TranSPHIRE: automated and feedback-optimized on-the-fly processing for cryo-EM

Stabrin, Markus; Schöenfeld, Fabian; Wagner, Tobias; Pospich, Sabrina; Gatsogiannis, Christos; Raunser, Stefan

doi:10.1038/s41467-020-19513-2

Cited by 70 publications

(70 citation statements)

References 49 publications

(89 reference statements)

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“…12437 images were acquired and 8663 of them were used for processing. Motion correction and CTF estimation have been performed in CTFFIND4 (31) and MotionCorr2 (32) during image acquisition using TranSPHIRE (33). The filament picking was performed using crYOLO (34).…”

Section: Methodsmentioning

confidence: 99%

“…After visual inspection, 378 non-tilted and 183 tilted micrographs were removed. For the remaining images, motion correction and CTF estimation have been performed in CTFFIND4 (31) and MotionCorr2 (32) during image acquisition using TranSPHIRE (33). Particle picking using crYOLO (34) resulted in 706 thousand non-tilted and 543 thousand tilted particles that were used in independent 3D refinements in Meridien.…”

Section: Methodsmentioning

confidence: 99%

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Mechanism of actin-dependent activation of nucleotidyl cyclase toxins from bacterial human pathogens

Belyy

Merino

Mechold

et al. 2021

Preprint

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Several bacterial human pathogens secrete nucleotidyl cyclase toxins, that are activated by interaction with actin of the eukaryotic host cells. However, the underlying molecular mechanism of this process which protects bacteria from self-intoxication is unclear. Here, we report structures of ExoY from Pseudomonas aeruginosa and Vibrio vulnificus in complex with their corresponding activators F-actin and profilin-G-actin. The structures reveal that in contrast to the apo state, two flexible regions become ordered and interact strongly with actin. The specific stabilization of these regions results in an allosteric stabilization of the distant nucleotide binding pocket and thereby to an activation of the enzyme. Differences in the sequence and conformation of the actin-binding regions are responsible for the selective binding to either F- or G-actin. This specificity can be biotechnologically modulated by exchanging these regions from one toxin to the other. Other bacterial nucleotidyl cyclases, such as the anthrax edema factor and CyaA from Bortedella pertussis, that bind to calmodulin undergo a similar disordered-to-ordered transition during activation, suggesting that the allosteric activation-by-stabilization mechanism of ExoY is paradigmatic for all bacterial nucleotidyl cyclase toxins.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mechanism of actin-dependent activation of nucleotidyl cyclase toxins from bacterial human pathogens

Belyy

Merino

Mechold

et al. 2021

Preprint

Self Cite

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show abstract

“…The details of all four data sets including pixel size, electron dose, exposure time, number of frames and defocus range are summarized in Table 1 . Data collection was monitored live using TranSPHIRE ( Stabrin et al, 2020 ), allowing for direct adjustments of data acquisition settings when necessary, i.e. defocus range or astigmatism.…”

Section: Methodsmentioning

confidence: 99%

Structural basis of TRPC4 regulation by calmodulin and pharmacological agents

Vinayagam

Quentin

Yu-Strzelczyk

et al. 2020

eLife

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Canonical transient receptor potential channels (TRPC) are involved in receptor-operated and/or store-operated Ca2+ signaling. Inhibition of TRPCs by small molecules was shown to be promising in treating renal diseases. In cells, the channels are regulated by calmodulin. Molecular details of both calmodulin and drug binding have remained elusive so far. Here we report structures of TRPC4 in complex with three pyridazinone-based inhibitors and calmodulin. The structures reveal that all the inhibitors bind to the same cavity of the voltage-sensing-like domain and allow us to describe how structural changes from the ligand binding site can be transmitted to the central ion-conducting pore of TRPC4. Calmodulin binds to the rib helix of TRPC4, which results in the ordering of a previously disordered region, fixing the channel in its closed conformation. This represents a novel calmodulin-induced regulatory mechanism of canonical TRP channels.

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“…Both datasets were monitored live with TranSPHIRE 44 to evaluate i.e. the defocus range and astigmatism.…”

Section: Methodsmentioning

confidence: 99%

Structure of a bacterial Rhs effector exported by the type VI secretion system

Guenther

Quentin

Ahmad

et al. 2021

Preprint

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The type VI secretion system (T6SS) is a widespread protein export apparatus found in Gram-negative bacteria. The majority of T6SSs deliver toxic effector proteins into competitor bacteria. Yet, the structure, function, and activation of many of these effectors remains poorly understood. Here, we present the structures of the T6SS effector RhsA from Pseudomonas protegens and its cognate T6SS spike protein, VgrG1, at 3.3 Å resolution. The structures reveal that the rearrangement hotspot (Rhs) repeats of RhsA assemble into a closed anticlockwise β-barrel spiral similar to that found in bacterial insecticidal Tc toxins and in metazoan teneurin proteins. We find that the C-terminal toxin domain of RhsA is autoproteolytically cleaved but remains inside the Rhs ′cocoon′ where, with the exception of three ordered structural elements, most of the toxin is disordered. The N-terminal ′plug′ domain is unique to T6SS Rhs proteins and resembles a champagne cork that seals the Rhs cocoon at one end while also mediating interactions with VgrG1. Interestingly, this domain is also autoproteolytically cleaved inside the cocoon but remains associated with it. We propose that mechanical force is required to remove the cleaved part of the plug, resulting in the release of the toxin domain as it is delivered into a susceptible bacterial cell by the T6SS.

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TranSPHIRE: automated and feedback-optimized on-the-fly processing for cryo-EM

Cited by 70 publications

References 49 publications

Mechanism of actin-dependent activation of nucleotidyl cyclase toxins from bacterial human pathogens

Mechanism of actin-dependent activation of nucleotidyl cyclase toxins from bacterial human pathogens

Structural basis of TRPC4 regulation by calmodulin and pharmacological agents

Structure of a bacterial Rhs effector exported by the type VI secretion system

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