2013-01-30 OBS 2008-07-09 PDBe PDBe 2008-07-10 2009-04-01 2013-01-23 EMD-1950

A lack of exact 4-fold symmetry in our deposited map

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 3dtp Alamo L, Wriggers W, Pinto A, Bartoli F, Salazar L, Zhao FQ, Craig R, Padron R Three-dimensional reconstruction of tarantula myosin filaments suggests how phosphorylation may regulate myosin activity. J.MOL.BIOL. 384 780 797 2008 18951904 doi:10.1016/j.jmb.2008.10.013

emd_1535.tif

emd_1535_1.tif

2 Myosin filaments from Tarantula striated muscle Polymer of a multiple myosin assembled over a paramyosin core protein Myosin II Myosin Type II Avicularia avicularia false polymer pinktoe tarantula Myofibrils Muscle Sarcomere Plunging in a liquid ethane ETHANE

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.

80 HOMEMADE PLUNGER 93 LAB6 BRIGHT FIELD 1950 88 1950 FLOOD BEAM 90 Eucentric

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

KODAK SO-163 FILM 2.0 35000 FEI/PHILIPS CM120T 23-OCT-2002 GATAN LIQUID NITROGEN 120 35000 OTHER 1008 8.47 14 Correlation

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

Situs 2.3 flexible REAL 1XYZ sr12 100 30 RIGHT HANDED 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 filament 7.0

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

helical Single particle reconstruction with a modification of the IHRSR method 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.

20 FSC 0.5

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