MYOFIBRILLAR STRUCTURE OF STRIATED MUSCLE

一个特殊的机会就在眼前， 分析肌肉中的运动分子，以阐明它们的作用， 在平衡的舞蹈动作中， 使整个肌肉充满活力的组件。 优次晶 为了让昆虫飞行肌肉（IFM）从巨大的水虫 Lethocerus给独特的X射线衍射（X射线）为每个 生理状态，并给出更清晰的电子显微镜薄片 (EM)比其他任何肌肉的肌球蛋白桥的图像。 肌球蛋白动力冲程的毫秒定时快照将 将机械数据与结构数据集成，协调EM 3D 用时间切片从快速冷冻的纤维重建（3-D） 同步辐射X射线衍射 新的三维方法提供了有力的 用于分析收缩肌肉中的横桥多样性的工具。 3-D EM断层扫描不仅支持图像平均， 识别非平均交叉桥结构，以及三维 用对应分析法对立交桥进行分类。 3-D 整个单位单元可以模拟全三维傅立叶以检查保真度 通过与自然X射线强度的比较， 通过拟合肌球蛋白头原子， 模型转换为3 - 4 nm分辨率的3-D，以查看（例如）如果电机动作 涉及轻链结构域双臂的4 - 12 nm摆动。 IFM的等长收缩可以通过拉伸触发1 - 2秒- 激活，或更长和更强的[Ca2 +]。 EM和X射线 两种活动状态之间的比较（时间分辨率10 - 50 ms） 并完成放松和僵硬的控制稳态。 同步动力冲程的时间分辨快照将 通过扰动具有突变长度的等距收缩求出 改变步骤。 长度步长引起短暂的2 - 10 ms响应， 力和结构，表明同步跨桥行动。 强约束的横桥的协调变形也将 寻求快速拉伸IFM在严谨，ADP和AMPPNP状态， 在那里，较慢的关闭率和规则的天桥结构应该有利于 在形状和角度上的一致和明显的变化。 过渡 到严谨的过桥形式，被认为是权力的最后阶段， 中风，将通过在快速扫描期间以0.2秒的间隔冷冻成像 ATP去除后的僵硬，以及沿着纤维的中途快速- 一端僵硬后冻结，另一端停留 在ATP缓冲液中松弛或激活。 早期~预力~阶段 将通过X射线和三维EM完全 分离（MgATP松弛）和一些每周连接的横桥 状态（乙二醇-AMPPNP冷或与Ca2 +; ADP-A1F4（-）与 Ca2+）

An exceptional opportunity is at hand for time-resolved structural analysis of motor molecules in muscle, to clarify their action as individual motors as well as in the balanced choreographic assemblies that animate whole muscles. Unexcelled paracrystalline order lets insect flight muscle (IFM) from the giant waterbug Lethocerus give distinctive X-ray diffraction (X-ray) for each physiological state, and give clearer thin-section electron microscope (EM) images of myosin crossbridges than any other muscle. Millisecond-timed snapshots of the myosin power stroke will integrate mechanical with structural data, coordinating EM 3D reconstructions (3-Ds) from quick-frozen fibers with time-slicing synchrotron X-ray diffraction. New methods for 3-Ds offer potent tools for analyzing crossbridge diversity in contracting muscle. 3-D EM tomography supports not only image averaging, but identification of non-averaged crossbridge structures, and 3-D classification of crossbridges by correspondence analysis. 3-Ds of the whole unit cell can simulate full 3-D Fouriers to check fidelity of thin-section Ems by comparison with native X-ray intensities. Crossbridge strain will be assessed by fitting myosin head atomic models into 3-Ds of 3-4 nm resolution, to see (e.g.) If motor action involves a 4-12 nm swing by the light-chain-domain lever-arm. Isometric contractions in IFM can be triggered for 1-2 s by stretch- activation, or longer and stronger by higher [Ca2+]. EM and X-ray comparisons between both active states (time resolution 10-50 ms) and control steady states of relaxed and rigor will be finished. Time-resolved snapshots of synchronized powerstrokes will be sought by perturbing isometric contractions with abrupt length change steps. Length steps elicit transient 2-10 ms responses in force and structure that indicate synchronized crossbridge action. Concerted deformation of strongly bound crossbridges will also be sought by quick-stretching IFM in rigor, ADP and AMPPNP states, where slower off-rates and regular crossbridge structure should favor uniform and conspicuous changes in shape and angle. The transition to rigor crossbridge form, considered the end-stage of the power stroke, will be imaged by freezing at 0.2s intervals during rapid rigorization after ATP removal, and also midway along fibers quick- frozen after being rigorized at one end while the other end stays relaxed or activated in an ATP buffer. Early ~pre-force~ stages in the crossbridge cycle will be sought by X-ray and 3-D EM of fully detached (MgATP-relaxed) and some weekly attached crossbridge states (glycol-AMPPNP cold or with Ca2+; ADP-A1F4(-) with Ca2+)