The β-Grasp Domain of Proteasomal ATPase Mpa Makes Critical Contacts with the Mycobacterium tuberculosis 20S Core Particle to Facilitate Degradation

Xiao, Xiansha; Feng, Xinwei; Yoo, Jin Hee; Kovach, Amanda; Darwin, K. Heran; H, Li

doi:10.1128/msphere.00274-22

Cited by 2 publications

(2 citation statements)

References 60 publications

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“…The success of the VAI Cryo-EM Core Facility supporting the research of several labs using single-particle cryo-EM has been clearly documented over the past few years. We have supported the work of labs interested in uncovering the molecular machinery behind eukaryotic DNA replication ( Yuan et al, 2020a ; Yuan et al, 2020b ), investigating the bacterial proteasome system in Mycobacterium tuberculosis ( Xiao et al, 2022 ), and determining the structure of membrane-embedded enzyme complexes, such as the oligosaccharyltransferase complex ( Bai and Li, 2019 ).…”

Section: Discussionmentioning

confidence: 81%

Best practice: setting up and operating a mid-sized cryo-EM facility

Meng,

Ratnayake,

Escobar Galvis

et al. 2023

Front. Mol. Biosci.

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Ever since the resolution revolution in 2013, cryo-electron microscopy (cryo-EM) has become a powerful methodology in structural biology that is especially suited to study the structure of large flexible molecular complexes. Since then, the need of setting up state-of-the-art cryo-EM facilities around the world has increased tremendously. Access to high-end cryo-EM instrumentation is however expensive and requires expertise. The establishment of large cryo-EM centers worldwide, many of which provide academic users free access for both data collection and user training, has been possible with the support of government agencies across the globe. In addition, many universities, and private institutions like the Van Andel Institute (VAI) have made significant investments to establish their own cryo-EM core facilities, ensuring on-site access to their researchers. This paper aims to serve as a blueprint for establishing a new mid-sized cryo-EM facility, as it provides key information based on our experience at VAI and discusses strategies used to optimize routine operation towards high performance and efficiency for single-particle cryo-EM. Information regarding initial planning, selection of equipment as well as the development of IT solutions that were required to improve data collection and analysis are included. In addition, we present an account of the most common issues affecting operation as well as the needs for maintenance over a 6-year period, which can help interested parties to estimate the long-term costs of running this type of facility. Lastly, a brief discussion on the pros and cons of establishing the facility is also included.

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Section: Discussionmentioning

confidence: 81%

Best practice: setting up and operating a mid-sized cryo-EM facility

Meng,

Ratnayake,

Escobar Galvis

et al. 2023

Front. Mol. Biosci.

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“…To build the molecular model for wild-type 20S CP, Mycobacterium tuberculosis 20S CP (PDB 8D6V) 27 was used as an initial template. For all the other datasets, the refined wild-type model was used as the template.…”

Section: Model Building and Refinementmentioning

confidence: 99%

Structural basis for allosteric regulation of the proteasome core particle

Turner,

Uday,

Velyvis

et al. 2024

Preprint

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Intracellular protein degradation is vital across all domains of life1. In eukaryotes, the ubiquitin proteasome system performs most non-lysosomal protein degradation and influences numerous cellular processes. Some bacteria, including the human pathogenMycobacterium tuberculosis(Mtb), encode a proteasome system that selectively degrades damaged or misfolded proteins crucial for the pathogen’s survival within host macrophages2–7. Consequently, the 20S core particle (CP), the central component of the proteasome system, has emerged as a viable target for tuberculosis treatment strategies2,8–10. Both eukaryotic andMtbproteasome systems are allosterically regulated11–13, yet the specific conformations involved have not been captured in high-resolution structures to date. Here we present the first structure ofMtb20S CP, and indeed any 20S CP, in an inactive state called 20SOFF, distinguished from the canonical active state, 20SON, by the conformation of switch helices I and II. The rearrangement of these helices collapses the S1 pocket, effectively inhibiting substrate binding. The switch helices are conserved and regulate the activity of HslV protease, the proteasome’s ancestral enzyme in bacteria, and a diverse family of serine/threonine protein phosphatases. Our results highlight the potential of harnessing allostery to develop therapeutics against the 20S CP inMtband eukaryotic systems.

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The β-Grasp Domain of Proteasomal ATPase Mpa Makes Critical Contacts with the Mycobacterium tuberculosis 20S Core Particle to Facilitate Degradation

Cited by 2 publications

References 60 publications

Best practice: setting up and operating a mid-sized cryo-EM facility

Best practice: setting up and operating a mid-sized cryo-EM facility

Structural basis for allosteric regulation of the proteasome core particle

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