PyTom: A python-based toolbox for localization of macromolecules in cryo-electron tomograms and subtomogram analysis

Hrabe, Thomas; Chen, Yuxiang; Pfeffer, Stefan; Cuellar, Luis Kuhn; Mangold, Ann-Victoria; Förster, Friedrich

doi:10.1016/j.jsb.2011.12.003

Cited by 235 publications

(245 citation statements)

References 44 publications

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“…EM data were analyzed using XMIPP (37) and the TOM toolbox (38). Fitting of densities simulated from atomic models was performed in PyTom (39) and analyzed using the TOM toolbox (38). All images of the resulting 3D reconstructions and atomic models were rendered in UCSF Chimera (40).…”

Section: Methodsmentioning

confidence: 99%

Structural characterization of the interaction of Ubp6 with the 26S proteasome

Aufderheide

Beck

Stengel

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediateenergy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation.conformational switching | proteolysis | proteostasis | quality control D egradation of proteins that are misfolded, damaged, or no longer needed is an essential element of cellular homeostasis. In eukaryotic cells, the ubiquitin-proteasome system (UPS) is the major pathway for regulated protein degradation (1). Proteins that are processed by the UPS are marked for destruction by polyubiquitin chains, which are recognized as a degradation signal by the 26S proteasome.The 26S proteasome consists of the core particle (CP), which degrades substrates into short peptides, and one or two 19S regulatory particles (RP), which associate with the ends of the cylinder-shaped CP to recruit substrates and prepare them for degradation (2, 3). Although the structure of the CP has been known for more than two decades (4, 5), the molecular architecture of the RP was unraveled by cryo-electron microscope (EM)-based approaches only recently (6-9). It comprises six RP triple A (AAA) ATPases (Rpt), 1-6, and 13 RP non-ATPases (Rpn), 1-3, 5-13, and 15. Similar to AAA-ATPases in prokaryotic ATP-dependent proteases, the Rpts form a hexameric ring that binds to the ends of the CP and is responsible for substrate unfolding and translocation into the CP. Unlike their prokaryotic counterparts, the Rpts are surrounded by non-ATPases. Apart from Rpn1, all Rpns form a cohesive struct...

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Section: Methodsmentioning

confidence: 99%

Structural characterization of the interaction of Ubp6 with the 26S proteasome

Aufderheide

Beck

Stengel

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

103

135

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“…For tomograms obtained from HeLa cell microsomes, cross correlation peaks in areas containing rough ER were visually inspected to identify true-positive matches. For the selected coordinates, unbinned subtomograms (200 3 voxels, object pixel: 0.288 nm) were reconstructed individually from the weighted backprojections and aligned to the template using PyTom 34 . With a second round of constrained principal component analysis, translocons including and lacking the OST complex were sorted.…”

Section: Methodsmentioning

confidence: 99%

Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon

et al. 2014

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In mammalian cells, proteins are typically translocated across the endoplasmic reticulum (ER) membrane in a co-translational mode by the ER protein translocon, comprising the proteinconducting channel Sec61 and additional complexes involved in nascent chain processing and translocation. As an integral component of the translocon, the oligosaccharyl-transferase complex (OST) catalyses co-translational N-glycosylation, one of the most common protein modifications in eukaryotic cells. Here we use cryoelectron tomography, cryoelectron microscopy single-particle analysis and small interfering RNA-mediated gene silencing to determine the overall structure, oligomeric state and position of OST in the native ER protein translocon of mammalian cells in unprecedented detail. The observed positioning of OST in close proximity to Sec61 provides a basis for understanding how protein translocation into the ER and glycosylation of nascent proteins are structurally coupled. The overall spatial organization of the native translocon, as determined here, serves as a reliable framework for further hypothesis-driven studies.

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“…A human tripeptidyl peptidase II homo-36-mer (EMD-2036) and homo-32-mer (EMD-2037) were used as templates after rescaling. Template matching, subtomogram averaging, and 3D auto-focus classification were carried out by PyTom (SI Materials and Methods) (31,32).…”

Section: Methodsmentioning

confidence: 99%

In situ structural studies of tripeptidyl peptidase II (TPPII) reveal spatial association with proteasomes

Fukuda

Beck

Plitzko

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Tripeptidyl peptidase II (TPPII) is a eukaryotic protease acting downstream of the 26S proteasome; it removes tripeptides from the degradation products released by the proteasome. Structural studies in vitro have revealed the basic architecture of TPPII, a twostranded linear polymer that assembles to form a spindle-shaped complex of ∼6 MDa. Dependent on protein concentration, TPPII has a distinct tendency for polymorphism. Therefore, its structure in vivo has remained unclear. To resolve this issue, we have scrutinized cryo-electron tomograms of rat hippocampal neurons for the occurrence and spatial distribution of TPPII by template matching. The quality of the tomograms recorded with the Volta phase plate enabled a detailed structural analysis of TPPII despite its low abundance. Two different assembly states (36-mers and 32-mers) coexist as well as occasional extended forms with longer strands. A distance analysis of the relative locations of TPPII and 26S proteasomes confirmed the visual impression that these two complexes spatially associate in agreement with TPPII's role in postproteasomal degradation.primary cultured neuronal cell | 26S proteasome | Volta phase plate | cryo-electron tomography | subtomogram averaging T he ubiquitin-proteasome system (UPS) is the main pathway of intracellular protein degradation in eukaryotic cells (1). In the UPS, the 26S proteasome, a 2.5-MDa multisubunit complex, degrades ubiquitylated proteins into oligopeptides with a size range of 4-25 residues (2, 3). The oligopeptides released by the 26S proteasome are further degraded by postproteasomal proteases (4, 5). One of the postproteasomal proteases that degrades oligopeptides longer than 15 residues is tripeptidyl peptidase II (TPPII) (6, 7). TPPII is a cytosolic serine protease of the subtilisin class (8) with an exopeptidase activity that removes tripeptides from free N-termini of peptides (9). TPPII has also been reported to be endowed with endopeptidase activity, but this activity is very low compared with its exopeptidase activity (10). Previously, only unfolded peptides had been reported to be degraded by TPPII (11). Tripeptides produced by TPPII are thought to be hydrolyzed further by an array of aminopeptidases (12). In addition to protein turnover, a role in MHC class 1 antigen presentation has also been proposed as one of the cellular functions of TPPII, but is still a subject of some controversy [for a review see Rockel et al. (11)]. A membrane-associated version of TPPII functions as a neuropeptidase inactivating the satiety hormone cholecystokinin-8 (CCK-8) by degrading into CCK-5 in the rat brain (13).Structural studies of isolated and purified TPPII revealed the existence of large (∼6 MDa) spindle-shaped complexes comprising two linear arrays of 20 monomers each in Drosophila melanogaster (14) and 18 monomers each in Homo sapiens (15). High-resolution structures of Drosophila TPPII (16) and human TPPII (15) have been obtained by hybrid approaches combining X-ray crystallography and EM single-particle analysis...

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PyTom: A python-based toolbox for localization of macromolecules in cryo-electron tomograms and subtomogram analysis

Cited by 235 publications

References 44 publications

Structural characterization of the interaction of Ubp6 with the 26S proteasome

Structural characterization of the interaction of Ubp6 with the 26S proteasome

Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon

In situ structural studies of tripeptidyl peptidase II (TPPII) reveal spatial association with proteasomes

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