National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM127440, R01GM092917, S10OD012272
United States
Citation
Journal: Nat Struct Mol Biol / Year: 2020 Title: Cryo-EM reveals the transition of Arp2/3 complex from inactive to nucleation-competent state. Authors: Mohammed Shaaban / Saikat Chowdhury / Brad J Nolen / Abstract: Arp2/3 complex, a crucial actin filament nucleator, undergoes structural rearrangements during activation by nucleation-promoting factors (NPFs). However, the conformational pathway leading to the ...Arp2/3 complex, a crucial actin filament nucleator, undergoes structural rearrangements during activation by nucleation-promoting factors (NPFs). However, the conformational pathway leading to the nucleation-competent state is unclear due to lack of high-resolution structures of the activated state. Here we report a ~3.9 Å resolution cryo-EM structure of activated Schizosaccharomyces pombe Arp2/3 complex bound to the S. pombe NPF Dip1 and attached to the end of the nucleated actin filament. The structure reveals global and local conformational changes that allow the two actin-related proteins in Arp2/3 complex to mimic a filamentous actin dimer and template nucleation. Activation occurs through a clamp-twisting mechanism, in which Dip1 forces two core subunits in Arp2/3 complex to pivot around one another, shifting half of the complex into a new activated position. By showing how Dip1 stimulates activation, the structure reveals how NPFs can activate Arp2/3 complex in diverse cellular processes.
History
Deposition
Mar 3, 2020
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Header (metadata) release
Apr 1, 2020
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Map release
Aug 12, 2020
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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: 9 / Number of copies: 1 / Formula: MG
Molecular weight
Theoretical: 24.305 Da
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
2.5 mg/mL
Buffer
pH: 8 Component:
Concentration
Formula
Name
50.0 mM
NaCl
sodium chloride
1.0 mM
MgCl2
magnesium chloride
1.0 mM
C10H16N5O13P3
adenosine triphosphate
10.0 mM
C4H11NO3
tris(hydroxymethyl)aminomethane
1.0 mM
C4H10O2S2
dithiothreitol
Grid
Model: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.019 kPa / Details: 20mA current
Vitrification
Cryogen name: ETHANE / Chamber humidity: 98 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER Details: 4 uL of sample was applied to freshly glow-discharged grid. Excess sample was manually blotted off with a dry Whatman No.1 filter paper for 5-7 seconds. Immediately after the blotting step ...Details: 4 uL of sample was applied to freshly glow-discharged grid. Excess sample was manually blotted off with a dry Whatman No.1 filter paper for 5-7 seconds. Immediately after the blotting step the sample containing grid was rapidly vitrified by plunge freezing into liquid ethane..
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Electron microscopy
Microscope
FEI TALOS ARCTICA
Details
Data were collected by combining untilted and tilted (20, 30 and 40 degrees) images. Stage shifting to the targeted exposure position was used for navigation during data acquisition.
Image recording
Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 3 / Number real images: 5309 / Average exposure time: 40.0 sec. / Average electron dose: 44.34 e/Å2 Details: Each micrograph was collected as dose-fractionated movies consisting of 62 fractions per movie.
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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