REL OBS PDBe PDBe PDBe 2008-07-09 2008-07-10 2009-04-01 2013-01-23 2013-01-30 EMD-1950 Alamo L Wriggers W Pinto A Bartoli F Salazar L Zhao F Craig R Padron R cryo-EM, thick filament, flexible docking, single particle reconstruction, Iterative Helical Real Space Reconstruction (IHRSR), Myosin regulation, myosin regulatory light chain, phosphorylation Alamo L Wriggers W Pinto A Bartoli F Salazar L Zhao FQ Craig R Padron R J.MOL.BIOL. 384 780 797 2008 18951904 doi:10.1016/j.jmb.2008.10.013 3dtp FULLOVERLAP Myosin filaments from Tarantula striated muscle Myosin filaments from Tarantula striated muscle Polymer of a multiple myosin assembled over a paramyosin core 2 Myosin II Avicularia avicularia pinktoe tarantula Muscle Myofibrils Sarcomere polymer false helical filament 7.0

100mM NaCl,3mM MgCl2,1mM EGTA, 5mM PIPES, 5mM NaH2PO4,1mM NaN3.

NEGATIVE

A 6 ul aliquot of native purified tarantula thick filaments suspension (Hidalgo et al. 2001) was applied to a 400 mesh grid coated with a holey carbon film that had been rendered hydrophilic by glow discharge in n-amylamine vapor for 3 minutes before use. After allowing the filaments to adsorb to the grid for 30 seconds, the grid was rinsed with the relaxing rinse, then placed in a humidity chamber (aprox. 80% relative humidity). 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.

Holey carbon grids 400 mesh

ETHANE 80 93 HOMEMADE PLUNGER

Vitrification instrument: Home-made plunger. 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.

Plunging in a liquid ethane FEI/PHILIPS CM120T FLOOD BEAM BRIGHT FIELD LAB6 120 2.0 1.95 1.95 35000.0 35000.0 GATAN LIQUID NITROGEN 88 90

Holey carbon grids Cryo preserved in Liquid ethane were observed in a Philips CM120 electron microscope under low dose conditions. Only filaments on thin carbon over holes were photographed

2002-10-23 KODAK SO-163 FILM OTHER 8.47 1008 14.0 Eucentric

There are 4 helices of myosin heads, rotated 30 degres, every 145 Angstroms. The filament segments were selected based on visual judgment of good helical order

100 30 C12 OTHER 20.0 FSC 0.5 CUT-OFF SPIDER

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.

1XYZ FLEXIBLE FIT Situs 2.3

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

Correlation REAL

emd_1535.tif

emd_1535_1.tif