Stewart M, Kensler R W
J Mol Biol. 1986 Dec 20;192(4):831-51. doi: 10.1016/0022-2836(86)90032-x.
The distribution of myosin heads on the surface of frog skeletal muscle thick filaments has been determined by computer processing of electron micrographs of isolated filaments stained with tannic acid and uranyl acetate. The heads are arranged in three strands but not in a strictly helical manner and so the structure has cylindrical symmetry. This accounts for the "forbidden" meridional reflections seen in diffraction patterns. Each layer-line therefore represents the sum of terms of Bessel orders 0, +/- 3, +/- 6, +/- 9 and so on. These terms interact so that, unlike a helical object without terms from overlapping Bessel orders, as the azimuth is changed, the amplitude on a layer-line at a particular radius varies substantially and its phase does not alter linearly. Consequently, a three-dimensional reconstruction cannot be produced from a single view. We have therefore used tilt series of three individual filaments to decompose the data on layer-lines 0 to 6 into terms of Bessel orders up to +/- 9 using a least-squares procedure. These data had a least-squares residual of 0.32 and enabled a three-dimensional reconstruction to be obtained at a nominal resolution of 6 nm. This showed, at a radius of about 10 nm, three strands of projecting morphological units with three units spaced along each strand every 42.9 nm axially. We have identified these units with pairs of myosin heads. Successive units along a strand are perturbed axially, azimuthally and radially from the positions expected if the structure was perfectly helical. This may simply be a consequence of steric restrictions in packing the heads on the thick filament surface, but could also reflect an underlying non-helical arrangement of myosin tails, which would be consistent with the thick filament shaft being constructed from three subfilaments in which the tails were arranged regularly. There was also material at a radius of about 6 nm spaced 42.9 nm axially, which we tentatively identified with accessory proteins. The filament shaft had a pronounced pattern of axial staining.
通过对用单宁酸和醋酸铀酰染色的分离细丝的电子显微照片进行计算机处理，确定了肌球蛋白头部在青蛙骨骼肌粗丝表面的分布。头部排列成三条链，但并非严格呈螺旋状，因此该结构具有圆柱对称性。这就解释了在衍射图案中看到的“禁阻”子午反射。因此，每条层线代表贝塞尔阶数0、±3、±6、±9等项的总和。这些项相互作用，所以与没有重叠贝塞尔阶数项的螺旋物体不同，随着方位角的变化，特定半径处层线上的振幅会有很大变化，并且其相位不会线性改变。因此，无法从单一视角生成三维重建图像。所以，我们使用了三根单独细丝的倾斜系列，通过最小二乘法程序将0至6层线的数据分解为高达±9的贝塞尔阶数项。这些数据的最小二乘残差为0.32，并使得能够以6纳米的标称分辨率获得三维重建图像。这显示在半径约10纳米处，有三条伸出的形态学单元链，每条链上沿轴向每42.9纳米有三个单元间隔排列。我们已将这些单元识别为成对的肌球蛋白头部。沿着一条链的连续单元在轴向、方位角和径向与如果结构是完美螺旋时预期的位置存在偏差。这可能仅仅是由于在粗丝表面包装头部时的空间限制导致的结果，但也可能反映了肌球蛋白尾部潜在的非螺旋排列，这与粗丝轴由三个亚丝构成且尾部规则排列的情况相符。在半径约6纳米处也有轴向间隔42.9纳米的物质，我们初步将其识别为辅助蛋白。细丝轴有明显的轴向染色图案。
