Selecting particles from digital micrographs is an essential step in single-particle electron cryomicroscopy (cryo-EM). As manual selection of complete datasets—typically comprising thousands of particles—is a tedious and time-consuming process, numerous automatic particle pickers have been developed. However, non-ideal datasets pose a challenge to particle picking. Here we present the particle picking software crYOLO which is based on the deep-learning object detection system You Only Look Once (YOLO). After training the network with 200–2500 particles per dataset it automatically recognizes particles with high recall and precision while reaching a speed of up to five micrographs per second. Further, we present a general crYOLO network able to pick from previously unseen datasets, allowing for completely automated on-the-fly cryo-EM data preprocessing during data acquisition. crYOLO is available as a standalone program under http://sphire.mpg.de/
 and is distributed as part of the image processing workflow in SPHIRE. Thorsten Wagner et al. present SPHIRE-crYOLO, a particle picking software for selecting particles from digital micrographs in cryoEM data. After training, the method automatically recognizes particles with high recall and precision, simplifying data pre-processing.

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SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM

Cryo–electron microscopy, or simply cryo-EM, refers mainly to three very different yet closely related techniques: electron crystallography, single-particle cryo-EM, and electron cryotomography. In the past few years, single-particle cryo-EM in particular has triggered a revolution in structural biology and has become a newly dominant discipline. This Review examines the fascinating story of its start and evolution over the past 40-plus years, delves into how and why the recent technological advances have been so groundbreaking, and briefly considers where the technique may be headed in the future.

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Single-particle cryo-EM—How did it get here and where will it go

Biological membranes are tricky to investigate. They are complex in terms of molecular composition and structure, functional over a wide range of time scales, and characterized by nonequilibrium conditions. Because of all of these features, simulations are a great technique to study biomembrane behavior. A significant part of the functional processes in biological membranes takes place at the molecular level; thus computer simulations are the method of choice to explore how their properties emerge from specific molecular features and how the interplay among the numerous molecules gives rise to function over spatial and time scales larger than the molecular ones. In this review, we focus on this broad theme. We discuss the current state-of-the-art of biomembrane simulations that, until now, have largely focused on a rather narrow picture of the complexity of the membranes. Given this, we also discuss the challenges that we should unravel in the foreseeable future. Numerous features such as the actin-cytosk...

Multiscale Simulations of Biological Membranes: The Challenge To Understand Biological Phenomena in a Living Substance.

The turnover of brain proteins is critical for organism survival, and its perturbations are linked to pathology. Nevertheless, protein lifetimes have been difficult to obtain in vivo. They are readily measured in vitro by feeding cells with isotopically labeled amino acids, followed by mass spectrometry analyses. In vivo proteins are generated from at least two sources: labeled amino acids from the diet, and non-labeled amino acids from the degradation of pre-existing proteins. This renders measurements difficult. Here we solved this problem rigorously with a workflow that combines mouse in vivo isotopic labeling, mass spectrometry, and mathematical modeling. We also established several independent approaches to test and validate the results. This enabled us to measure the accurate lifetimes of ~3500 brain proteins. The high precision of our data provided a large set of biologically significant observations, including pathway-, organelle-, organ-, or cell-specific effects, along with a comprehensive catalog of extremely long-lived proteins (ELLPs).

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Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions

Selecting particles from digital micrographs is an essential step in single particle electron cryomicroscopy (cryo-EM). Since manual selection is a tedious and time-consuming process, many automatic particle pickers have been developed. However, they have problems especially with non-ideal datasets. Here, we present a novel automated particle picking software called crYOLO, which is based on the deep learning object detection system “You Only Look Once” (YOLO). After training the network with 500 – 2,500 particles per dataset, it automatically selects the particles with high accuracy and precision reaching a speed of up to six micrographs per second. Importantly, we show that crYOLO can be trained to select previously unseen datasets paving the way for completely automated “on-the-fly” cryo-EM data pre-processing during data acquisition. CrYOLO is available as standalone program under http://sphire.mpg.de/ and will be part of the image processing workflow in SPHIRE.

/pdf/sphire-cryolo-a-fast-and-well-centering-automated-particle-4ktd316nfx.pdf

SPHIRE-crYOLO: A fast and well-centering automated particle picker for cryo-EM

Canonical transient receptor channels (TRPC) are non-selective cation channels. They are involved in receptor-operated Ca2+ signaling and have been proposed to act as store-operated channels (SOC). Their malfunction is related to cardiomyopathies and their modulation by small molecules has been shown to be effective against renal cancer cells. The molecular mechanism underlying the complex activation and regulation is poorly understood. Here, we report the electron cryo-microscopy structure of zebrafish TRPC4 in its unliganded (apo), closed state at an overall resolution of 3.6 A. The structure reveals the molecular architecture of the cation conducting pore, including the selectivity filter and lower gate. The cytoplasmic domain contains two key hubs that have been shown to interact with modulating proteins. Structural comparisons with other TRP channels give novel insights into the general architecture and domain organization of this superfamily of channels and help to understand their function and pharmacology.

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Electron cryo-microscopy structure of the canonical TRPC4 ion channel.

Archaeal photoreceptors consist of sensory rhodopsins in complex with their cognate transducers. After light excitation, a two-component signaling chain is activated, which is homologous to the chemotactic signaling cascades in enterobacteria. The latter system has been studied in detail. From structural and functional studies, a picture emerges which includes stable signaling complexes, which assemble to receptor arrays displaying hexagonal structural elements. At this higher order structural level, signal amplification and sensory adaptation occur. Here, we describe electron microscopy data, which show that also the archaeal phototaxis receptors sensory rhodopsin I and II in complex with their cognate transducers can form hexagonal lattices even in the presence of a detergent. This result could be confirmed by molecular dynamics calculations, which revealed similar structural elements. Calculations of the global modes of motion displayed one mode, which resembles the "U"-"V" transition of the NpSRII:NpHtrII complex, which was previously argued to represent a functionally relevant global conformational change accompanying the activation process [Ishchenko et al. (2013) J. Photochem. Photobiol. B 123, 55-58]. A model of cooperativity at the transmembrane level is discussed.

Sensory Rhodopsin I and Sensory Rhodopsin II Form Trimers of Dimers in Complex with their Cognate Transducers

Two-component systems (TCS) are widespread signaling systems present in all domains of life TCS typically consist of a signal receptor/transducer and a response regulator The receptors (histidine kinases, chemoreceptors and photoreceptors) are often embedded in the membrane and have a similar modular structure Chemoreceptors were shown to function in highly ordered arrays, with trimers of dimers being the smallest functional unit However, much less is known about photoreceptors Here, we use small-angle scattering (SAS) to show that detergent-solubilized sensory rhodopsin II in complex with its cognate transducer forms dimers at low salt concentration, which associate into trimers of dimers at higher buffer molarities We then fit an atomistic model of the whole complex into the SAS data The obtained results suggest that the trimer of dimers is "tripod"-shaped and that the contacts between the dimers occur only through their cytoplasmic regions, whereas the transmembrane regions remain unconnected

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Molecular model of a sensor of two-component signaling system

Canonical transient receptor channels (TRPC) are non-selective cation channels. They are involved in receptor-operated Ca2+ signaling and have been proposed to act as store-operated channels (SOC). Their malfunction is related to cardiomyopathies and their modulation by small molecules has been shown to be effective against renal cancer cells. The molecular mechanism underlying the complex activation and regulation is poorly understood. Here, we report the electron cryo-microscopy structure of zebrafish TRPC4 in its unliganded (apo), closed state at an overall resolution of 0.36 nm. The structure reveals the molecular architecture of the cation conducting pore, including the selectivity filter and lower gate. The cytoplasmic domain contains two key hubs that have been shown to interact with modulating proteins. Structural comparisons with other TRP channels give novel insights into the general architecture and domain organization of this superfamily of channels and help to understand their function and pharmacology.

https://www.biorxiv.org/content/early/2018/03/14/280503.full.pdf

Arne Bothe

Papers

Electron cryo-microscopy structure of the canonical TRPC4 ion channel.

Sensory Rhodopsin I and Sensory Rhodopsin II Form Trimers of Dimers in Complex with their Cognate Transducers

Molecular model of a sensor of two-component signaling system

Electron cryo-microscopy structure of the canonical TRPC4 ion channel