National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01-GM07391
United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
T32-GM008275
United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)
F31-HL146077
United States
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2023 Title: Transition State of Arp2/3 Complex Activation by Actin-Bound Dimeric Nucleation-Promoting Factor. Authors: Trevor van Eeuwen / Malgorzata Boczkowska / Grzegorz Rebowski / Peter J Carman / Fred E Fregoso / Roberto Dominguez / Abstract: Arp2/3 complex generates branched actin networks that drive fundamental processes such as cell motility and cytokinesis. The complex comprises seven proteins, including actin-related proteins (Arps) ...Arp2/3 complex generates branched actin networks that drive fundamental processes such as cell motility and cytokinesis. The complex comprises seven proteins, including actin-related proteins (Arps) 2 and 3 and five scaffolding proteins (ArpC1-ArpC5) that mediate interactions with a pre-existing (mother) actin filament at the branch junction. Arp2/3 complex exists in two main conformations, inactive with the Arps interacting end-to-end and active with the Arps interacting side-by-side like subunits of the short-pitch helix of the actin filament. Several cofactors drive the transition toward the active state, including ATP binding to the Arps, WASP-family nucleation-promoting factors (NPFs), actin monomers, and binding of Arp2/3 complex to the mother filament. The precise contribution of each cofactor to activation is poorly understood. We report the 3.32-Å resolution cryo-electron microscopy structure of a transition state of Arp2/3 complex activation with bound constitutively dimeric NPF. Arp2/3 complex-binding region of the NPF N-WASP was fused C-terminally to the α and β subunits of the CapZ heterodimer. One arm of the NPF dimer binds Arp2 and the other binds actin and Arp3. The conformation of the complex is intermediate between those of inactive and active Arp2/3 complex. Arp2, Arp3, and actin also adopt intermediate conformations between monomeric (G-actin) and filamentous (F-actin) states, but only actin hydrolyzes ATP. In solution, the transition complex is kinetically shifted toward the short-pitch conformation and has higher affinity for F-actin than inactive Arp2/3 complex. The results reveal how all the activating cofactors contribute in a coordinated manner toward Arp2/3 complex activation.
Cryogen name: ETHANE / Instrument: LEICA EM CPC Details: Grids were manually blotted for 3 seconds with Whatman 41 filter paper and manually plunged using a Leica EM CPC manual plunger..
Details
This sample was monodisperse.
-
Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Slit width: 20 eV
Details
Data were collected in super-resolution mode with an illuminated area of 1.01 um, nominal dose of 40 e-/A^2, a dose rate of 4.87 e-/s/pixel, and 2 or 5 exposures per hole by image shift.
Image recording
Film or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 9661 / Average exposure time: 2.6 sec. / Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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