Centro de Biología Estructural, Instituto Venezolano de Investigaciones Científicas-IVIC, Apdo. 20632, Caracas 1020A, Venezuela.
J Struct Biol. 2012 Dec;180(3):469-78. doi: 10.1016/j.jsb.2012.08.010. Epub 2012 Sep 7.
Electron microscopy (EM) studies of 2D crystals of smooth muscle myosin molecules have shown that in the inactive state the two heads of a myosin molecule interact asymmetrically forming a myosin interacting-heads motif. This suggested that inactivation of the two heads occurs by blocking of the actin-binding site of one (free head) and the ATP hydrolysis site of the other (blocked head). This motif has been found by EM of isolated negatively stained myosin molecules of unregulated (vertebrate skeletal and cardiac muscle) and regulated (invertebrate striated and vertebrate smooth muscle) myosins, and nonmuscle myosin. The same motif has also been found in 3D-reconstructions of frozen-hydrated (tarantula, Limulus, scallop) and negatively stained (scallop, vertebrate cardiac) isolated thick filaments. We are carrying out studies of isolated thick filaments from other species to assess how general this myosin interacting-heads motif is. Here, using EM, we have visualized isolated, negatively stained thick filaments from scorpion striated muscle. We modified the iterative helical real space reconstruction (IHRSR) method to include filament tilt, and band-pass filtered the aligned segments before averaging, achieving a 3.3 nm resolution 3D-reconstruction. This reconstruction revealed the presence of the myosin interacting-heads motif (adding to evidence that is widely spread), together with 12 subfilaments in the filament backbone. This demonstrates that conventional negative staining and imaging can be used to detect the presence of the myosin interacting-heads motif in helically ordered thick filaments from different species and muscle types, thus avoiding the use of less accessible cryo-EM and low electron-dose procedures.
电子显微镜（EM）研究二维晶体平滑肌肌球蛋白分子表明，在非活跃状态下，肌球蛋白分子的两个头部不对称地相互作用，形成肌球蛋白相互作用头部模体。这表明两个头部的失活是通过阻止一个（自由头部）的肌动蛋白结合位点和另一个（阻塞头部）的 ATP 水解位点来实现的。这个模体已经通过 EM 观察到了分离的未调节（脊椎动物骨骼肌和心肌）和调节（无脊椎动物横纹肌和脊椎动物平滑肌）肌球蛋白以及非肌肉肌球蛋白的负染肌球蛋白分子。同样的模体也在冷冻水合（狼蛛、鲎、扇贝）和负染（扇贝、脊椎动物心脏）分离的粗丝的 3D 重建中被发现。我们正在对其他物种的分离粗丝进行研究，以评估这种肌球蛋白相互作用头部模体的普遍性。在这里，我们使用 EM 可视化了来自蝎子横纹肌的分离的、负染的粗丝。我们修改了迭代螺旋实空间重建（IHRSR）方法，包括细丝倾斜，并在平均之前对对齐的片段进行带通滤波，实现了 3.3nm 分辨率的 3D 重建。该重建揭示了肌球蛋白相互作用头部模体的存在（增加了广泛传播的证据），以及细丝骨架中的 12 个亚丝。这表明，常规的负染和成像可以用于检测来自不同物种和肌肉类型的螺旋有序粗丝中肌球蛋白相互作用头部模体的存在，从而避免使用不太容易获得的冷冻 EM 和低电子剂量的方法。
