MYOFIBRILLAR STRUCTURE OF STRIATED MUSCLE

一个绝佳的机会就在眼前，时间分辨的结构 分析肌肉中的运动分子，以阐明它们的作用 个人马达以及平衡编排 使整个肌肉充满活力的集合。无与伦比的准晶 命令让昆虫从巨大的水虫那里获得飞行肌肉(IFM) Lethocerus给出了每个人独特的X射线衍射(X光) 生理状态，并提供更清晰的薄片电子显微镜 (EM)肌球蛋白跨越任何其他肌肉的图像。 肌球蛋白力量中风的毫秒级快照将 将机械数据与结构数据相结合，协调EM 3D 基于时间切片的快速冷冻纤维重建(3D) 同步辐射X射线衍射仪。3D的新方法提供了强有力的 用于分析收缩肌肉中交叉桥多样性的工具。3-D EM断层扫描不仅支持图像平均，而且 非平均立交桥结构的识别和三维 应用对应分析对道口桥进行分类。3-D，共 整个单元格可以模拟完整的3-D傅里叶来检查保真度 通过与自然X射线强度进行比较，确定了薄层EMS的强度。 Crossbridge菌株将通过安装肌球蛋白头部原子进行评估 3-4纳米分辨率的3-D模型，以查看(例如)如果马达动作 涉及轻链结构域杠杆臂4-12 nm的摆动。 1-2岁S伸展可触发IFM等长收缩 高[Ca~(2+)]时激活时间更长、强度更强。电子显微镜和X射线 两种活动状态之间的比较(时间分辨率为10-50毫秒) 和控制的放松和严谨的稳定状态将完成。 同步电击的时间分辨快照将是 通过摄动具有突然长度的等长收缩来寻找 更改步骤。长度步长在以下时间内引发2-10毫秒的瞬时响应 指示同步交叉桥动作的力和结构。 强约束立交桥的协调变形也将 由快速拉伸IFM在Rigor、ADP和AMPPNP州寻求， 较慢的折扣率和规则的交叉桥结构应有利于 在形状和角度上均匀而明显的变化。过渡时期 以严谨的横桥形式，被认为是力量的末期 中风，将通过在快速期间以0.2s的间隔冻结来成像 去除ATP后的僵化，以及纤维的中途快速- 一端僵化后冻结，另一端保持不变 在ATP缓冲器中放松或激活。早期~力前~阶段 交叉桥周期将通过X光和3-D EM来寻找 分离(镁ATP-放松)和一些每周附加的交叉桥 状态(乙二醇-AMPPNP冷的或有钙的；ADP-A1F4(-)有 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+)