National Institutes of Health/Office of the Director
5DP5OD017885
米国
引用
ジャーナル: Nat Nanotechnol / 年: 2018 タイトル: Controllable molecular motors engineered from myosin and RNA. 著者: Tosan Omabegho / Pinar S Gurel / Clarence Y Cheng / Laura Y Kim / Paul V Ruijgrok / Rhiju Das / Gregory M Alushin / Zev Bryant / 要旨: Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems or in living cells . ...Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems or in living cells . Previously, synthetic nucleic acid motors and modified natural protein motors have been developed in separate complementary strategies to achieve tunable and controllable motor function. Integrating protein and nucleic-acid components to form engineered nucleoprotein motors may enable additional sophisticated functionalities. However, this potential has only begun to be explored in pioneering work harnessing DNA scaffolds to dictate the spacing, number and composition of tethered protein motors . Here, we describe myosin motors that incorporate RNA lever arms, forming hybrid assemblies in which conformational changes in the protein motor domain are amplified and redirected by nucleic acid structures. The RNA lever arm geometry determines the speed and direction of motor transport and can be dynamically controlled using programmed transitions in the lever arm structure . We have characterized the hybrid motors using in vitro motility assays, single-molecule tracking, cryo-electron microscopy and structural probing . Our designs include nucleoprotein motors that reversibly change direction in response to oligonucleotides that drive strand-displacement reactions. In multimeric assemblies, the controllable motors walk processively along actin filaments at speeds of 10-20 nm s. Finally, to illustrate the potential for multiplexed addressable control, we demonstrate sequence-specific responses of RNA variants to oligonucleotide signals.
凍結剤: ETHANE / チャンバー内湿度: 95 % / チャンバー内温度: 298 K / 装置: LEICA EM GP 詳細: Sample was applied to a glow-discharged holey carbon grid. 3 uL actin was incubated for 60 seconds. 3 uL of myosin VI was added and incubated for 60 seconds. 3 uL solution was removed. An ...詳細: Sample was applied to a glow-discharged holey carbon grid. 3 uL actin was incubated for 60 seconds. 3 uL of myosin VI was added and incubated for 60 seconds. 3 uL solution was removed. An additional 3 uL of myosin VI was applied. After 60 seconds, 3 uL solution was removed, and the grid was blotted for 3 seconds from the backside with filter paper..
詳細
0.45 mg/mL myosin VI was added to 0.025 mg/mL actin
タイプ: NOT APPLICABLE / ソフトウェア - 名称: FREALIGN (ver. 9.11)
FSC曲線 (解像度の算出)
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原子モデル構築 1
詳細
Initial models were assembled from 8 actins (3J8A) and 6 myosins (2BKI) through rigid body docking in Chimera, followed by flexible fitting with DireX. Resulting models were subjected to MDFF.
精密化
プロトコル: FLEXIBLE FIT / 温度因子: 150
得られたモデル
PDB-6bnp: CryoEM structure of MyosinVI-actin complex in the rigor (nucleotide-free) state
PDB-6bnv: CryoEM structure of MyosinVI-actin complex in the rigor (nucleotide-free) state, backbone-averaged with side chains truncated to alanine