against those expected for perfect helical symmetry. These procedures improved the resolution of the filament reconstruction from 30 Å to 13 Å. This approach could be useful in other helical filaments with a large axial rise and/or flexible subunits.
History
DepositionNov 12, 2015-
Header (metadata) releaseDec 30, 2015-
Map releaseDec 30, 2015-
UpdateDec 30, 2015-
Current statusDec 30, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.005
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.005
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6514.gif

Downloads & links

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Map

FileDownload / File: emd_6514.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of Tarantula thick filament at 30 Angstrom resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.74 Å/pix.
x 240 pix.
= 658.56 Å		2.74 Å/pix.
x 240 pix.
= 658.56 Å		2.74 Å/pix.
x 240 pix.
= 658.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.744 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.005 / Movie #1: 0.005
Minimum - Maximum-0.01001488 - 0.06250802
Average (Standard dev.)0.00171727 (±0.00621097)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 658.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.7442.7442.744
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z658.560658.560658.560
α/β/γ90.00090.00090.000
start NX/NY/NZ-300-64
NX/NY/NZ6161129
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0100.0630.002

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Supplemental data

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Sample components

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Entire : tarantula thick filament

EntireName: tarantula thick filament
Components
  • Sample: tarantula thick filament
  • Protein or peptide: thick filament

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Supramolecule #1000: tarantula thick filament

SupramoleculeName: tarantula thick filament / type: sample / ID: 1000 / Oligomeric state: helical filaments of myosin II / Number unique components: 1
Molecular weightExperimental: 520 KDa / Theoretical: 520 KDa / Method: Sedimentation

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Macromolecule #1: thick filament

MacromoleculeName: thick filament / type: protein_or_peptide / ID: 1 / Name.synonym: myosin filament / Number of copies: 18 / Oligomeric state: helical filament / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Grammostola rosea (Chilean rose tarantula) / synonym: tarantula / Tissue: leg / Cell: striated muscle / Organelle: myofibril / Location in cell: cytoskeleton
Molecular weightExperimental: 520 KDa / Theoretical: 520 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 7
Details: 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 5 mM PIPES, 5 mM NaH2PO4, 1 mM NaN3
GridDetails: Quantifoil (R2/2) 200 mesh grids, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 85 K / Instrument: HOMEMADE PLUNGER / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateApr 13, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 690 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 109000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 59000
Sample stageSpecimen holder: Nitrogen-cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsCryo-EM images of tarantula thick filaments were processed using SPIDER and IHRSR
CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Helical parameters - Δz: 145 Å
Applied symmetry - Helical parameters - Δ&Phi: 30 °
Applied symmetry - Helical parameters - Axial symmetry: C4 (4 fold cyclic)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: OTHER / Software - Name: SPIDER, IHRSR
Details: Particles were not pre-centered. All particles were used for reconstruction.
Number images used: 3174

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