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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
DescriptorMYOSIN 2 HEAVY CHAIN STRIATED MUSCLE
MYOSIN 2 ESSENTIAL LIGHT 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
Specimen sourceAphonopelma / arthropod / tarantula
MethodElectron microscopy (20 Å resolution / Filament / Helical)
AuthorsAlamo, L. / Qi, D. / Wriggers, W. / Pinto, A. / Zhu, J. / Bilbao, A. / Gillilan, R.E. / Hu, S. / Padron, R.
CitationJ. Mol. Biol., 2016, 428, 1142-1164

primary. J. Mol. Biol., 2016, 428, 1142-1164 Yorodumi Papers
Conserved Intramolecular Interactions Maintain Myosin Interacting-Heads Motifs Explaining Tarantula Muscle Super-Relaxed State Structural Basis.
Lorenzo Alamo / Dan Qi / Willy Wriggers / Antonio Pinto / Jingui Zhu / Aivett Bilbao / Richard E Gillilan / Songnian Hu / Raúl Padrón

#1. J.Mol.Biol., 2008, 384, 780-797 Yorodumi Papers
Three-dimensional reconstruction of tarantula myosin filaments suggests how phosphorylation may regulate myosin activity.
Alamo, L. / Wriggers, W. / Pinto, A. / Bartoli, F. / Salazar, L. / Zhao, F.Q. / Craig, R. / Padron, R.

#2. BMC Genomics, 2009, 10, 117-117 Yorodumi Papers
Analysis of tarantula skeletal muscle protein sequences and identification of transcriptional isoforms.
Zhu, J. / Sun, Y. / Zhao, F.Q. / Yu, J. / Craig, R. / Hu, S.

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

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Components

#1: Polypeptide(L)
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 / arthropod

Cellular component

Molecular function

#2: Polypeptide(L)
MYOSIN 2 ESSENTIAL LIGHT CHAIN STRIATED MUSCLE


Mass: 17628.104 Da / Num. of mol.: 4 / Fragment: ESSENTIAL LIGHT CHAIN (ELC) / Source: (natural) Aphonopelma / arthropod

Molecular function

#3: Polypeptide(L)
MYOSIN 2 REGULATORY LIGHT CHAIN STRIATED MUSCLE


Mass: 21794.281 Da / Num. of mol.: 4 / Fragment: REGULATORY LIGHT CHAIN (RLC) / Source: (natural) Aphonopelma / arthropod

Molecular function

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / Reconstruction method: HELICAL

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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 FIELD / 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
2SPIDERRECONSTRUCTION
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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