Three-dimensional structure of the myosin V inhibited state by cryoelectron tomography

Unconventional myosin V (myoV) is an actin-based molecular motor that has a key function in organelle and mRNA transport, as well as in membrane trafficking. MyoV was the first member of the myosin superfamily shown to be processive, meaning that a single motor protein can 'walk' hand-over-hand along an actin filament for many steps before detaching. Full-length myoV has a low actin-activated MgATPase activity at low [Ca2+], whereas expressed constructs lacking the cargo-binding domain have a high activity regardless of [Ca2+] (refs 5-7). Hydrodynamic data and electron micrographs indicate that the active state is extended, whereas the inactive state is compact. Here we show the first three-dimensional structure of the myoV inactive state. Each myoV molecule consists of two heads that contain an amino-terminal motor domain followed by a lever arm that binds six calmodulins. The heads are followed by a coiled-coil dimerization domain (S2) and a carboxy-terminal globular cargo-binding domain. In the inactive structure, bending of myoV at the head-S2 junction places the cargo-binding domain near the motor domain's ATP-binding pocket, indicating that ATPase inhibition might occur through decreased rates of nucleotide exchange. The actin-binding interfaces are unobstructed, and the lever arm is oriented in a position typical of strong actin-binding states. This structure indicates that motor recycling after cargo delivery might occur through transport on actively treadmilling actin filaments rather than by diffusion.     

Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization

The structural change that generates force and motion in actomyosin motility has been proposed to be tilting of the myosin light chain domain, which serves as a lever arm. Several experimental approaches have provided support for the lever arm hypothesis; however, the extent and timing of tilting motions are not well defined in the motor protein complex of functioning actomyosin. Here we report three-dimensional measurements of the structural dynamics of the light chain domain of brain myosin V using a single-molecule fluorescence polarization technique that determines the orientation of individual protein domains with 20-40-ms time resolution. Single fluorescent calmodulin light chains tilted back and forth between two well-defined angles as the myosin molecule processively translocated along actin. The results provide evidence for lever arm rotation of the calmodulin-binding domain in myosin V, and support a 'hand-over-hand' mechanism for the translocation of double-headed myosin V molecules along actin filaments. The technique is applicable to the study of real-time structural changes in other biological systems.     

Molecular engineering of a backwards-moving myosin motor

All members of the diverse myosin superfamily have a highly conserved globular motor domain that contains the actin- and nucleotide-binding sites and produces force and movement. The light-chain-binding domain connects the motor domain to a variety of functionally specialized tail domains and amplifies small structural changes in the motor domain through rotation of a lever arm. Myosins move on polarized actin filaments either forwards to the barbed (+) or backwards to the pointed (-) end. Here, we describe the engineering of an artificial backwards-moving myosin from three pre-existing molecular building blocks. These blocks are: a forward-moving class I myosin motor domain, a directional inverter formed by a four-helix bundle segment of human guanylate-binding protein-1 and an artificial lever arm formed by two alpha-actinin repeats. Our results prove that reverse-direction movement of myosins can be achieved simply by rotating the direction of the lever arm 180 degrees.     

A structural change in the kinesin motor protein that drives motility

Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of an approximately 15-amino-acid region (the neck linker) in kinesin using electron paramagnetic resonance, fluorescence resonance energy transfer, pre-steady state kinetics and cryo-electron microscopy. This region becomes immobilized and extended towards the microtubule 'plus' end when kinesin binds microtubules and ATP, and reverts to a more mobile conformation when gamma-phosphate is released after nucleotide hydrolysis. This conformational change explains both the direction of kinesin motion and processive movement by the kinesin dimer.     

The core of the motor domain determines the direction of myosin movement

Myosins constitute a superfamily of at least 18 known classes of molecular motors that move along actin filaments. Myosins move towards the plus end of F-actin filaments; however, it was shown recently that a certain class of myosin, class VI myosin, moves towards the opposite end of F-actin, that is, in the minus direction. As there is a large, unique insertion in the myosin VI head domain between the motor domain and the light-chain-binding domain (the lever arm), it was thought that this insertion alters the angle of the lever-arm switch movement, thereby changing the direction of motility. Here we determine the direction of motility of chimaeric myosins that comprise the motor domain and the lever-arm domain (containing an insert) from myosins that have movement in the opposite direction. The results show that the motor core domain, but neither the large insert nor the converter domain, determines the direction of myosin motility.     

Atomic model of a myosin filament in the relaxed state

Contraction of muscle involves the cyclic interaction of myosin heads on the thick filaments with actin subunits in the thin filaments. Muscles relax when this interaction is blocked by molecular switches on either or both filaments. Insight into the relaxed (switched OFF) structure of myosin has come from electron microscopic studies of smooth muscle myosin molecules, which are regulated by phosphorylation. These studies suggest that the OFF state is achieved by an asymmetric, intramolecular interaction between the actin-binding region of one head and the converter region of the other, switching both heads off. Although this is a plausible model for relaxation based on isolated myosin molecules, it does not reveal whether this structure is present in native myosin filaments. Here we analyse the structure of a phosphorylation-regulated striated muscle thick filament using cryo-electron microscopy. Three-dimensional reconstruction and atomic fitting studies suggest that the 'interacting-head' structure is also present in the filament, and that it may underlie the relaxed state of thick filaments in both smooth and myosin-regulated striated muscles over a wide range of species.     

The structure of the myosin VI motor reveals the mechanism of directionality reversal

Here we solve a 2.4-A structure of a truncated version of the reverse-direction myosin motor, myosin VI, that contains the motor domain and binding sites for two calmodulin molecules. The structure reveals only minor differences in the motor domain from that in plus-end directed myosins, with the exception of two unique inserts. The first is near the nucleotide-binding pocket and alters the rates of nucleotide association and dissociation. The second unique insert forms an integral part of the myosin VI converter domain along with a calmodulin bound to a novel target motif within the insert. This serves to redirect the effective 'lever arm' of myosin VI, which includes a second calmodulin bound to an 'IQ motif', towards the pointed (minus) end of the actin filament. This repositioning largely accounts for the reverse directionality of this class of myosin motors. We propose a model incorporating a kinesin-like uncoupling/docking mechanism to provide a full explanation of the movements of myosin VI.     

Electron cryo-microscopy shows how strong binding of myosin to actin releases nucleotide

Muscle contraction involves the cyclic interaction of the myosin cross-bridges with the actin filament, which is coupled to steps in the hydrolysis of ATP. While bound to actin each cross-bridge undergoes a conformational change, often referred to as the "power stroke", which moves the actin filament past the myosin filaments; this is associated with the release of the products of ATP hydrolysis and a stronger binding of myosin to actin. The association of a new ATP molecule weakens the binding again, and the attached cross-bridge rapidly dissociates from actin. The nucleotide is then hydrolysed, the conformational change reverses, and the myosin cross-bridge reattaches to actin. X-ray crystallography has determined the structural basis of the power stroke, but it is still not clear why the binding of actin weakens that of the nucleotide and vice versa. Here we describe, by fitting atomic models of actin and the myosin cross-bridge into high-resolution electron cryo-microscopy three-dimensional reconstructions, the molecular basis of this linkage. The closing of the actin-binding cleft when actin binds is structurally coupled to the opening of the nucleotide-binding pocket.     

Video imaging of walking myosin V by high-speed atomic force microscopy

The dynamic behaviour of myosin V molecules translocating along actin filaments has been mainly studied by optical microscopy. The processive hand-over-hand movement coupled with hydrolysis of adenosine triphosphate was thereby demonstrated. However, the protein molecules themselves are invisible in the observations and have therefore been visualized by electron microscopy in the stationary states. The concomitant assessment of structure and dynamics has been unfeasible, a situation prevailing throughout biological research. Here we directly visualize myosin V molecules walking along actin tracks, using high-speed atomic force microscopy. The high-resolution movies not only provide corroborative 'visual evidence' for previously speculated or demonstrated molecular behaviours, including lever-arm swing, but also reveal more detailed behaviours of the molecules, leading to a comprehensive understanding of the motor mechanism. Our direct and dynamic high-resolution visualization is a powerful new approach to studying the structure and dynamics of biomolecules in action.     

Regulated degradation of a class V myosin receptor directs movement of the yeast vacuole

Normal cellular function requires that organelles be positioned in specific locations. The direction in which molecular motors move organelles is based in part on the polarity of microtubules and actin filaments. However, this alone does not determine the intracellular destination of organelles. For example, the yeast class V myosin, Myo2p, moves several organelles to distinct locations during the cell cycle. Thus the movement of each type of Myo2p cargo must be regulated uniquely. Here we report a regulatory mechanism that specifically provides directionality to vacuole movement. The vacuole-specific Myo2p receptor, Vac17p, has a key function in this process. Vac17p binds simultaneously to Myo2p and to Vac8p, a vacuolar membrane protein. The transport complex, Myo2p-Vac17p-Vac8p, moves the vacuole to the bud, and is then disrupted through the degradation of Vac17p. The vacuole is ultimately deposited near the centre of the bud. Removal of a PEST sequence (a potential signal for rapid protein degradation) within Vac17p causes its stabilization and the subsequent 'backward' movement of vacuoles, which mis-targets them to the neck between the mother cell and the bud. Thus the regulated disruption of this transport complex places the vacuole in its proper location. This may be a general mechanism whereby organelles are deposited at their terminal destination.     

直流电动机制动状态的Matlab仿真

以直流电动机的能耗制动和反接制动为例,利用Matlab仿真环境Simulink中的电力系统工具箱对电动机运行状态进行仿真.通过调节电路,根据仿真结果可以找到最佳运行参数.     

第二类Pфchl-Teller势场中相对论粒子任意l波束缚态解

在球坐标系中研究了具有离心项的第二类Pschl-Teller型标量势与矢量势的Klein-Gordon方程和Dirac方程.在标量势等于矢量势的条件下,运用合适的指数近似将具有离心项的径向Klein-Gordon方程和Dirac方程转化成超几何微分方程,从而获得了系统的任意l波Klein-Gordon方程和Dirac方程解析束缚态径向波函数.最后,我们对l=0这一特殊情况进行了简单讨论.     

基于DSP控制芯片的直流无刷电机控制系统研究

对直流无刷电动机控制系统进行了总体上的设计,并给出了基于Matlab的系统仿真结果,同时给出了以TMS320F240作为控制芯片的控制系统的软硬件实现方法.     

基于DSP控制芯片的直流无刷电机控制系统研究

对直流无刷电动机控制系统进行了总体上的设计,并给出了基于Matlab的系统仿真结果,同时给出了以TMS320F240作为控制芯片的控制系统的软硬件实现方法。     

基于电流解耦的异步电机V/F控制补偿方法

针对异步电机恒压频比(V/F)控制低速性能不理想的问题,提出了一种新颖的定子电阻压降的补偿方法.该方法在对定子电流进行解耦的基础上,根据异步电机低频运行时简化的等值电路和矢量图,采用一种基于力矩电流的标量补偿方法对定子电阻压降进行补偿,并结合转差频率补偿,以实现电机低频时的自动转矩提升.该方法有效改善了异步电机V/F控制时的低速性能,保证低频运行时依然能获得额定磁通和相应的转矩.基于Saber软件的仿真结果表明:采用补偿方法后,电机能够带额定负载稳定运行在2 Hz工况下,电机的带负载能力有明显的提高,补偿后的机械特性略有上扬.     

Measurement of the internal state of a single atom without energy exchange

A measurement necessarily changes the quantum state being measured, a phenomenon known as back-action. Real measurements, however, almost always cause a much stronger back-action than is required by the laws of quantum mechanics. Quantum non-demolition measurements have been devised that keep the additional back-action entirely within observables other than the one being measured. However, this back-action on other observables often imposes its own constraints. In particular, free-space optical detection methods for single atoms and ions (such as the shelving technique, a sensitive and well-developed method) inevitably require spontaneous scattering, even in the dispersive regime. This causes irreversible energy exchange (heating), which is a limitation in atom-based quantum information processing, where it obviates straightforward reuse of the qubit. No such energy exchange is required by quantum mechanics. Here we experimentally demonstrate optical detection of an atomic qubit with significantly less than one spontaneous scattering event. We measure the transmission and reflection of an optical cavity containing the atom. In addition to the qubit detection itself, we quantitatively measure how much spontaneous scattering has occurred. This allows us to relate the information gained to the amount of spontaneous emission, and we obtain a detection error below 10 per cent while scattering less than 0.2 photons on average. Furthermore, we perform a quantum Zeno-type experiment to quantify the measurement back-action, and find that every incident photon leads to an almost complete state collapse. Together, these results constitute a full experimental characterization of a quantum measurement in the 'energy exchange-free' regime below a single spontaneous emission event. Besides its fundamental interest, this approach could significantly simplify proposed neutral-atom quantum computation schemes, and may enable sensitive detection of molecules and atoms lacking closed transitions.     

无刷直流电机神经网络控制器设计

无刷直流电动机在高性能驱动应用中具有良好的调整性能、高电压、高转矩、高体积比以及无电刷的优点。为了辨识、控制运行在高性能驱动环境下的无刷直流电机,本文设计了一种多层神经网络控制器。这个神经网络既能适时辨识系统的动态性能,又能控制电机使速度和位置跟踪预先选择的轨迹,且跟踪精度较高。在控制中,系统参数漂移和外界干扰可在任意时刻得到补偿,神经网络实现了自适应控制功能,仿真结果表明这种神经网络是有效的。     

关于Ramsey数下界的一个注记

Ramsey理论是组合论中的一个重要内容，但确定Ramsey数R(k，f)是非常困难的．给出了Ramsey数R(k1，k2，…，km)的一个下界公式；同时也指出了2002年《数学的实践与认识》上某论文中的一些错误。     

点可区别全色数的一个界

图G的一个正常全染色被称作点可区别全染色,如果G中任意两个点的色集合不同,其中每个点的色集合包含该点及其关联边的颜色。在点可区别全色数界(χvt(G)≤|V(G)|+2)的基础上,应用概率的方法得到了阶数为n,且无孤立边的简单图G的点可区别全色数的一个较小上界。