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- PDB-3jbh: TWO HEAVY MEROMYOSIN INTERACTING-HEADS MOTIFS FLEXIBLE DOCKED INT... -

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Entry
Database: PDB / ID: 3jbh
TitleTWO HEAVY MEROMYOSIN INTERACTING-HEADS MOTIFS FLEXIBLE DOCKED INTO TARANTULA THICK FILAMENT 3D-MAP ALLOWS IN DEPTH STUDY OF INTRA- AND INTERMOLECULAR INTERACTIONS
Components
  • MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
  • MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
  • MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
KeywordsCONTRACTILE PROTEIN / MYOSIN INTERACTING-HEADS MOTIF / CRYO-EM / THICK FILAMENT / FLEXIBLE DOCKING / SINGLE PARTICLE RECONSTRUCTION / ITERATIVE HELICAL REAL SPACE RECONSTRUCTION (IHRSR) / INTER- AND INTRAMOLECULAR INTERACTIONS / MYOSIN REGULATION / SUPER-RELAXATION / STRIATED MUSCLE / TARANTULA
Function / homologyP-loop containing nucleoside triphosphate hydrolase / Myosin S1 fragment, N-terminal / Myosin N-terminal SH3-like domain / Myosin tail / Myosin head (motor domain) / Kinesin motor domain superfamily / Myosin light chain alkali / EF-hand calcium-binding domain. / Myosin IQ motif-containing domain superfamily / EF-Hand 1, calcium-binding site ...P-loop containing nucleoside triphosphate hydrolase / Myosin S1 fragment, N-terminal / Myosin N-terminal SH3-like domain / Myosin tail / Myosin head (motor domain) / Kinesin motor domain superfamily / Myosin light chain alkali / EF-hand calcium-binding domain. / Myosin IQ motif-containing domain superfamily / EF-Hand 1, calcium-binding site / EF-hand domain pair / Myosin, N-terminal, SH3-like / EF-hand domain pair / Myosin tail / EF-hand domain / Myosin head, motor domain / IQ motif, EF-hand binding site / IQ motif profile. / EF-hand calcium-binding domain profile. / Myosin motor domain profile. / Myosin N-terminal SH3-like domain profile. / EF-hand domain / myosin complex / microtubule motor activity / microtubule-based movement / actin filament binding / microtubule binding / calcium ion binding / ATP binding / Myosin 2 heavy chain striated muscle / Myosin 2 essential light chain striated muscle / Myosin 2 regulatory light chain striated muscle
Function and homology information
Specimen sourceAphonopelma (spider)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / 20 Å resolution
AuthorsAlamo, L. / Qi, D. / Wriggers, W. / Pinto, A. / Zhu, J. / Bilbao, A. / Gillilan, R.E. / Hu, S. / Padron, R.
CitationJournal: BMC Genomics / Year: 2009
Title: Analysis of tarantula skeletal muscle protein sequences and identification of transcriptional isoforms.
Authors: Jingui Zhu / Yongqiao Sun / Fa-Qing Zhao / Jun Yu / Roger Craig / Songnian Hu
Abstract: BACKGROUND: Tarantula has been used as a model system for studying skeletal muscle structure and function, yet data on the genes expressed in tarantula muscle are lacking.RESULTS: We constructed a ...BACKGROUND: Tarantula has been used as a model system for studying skeletal muscle structure and function, yet data on the genes expressed in tarantula muscle are lacking.
RESULTS: We constructed a cDNA library from Aphonopelma sp. (Tarantula) skeletal muscle and got 2507 high-quality 5'ESTs (expressed sequence tags) from randomly picked clones. EST analysis showed 305 unigenes, among which 81 had more than 2 ESTs. Twenty abundant unigenes had matches to skeletal muscle-related genes including actin, myosin, tropomyosin, troponin-I, T and C, paramyosin, muscle LIM protein, muscle protein 20, a-actinin and tandem Ig/Fn motifs (found in giant sarcomere-related proteins). Matches to myosin light chain kinase and calponin were also identified. These results support the existence of both actin-linked and myosin-linked regulation in tarantula skeletal muscle. We have predicted full-length as well as partial cDNA sequences both experimentally and computationally for myosin heavy and light chains, actin, tropomyosin, and troponin-I, T and C, and have deduced the putative peptides. A preliminary analysis of the structural and functional properties was also carried out. Sequence similarities suggested multiple isoforms of most myofibrillar proteins, supporting the generality of multiple isoforms known from previous muscle sequence studies. This may be related to a mix of muscle fiber types.
CONCLUSION: The present study serves as a basis for defining the transcriptome of tarantula skeletal muscle, for future in vitro expression of tarantula proteins, and for interpreting structural and functional observations in this model species.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Sep 1, 2015 / Release: Mar 9, 2016
RevisionDateData content typeGroupProviderType
1.0Mar 9, 2016Structure modelrepositoryInitial release
1.1Dec 14, 2016Structure modelDatabase references

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Assembly

Deposited unit
A: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
B: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
C: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
D: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
E: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
F: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
G: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
H: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
I: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
J: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
K: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
L: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE


Theoretical massNumber of molelcules
Total (without water)1,055,85912
Polyers1,055,85912
Non-polymers00
Water0
1
A: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
B: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
C: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
D: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
E: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
F: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
G: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
H: MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
I: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
J: MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE
K: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
L: MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE
x 20


Theoretical massNumber of molelcules
Total (without water)21,117,173240
Polyers21,117,173240
Non-polymers00
Water0
TypeNameSymmetry operationNumber
helical symmetry operation20
2


  • idetical with deposited unit in distinct coordinate
  • helical asymmetric unit
TypeNameSymmetry operationNumber
helical symmetry operation1
3


  • idetical with deposited unit in distinct coordinate
  • helical asymmetric unit, std helical frame
TypeNameSymmetry operationNumber
transform to helical frame1
Helical symmetryCircular symmetry: 4 / Dyad axis: no / N subunits divisor: 1 / Number of operations: 1 / Rise per n subunits: 100 Å / Rotation per n subunits: 30 deg.

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Components

#1: Protein/peptide
MYOSIN 2 HEAVY CHAIN STRIATED MUSCLE


Mass: 224542.281 Da / Num. of mol.: 4 / Fragment: SUBFRAGMENT 1 (S1) AND SUBFRAGMENT 2 (S2) / Source: (natural) Aphonopelma (spider) / Tissue: LEG MUSCLE / References: UniProt: A0A140UGH3*PLUS
#2: Protein/peptide
MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE


Mass: 17628.104 Da / Num. of mol.: 4 / Fragment: ESSENTIAL LIGHT CHAIN (ELC) / Source: (natural) Aphonopelma (spider) / Tissue: LEG MUSCLE / References: UniProt: A0A140UGH4*PLUS
#3: Protein/peptide
MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE


Mass: 21794.281 Da / Num. of mol.: 4 / Fragment: REGULATORY LIGHT CHAIN (RLC) / Source: (natural) Aphonopelma (spider) / Tissue: LEG MUSCLE / References: UniProt: A0A140UGH5*PLUS

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / Reconstruction method: helical reconstruction

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Sample preparation

Component
IDNameTypeDetailsParent ID
1Myosin filaments from Tarantula striated muscleCOMPLEXPolymer of a multiple myosin assembled over a paramyosin core0
2MYOSIN II1
Buffer solutionName: 100mM NaCl, 3mM MgCl2, 1mM EGTA, 5mM PIPES, 5mM NaH2PO4, 1mM NaN3
Details: 100mM NaCl, 3mM MgCl2, 1mM EGTA, 5mM PIPES, 5mM NaH2PO4, 1mM NaN3
pH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON GRIDS.
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temp: 93 K / Humidity: 80 %
Details: Blotting was performed from one side of the grid till a thin sample film on it using Whatman No 42 filter paper, then the grid was immediately plunged under gravity into liquid ethane cooled by liquid nitrogen. Grids were stored under liquid nitrogen.
Method: Blotting was performed from one side of the grid till a thin sample film on it using Whatman No 42 filter paper, then the grid was immediately plunged under gravity into liquid ethane cooled by liquid nitrogen. Grids were stored under liquid nitrogen.

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Electron microscopy imaging

MicroscopyMicroscope model: FEI/PHILIPS CM120T / Date: Oct 23, 2002 / Details: low dose
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 35000 / Calibrated magnification: 35000 / Nominal defocus max: 1950 nm / Nominal defocus min: 1950 nm / Cs: 2 mm
Specimen holderTemperature: 88 kelvins / Temperature (max): 90 kelvins / Temperature (min): 88 kelvins
Image recordingFilm or detector model: KODAK SO-163 FILM
Image scansNumber digital images: 1008
RadiationDiffraction protocol: SINGLE WAVELENGTH / Monochromatic or laue m l: M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1Situs2.3model fitting
2SPIDER3D reconstruction
Helical symmertyAngular rotation/subunit: 30 deg. / Axial rise/subunit: 100 Å / Axial symmetry: C4
3D reconstructionMethod: Single particle reconstruction with a modification of the IHRSR method
Resolution: 20 Å / Resolution method: FSC 0.5 CUT-OFF / Nominal pixel size: 2.48 / Actual pixel size: 2.48
Details: Three-dimensional single particle reconstruction was carried out by a modification of the IHRSR method, using SPIDER. Low-dose electron micrographs of 1008 frozen-hydrated thick filaments halves ere digitized at 0.248 nm per pixel using a Nikon Super Coolscan 8000 ED scanner. Filaments were aligned with the bare zone at the top, to ensure correct polarity in subsequent steps. A total of 15,504 segments, each 62 nm long, with an overlap of 55.8 nm, and containing aprox. 40,000 unique pairs of interacting myosin heads went into the reconstruction. As an initial reference model we used the tarantula negatively stained 3D-map, which was axially rotated, axially shifted and also out of plane tilted up to plus-minus12deg. for projection matching, giving a total of 4,095 projections (13 tilted projections plus-minus12deg. every 2deg., 45 reference rotated projections (0-90 degrees, 2deg. rotation angle), and 7 image axial shifts of 2.2 nm. The resulting 3D-map combines about 10,700 out of 15,504 filament segments, a yield of 69 percent of included segments. There are 4 helices of myosin heads, rotated 30 degrees, every 145 Angstroms. The filament segments were selected based on visual judgement of good helical order.
Symmetry type: HELICAL
Atomic model buildingDetails: METHOD--FLEXIBLE FITTING DETAILS--Protocol- Flexible Fitting. The flexible docking procedure is based on a connected (motion capture) network of identified features within the atomic model. The atomic model is allowed to move according to displacements tracked by 31 control points defined by the network, in order to find the best match to the cryo-EM map
Ref protocol: FLEXIBLE FIT / Ref space: REAL / Target criteria: Correlation
Atomic model buildingPDB-ID: 3DTP
Number of atoms included #LASTProtein: 41968 / Nucleic acid: 0 / Ligand: 0 / Solvent: 0 / Total: 41968

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