Cryo-EM Structure of Actin Filaments from Zea mays Pollen

Actins are among the most abundant and conserved proteins in eukaryotic cells, where they form filamentous structures that perform vital roles in key cellular processes. Although large amounts of data on the biochemical activities, dynamic behaviors, and important cellular functions of plant actin filaments have accumulated, their structural basis is elusive. Here, we report a 3.9 Å structure of the plant actin filament (ZMPA) from Zea mays pollen using cryo-electron microscopy. The structure shows a right-handed, double-stranded (two strands running parallel to each other) and staggered architecture that is stabilized by intra- and interstrand interactions. While the overall structure resembles that of other actin filaments, its DNase I-binding loop (D-loop) bends further outward, adopting an open conformation similar to that of the jasplakinolide- or Beryllium fluoride (BeFx)-stabilized rabbit skeleton muscle actin (RSMA) filament. Single-molecule magnetic tweezer analysis revealed that the ZMPA filament can resist a greater stretching force than the RSMA filament. Overall, these data provide evidence that plant actin filaments have greater stability than animal actin filaments, which is important for their roles as tracks for long-distance vesicle and organelle transportation.

Methods
The structural data (Supplemental Movies 1 and 2) were from the structure of Zea mays pollen actin filaments that we determined by cryo-electron microscopy. These movies were made by Chimera software. The stretching data (Supplemental Movies 3, 4, 5 and 6) were from the single-molecular magnetic tweezer analysis of actin filaments in our study. These movies were made by Wondershare and MATLAB.

肌动蛋白（Actins）是真核细胞中丰度最高且最为保守的蛋白质之一，可聚合形成丝状结构，在关键细胞进程中发挥至关重要的作用。尽管针对植物肌动蛋白丝的生化活性、动态行为与重要细胞功能已积累了海量研究数据，但其结构基础仍不甚明晰。本研究报道了利用冷冻电镜（cryo-electron microscopy）解析的玉米（Zea mays）花粉来源植物肌动蛋白丝（ZMPA）的3.9 Å分辨率结构。该结构呈现右手螺旋双链架构：两条链彼此平行排布且相互交错，通过链内与链间相互作用实现稳定。尽管整体结构与其他肌动蛋白丝相似，但其DNA酶I结合环（DNase I-binding loop）向外弯折程度更大，呈现出类似经jasplakinolide或氟化铍（beryllium fluoride, BeFx）稳定的兔骨骼肌肌动蛋白（rabbit skeleton muscle actin, RSMA）丝的开放构象。单分子磁镊分析（single-molecule magnetic tweezer analysis）结果显示，植物肌动蛋白丝（ZMPA）相较于兔骨骼肌肌动蛋白（RSMA）丝可承受更大的拉伸力。综上，这些数据表明植物肌动蛋白丝比动物肌动蛋白丝具有更高的稳定性，这对于其作为长距离囊泡与细胞器运输轨道的功能至关重要。

实验方法
本研究的结构数据（补充视频1与2）来自我们通过冷冻电镜解析的玉米花粉肌动蛋白丝结构，这些视频由Chimera软件制作完成。拉伸实验相关数据（补充视频3、4、5与6）来自本研究中肌动蛋白丝的单分子磁镊分析，相关视频由Wondershare与MATLAB软件制作完成。