cryoEM Administrative Supplement for Equipment

摘要 从Direct购买革命性的新相机Apollo需要资金 这将使收集目前获得的大量图像数据成为可能 以“电影”的形式。收集大量数据的能力将拓宽 国际和平研究所R35拨款中可解决的问题目前侧重于获得原子解析 来自横纹肌的粗丝图像。一般来说，粗丝来自所有的条纹 肌肉有四个结构不同的区域：(1)中央有一个160纳米长的裸露区域， 肌球蛋白的尾巴相互平行排列，(2)A带出现在肌球蛋白的位置 分子以螺旋或准螺旋结构排列，肌球蛋白尾部被包裹在 平行排列，(3)锥形末端，在此连接细丝，例如TiN，连接 粗丝至Z盘，以及(4)无脊椎动物粗丝A带特征 尤其是脊椎动物，它们没有遵循肌球蛋白分子的排列。这个 肌球蛋白在无脊椎动物粗丝A带中的螺旋排列有助于原子 使用所有肌球蛋白轴向重复序列的肌球蛋白尾部和头部的结构 从裸露区域的末端到锥形末端的起点。然而，赤裸裸的区域， 从裸区过渡到螺旋排列，锥形末端以及附加特征 不遵循肌球蛋白螺旋对称性的蛋白在厚壁蛋白的组装中起着重要的作用 细丝。特征，如无脊椎动物粗丝的副肌球蛋白不符合 肌球蛋白在A带中呈螺旋对称性，在低温电子显微镜重建中表现不佳。 副肌球蛋白与其他非肌球蛋白结合可能起决定厚度的作用 无脊椎动物的细丝长度。肌球蛋白、肌球蛋白结合蛋白C和其他非肌球蛋白 脊椎动物的粗丝也不会沿着整个A带遵循规律的图案。 获得能够在一秒钟内录制60帧电影的速度快得多的摄像机， 将提供获取这些功能的高分辨率3-D图像所需的工具 在通过强制螺旋对称解决的结构中，即使有，也表现得很好。

ABSTRACT Funds are requested for the purchase of a revolutionary new camera, the Apollo, from Direct Electron Ltd. that would enable the collection of large amounts of image data, currently obtained in the form of “movies”. The ability to collect large amounts of data would broaden the types of questions addressable in the PI's R35 grant currently focused on obtaining atomic resolution images of thick filaments from striated muscle. Generally, thick filaments from all striated muscles have four structurally distinct regions: (1) a 160 nm long bare zone in the center where myosin tails are packed antiparallel to each other, (2) an A-band follows where myosin molecules are arranged in a helical or quasi helical structure with the myosin tails packed in a parallel arrangement, (3) a tapered end where the connecting filament, e.g. titin, connects the thick filament to the Z-disk, and (4) features along the thick filament A-band from invertebrates in particular, but also from vertebrates, that fail to follow the myosin molecule arrangement. The helical arrangement of myosin in the A-band of invertebrate thick filaments facilitates an atomic structure of the myosin tail and head arrangement by using all of the myosin axial repeats from the end of the bare zone to the beginning of the tapered ends. However, the bare zone, the transition from bare zone to helical arrangement, the tapered ends as well as additional features that do not follow the myosin helical symmetry perform important roles in the assembly of thick filaments. Features, such as the paramyosin of invertebrate thick filaments fail to follow the myosin helical symmetry in the A-band and are poorly represented in cryoEM reconstructions. Paramyosin in concert with other non-myosin proteins may function to determine the thick filament length in invertebrates. Titin, myosin binding protein C and other non-myosin proteins of vertebrate thick filaments also do not follow a regular pattern along the entire A-band. Acquisition of a much faster camera capable of recording a 60-frame movie in a single second, will provide the tool needed to obtain high resolution 3-D images of these features that are not well represented if at all in structures solved by enforcing helical symmetry.