National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01-GM114627
United States
Citation
Journal: Nat Chem Biol / Year: 2021 Title: Optical control of fast and processive engineered myosins in vitro and in living cells. Authors: Paul V Ruijgrok / Rajarshi P Ghosh / Sasha Zemsky / Muneaki Nakamura / Rui Gong / Lin Ning / Robert Chen / Vipul T Vachharajani / Alexander E Chu / Namrata Anand / Raphael R Eguchi / Po-Ssu ...Authors: Paul V Ruijgrok / Rajarshi P Ghosh / Sasha Zemsky / Muneaki Nakamura / Rui Gong / Lin Ning / Robert Chen / Vipul T Vachharajani / Alexander E Chu / Namrata Anand / Raphael R Eguchi / Po-Ssu Huang / Michael Z Lin / Gregory M Alushin / Jan T Liphardt / Zev Bryant / Abstract: Precision tools for spatiotemporal control of cytoskeletal motor function are needed to dissect fundamental biological processes ranging from intracellular transport to cell migration and division. ...Precision tools for spatiotemporal control of cytoskeletal motor function are needed to dissect fundamental biological processes ranging from intracellular transport to cell migration and division. Direct optical control of motor speed and direction is one promising approach, but it remains a challenge to engineer controllable motors with desirable properties such as the speed and processivity required for transport applications in living cells. Here, we develop engineered myosin motors that combine large optical modulation depths with high velocities, and create processive myosin motors with optically controllable directionality. We characterize the performance of the motors using in vitro motility assays, single-molecule tracking and live-cell imaging. Bidirectional processive motors move efficiently toward the tips of cellular protrusions in the presence of blue light, and can transport molecular cargo in cells. Robust gearshifting myosins will further enable programmable transport in contexts ranging from in vitro active matter reconstitutions to microfabricated systems that harness molecular propulsion.
History
Deposition
Oct 5, 2020
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Header (metadata) release
Jan 13, 2021
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Map release
Jan 13, 2021
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Update
Mar 6, 2024
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Current status
Mar 6, 2024
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Name: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 3 / Formula: MG
Molecular weight
Theoretical: 24.305 Da
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Experimental details
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Structure determination
Method
cryo EM
Processing
helical reconstruction
Aggregation state
filament
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Sample preparation
Buffer
pH: 7 Component:
Concentration
Formula
Name
10.0 mM
C3N2H4
Imidazole
50.0 mM
KCl
Potassium chloride
1.0 mM
MgCl2
Magnesium chloride
1.0 mM
C14H24N2O10
EGTA
0.5 mM
C4H10O2S2
DTT
0.01 weight/volume
NaN3
sodium azide
Details: 10 mM imidazole pH 7.0,50 mM KCl,1mM MgCl2, 1mM EGTA, 0.5 mM DTT, 0.01% NaN3
Grid
Model: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: OTHER
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: LEICA EM GP
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 67.12 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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