Accelerated 2D Classification With ISAC Using GPUs

Schöenfeld, Fabian; Stabrin, Markus; Shaikh, Tanvir R.; Wagner, Tobias; Raunser, Stefan

doi:10.3389/fmolb.2022.919994

Cited by 6 publications

(3 citation statements)

References 20 publications

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“…Using customized scripts, each subtomogram was rotated to orient the thin filaments parallel to the X-Y plane using the previous angles from the tracing. Then, the central slab of 100 slices was projected and used as an input for 2D classification with ISAC [57][58][59] . The classes that did not show a clear presence of thin filaments were discarded and the remaining segments were re-extracted as subtomograms and processed in RELION 3.1 (refs.…”

Section: Thin Filament Processing Pipeline Model Building and Visuali...mentioning

confidence: 99%

Structure of the native myosin filament in the relaxed cardiac sarcomere

Tamborrini,

Wang,

Wagner

et al. 2023

Nature

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The thick filament is a key component of sarcomeres, the basic units of striated muscle1. Alterations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases2. Despite the central importance of the thick filament, its molecular organization remains unclear. Here we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by cryo-electron tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin, titin and myosin-binding protein C (MyBP-C). The arrangement of myosin molecules is dependent on their position along the filament, suggesting specialized capacities in terms of strain susceptibility and force generation. Three pairs of titin-α and titin-β chains run axially along the filament, intertwining with myosin tails and probably orchestrating the length-dependent activation of the sarcomere. Notably, whereas the three titin-α chains run along the entire length of the thick filament, titin-β chains do not. The structure also demonstrates that MyBP-C bridges thin and thick filaments, with its carboxy-terminal region binding to the myosin tails and directly stabilizing the OFF state of the myosin heads in an unforeseen manner. These results provide a foundation for future research investigating muscle disorders involving sarcomeric components.

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Section: Thin Filament Processing Pipeline Model Building and Visuali...mentioning

confidence: 99%

Structure of the native myosin filament in the relaxed cardiac sarcomere

Tamborrini,

Wang,

Wagner

et al. 2023

Nature

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“…The used processing strategy was essentially the same as reported in our previous work 8 . Briefly, the extracted particles were first 2D classified using ISAC2 (refs 64, 65 ) in helical mode and all non-protein picks were discarded. The particles were then refined using meridien alpha, which imposes helical restraints to limit particle shifts to the helical rise (set to 27.5 Å) to prevent particle duplication, but does not apply helical symmetry 55 .…”

Section: Methodsmentioning

confidence: 99%

Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments

Oosterheert

Blanc

Roy

et al. 2023

Preprint

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The release of inorganic phosphate (Pi) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. However, the molecular mechanisms underlying Pi release from both the core and barbed end of actin filaments remain unclear. Here, we combine cryo-EM with molecular dynamics simulations and in vitro reconstitution to demonstrate how actin releases Pi through a molecular backdoor. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and only opens transiently through concerted backbone movements and rotameric rearrangements of residues close to the nucleotide binding pocket. This mechanism explains why Pi escapes rapidly from the filament end and yet slowly from internal actin subunits. In an actin variant associated with nemaline myopathy, the backdoor is predominantly open in filament-core subunits, resulting in greatly accelerated Pi release after polymerization and filaments with drastically shortened ADP-Pi caps. This demonstrates that the Pi release rate from F-actin is controlled by steric hindrance through the backdoor rather than by the disruption of the ionic bond between Pi and Mg2+ at the nucleotide-binding site. Our results provide the molecular basis for Pi release from actin and exemplify how a single, disease-linked point mutation distorts the nucleotide state distribution and atomic structure of the actin filament.

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“…Particles in representative micrographs were manually picked to train a model, which was then used for automated particle picking in crYOLO 1.8.1 83 , selecting 1,259,079 particles with a box size of 384 ×384 pixels. Subsequently, 2D classification was performed using the iterative stable alignment clustering method (ISAC) with 200 particles/class 84 , 85 . After manually sorting out the “low quality” 2D classes, 605,359 particles remained.…”

Section: Methodsmentioning

confidence: 99%

Structure of the peroxisomal Pex1/Pex6 ATPase complex bound to a substrate

Rüttermann,

Koci,

Lill

et al. 2023

Nat Commun

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The double-ring AAA+ ATPase Pex1/Pex6 is required for peroxisomal receptor recycling and is essential for peroxisome formation. Pex1/Pex6 mutations cause severe peroxisome associated developmental disorders. Despite its pathophysiological importance, mechanistic details of the heterohexamer are not yet available. Here, we report cryoEM structures of Pex1/Pex6 from Saccharomyces cerevisiae, with an endogenous protein substrate trapped in the central pore of the catalytically active second ring (D2). Pairs of Pex1/Pex6(D2) subdomains engage the substrate via a staircase of pore-1 loops with distinct properties. The first ring (D1) is catalytically inactive but undergoes significant conformational changes resulting in alternate widening and narrowing of its pore. These events are fueled by ATP hydrolysis in the D2 ring and disengagement of a “twin-seam” Pex1/Pex6(D2) heterodimer from the staircase. Mechanical forces are propagated in a unique manner along Pex1/Pex6 interfaces that are not available in homo-oligomeric AAA-ATPases. Our structural analysis reveals the mechanisms of how Pex1 and Pex6 coordinate to achieve substrate translocation.

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Accelerated 2D Classification With ISAC Using GPUs

Cited by 6 publications

References 20 publications

Structure of the native myosin filament in the relaxed cardiac sarcomere

Structure of the native myosin filament in the relaxed cardiac sarcomere

Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments

Structure of the peroxisomal Pex1/Pex6 ATPase complex bound to a substrate

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