Setting Up Parallel Illumination on the Talos Arctica for High-Resolution Data Collection

Herzik, Mark A.

doi:10.1007/978-1-0716-0966-8_6

Cited by 25 publications

(13 citation statements)

References 18 publications

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“…Vitrified samples were imaged using a Talos Arctica (ThermoFisher, operated at 200 keV) equipped with a BioQuantum energy filter (Gatan) and a K3 direct electron detector (Gatan). The microscope was subjected to stringent alignment procedures, including coma-free alignment and parallel illumination ( 36 ). High-throughput imaging was achieved using a 3-by-3 image shift in SerialEM ( 37 ).…”

Section: Methodsmentioning

confidence: 99%

Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM

Park

Tsai

Chen

et al. 2022

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

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CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the transposase component, TnsB, remains uncharacterized. Using cryo-electron microscopy (cryo-EM) structure determination, we reveal the conformation of TnsB during transposon integration for the type V-K CAST system from Scytonema hofmanni (ShCAST). Our structure of TnsB is a tetramer, revealing strong mechanistic relationships with the overall architecture of RNaseH transposases/integrases in general, and in particular the MuA transposase from bacteriophage Mu. However, key structural differences in the C-terminal domains indicate that TnsB’s tetrameric architecture is stabilized by a different set of protein–protein interactions compared with MuA. We describe the base-specific interactions along the TnsB binding site, which explain how different CAST elements can function on cognate mobile elements independent of one another. We observe that melting of the 5′ nontransferred strand of the transposon end is a structural feature stabilized by TnsB and furthermore is crucial for donor–DNA integration. Although not observed in the TnsB strand-transfer complex, the C-terminal end of TnsB serves a crucial role in transposase recruitment to the target site. The C-terminal end of TnsB adopts a short, structured 15-residue “hook” that decorates TnsC filaments. Unlike full-length TnsB, C-terminal fragments do not appear to stimulate filament disassembly using two different assays, suggesting that additional interactions between TnsB and TnsC are required for redistributing TnsC to appropriate targets. The structural information presented here will help guide future work in modifying these important systems as programmable gene integration tools.

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Section: Methodsmentioning

confidence: 99%

Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM

Park

Tsai

Chen

et al. 2022

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

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“…All cryo-EM data were collected on a 200 keV Thermo Fisher Scientific G3 Talos Arctica equipped with a Gatan K3 direct-electron detector. The microscope was aligned using a cross grating replica 2160 mm (TedPella) TEM grid and parallel illumination was obtained by adjusting the C2 lens in diffraction mode at 850 mm 70 . Coma Free alignment was done in SerialEM (version 3.8) 71 , 72 and verified by acquiring 3 × 3 Zemlin Tableau 73 .…”

Section: Methodsmentioning

confidence: 99%

Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria

et al. 2022

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How bacteria sense and respond to nitrogen levels are central questions in microbial physiology. In Gram-positive bacteria, nitrogen homeostasis is controlled by an operon encoding glutamine synthetase (GS), a dodecameric machine that assimilates ammonium into glutamine, and the GlnR repressor. GlnR detects nitrogen excess indirectly by binding glutamine-feedback-inhibited-GS (FBI-GS), which activates its transcription-repression function. The molecular mechanisms behind this regulatory circuitry, however, are unknown. Here we describe biochemical and structural analyses of GS and FBI-GS-GlnR complexes from pathogenic and non-pathogenic Gram-positive bacteria. The structures show FBI-GS binds the GlnR C-terminal domain within its active-site cavity, juxtaposing two GlnR monomers to form a DNA-binding-competent GlnR dimer. The FBI-GS-GlnR interaction stabilizes the inactive GS conformation. Strikingly, this interaction also favors a remarkable dodecamer to tetradecamer transition in some GS, breaking the paradigm that all bacterial GS are dodecamers. These data thus unveil unique structural mechanisms of transcription and enzymatic regulation.

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“…Cryo-EM data were collected on a Thermo-Fisher Talos Arctica transmission electron microscope operating at 200 keV using parallel illumination conditions 23 . Micrographs were acquired with a Gatan K2 Summit direct electron detector with a total electron exposure of 50e − /Å 2 as a 97-frame dose-fractionated movie during a 9.7 s exposure time.…”

Section: Methodsmentioning

confidence: 99%

Cryo-EM structure of hexameric yeast Lon (PIM1) highlights importance of conserved structural elements

Yang

Song

Wiseman

et al. 2021

Preprint

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Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphologic perturbations. Although structures of bacterial and human Lon protease reveal a hexameric assembly, PIM1 was speculated to form a heptameric assembly, and is uniquely characterized by a ~50 residue insertion between the ATPase and protease domains. To understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-EM structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrate to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a ~15 residue C-terminal extension. These additional C-terminal residues form an alpha-helix that is located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1’s enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.

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Setting Up Parallel Illumination on the Talos Arctica for High-Resolution Data Collection

Cited by 25 publications

References 18 publications

Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM

Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM

Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria

Cryo-EM structure of hexameric yeast Lon (PIM1) highlights importance of conserved structural elements

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