Venkata P. Dandey

Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy.

Significance Invading pathogens and other danger-associated signals are recognized by the innate immune system. Subsequently, the eukaryotic protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)] assembles to long filaments, which might serve to amplify the signal and activate an inflammatory response. We have determined the structure of the mouse ASC filament at atomic resolution. The pyrin domain of ASC forms the helical filament core, and the CARD, thus far elusive to experimental observation, is flexibly unfolded on the filament periphery. The integration of data from two structural methods, cryo-electron microscopy and solid-state NMR spectroscopy, opens perspectives for structural studies of inflammasomes and related molecular assemblies. Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.

2dx_automator: Implementation of a semiautomatic high-throughput high-resolution cryo-electron crystallography pipeline

The introduction of direct electron detectors (DED) to cryo-electron microscopy has tremendously increased the signal-to-noise ratio (SNR) and quality of the recorded images. We discuss the optimal use of DEDs for cryo-electron crystallography, introduce a new automatic image processing pipeline, and demonstrate the vast improvement in the resolution achieved by the use of both together, especially for highly tilted samples. The new processing pipeline (now included in the software package 2dx) exploits the high SNR and frame readout frequency of DEDs to automatically correct for beam-induced sample movement, and reliably processes individual crystal images without human interaction as data are being acquired. A new graphical user interface (GUI) condenses all information required for quality assessment in one window, allowing the imaging conditions to be verified and adjusted during the data collection session. With this new pipeline an automatically generated unit cell projection map of each recorded 2D crystal is available less than 5 min after the image was recorded. The entire processing procedure yielded a three-dimensional reconstruction of the 2D-crystallized ion-channel membrane protein MloK1 with a much-improved resolution of 5Å in-plane and 7Å in the z-direction, within 2 days of data acquisition and simultaneous processing. The results obtained are superior to those delivered by conventional photographic film-based methodology of the same sample, and demonstrate the importance of drift-correction.

Spotiton: New features and applications

We present an update describing new features and applications of Spotiton, a novel instrument for vitrifying samples for cryoEM. We have used Spotiton to prepare several test specimens that can be reconstructed using routine single particle analysis to ∼3 Å resolution, indicating that the process has no apparent deleterious effect on the sample integrity. The system is now in routine and continuous use in our lab and has been used to successfully vitrify a wide variety of samples.

Optimizing “self-wicking” nanowire grids

We have developed a self-blotting TEM grid for use with a novel instrument for vitrifying samples for cryo-electron microscopy (cryoEM). Nanowires are grown on the copper surface of the grid using a simple chemical reaction and the opposite smooth side is used to adhere to a holey sample substrate support, for example carbon or gold. When small volumes of sample are applied to the nanowire grids the wires effectively act as blotting paper to rapidly wick away the liquid, leaving behind a thin film. In this technical note, we present a detailed description of how we make these grids using a variety of substrates fenestrated with either lacey or regularly spaced holes. We explain how we characterize the quality of the grids and we describe their behavior under a variety of conditions.

Exploring the Interactome: Microfluidic Isolation of Proteins and Interacting Partners for Quantitative Analysis by Electron Microscopy

Multimolecular protein complexes are important for many cellular processes. However, the stochastic nature of the cellular interactome makes the experimental detection of complex protein assemblies difficult and quantitative analysis at the single molecule level essential. Here, we present a fast and simple microfluidic method for (i) the quantitative isolation of endogenous levels of untagged protein complexes from minute volumes of cell lysates under close to physiological conditions and (ii) the labeling of specific components constituting these complexes. The method presented uses specific antibodies that are conjugated via a photocleavable linker to magnetic beads that are trapped in microcapillaries to immobilize the target proteins. Proteins are released by photocleavage, eluted, and subsequently analyzed by quantitative transmission electron microscopy at the single molecule level. Additionally, before photocleavage, immunogold can be employed to label proteins that interact with the primary target protein. Thus, the presented method provides a new way to study the interactome and, in combination with single molecule transmission electron microscopy, to structurally characterize the large, dynamic, heterogeneous multimolecular protein complexes formed.

Routine single particle CryoEM sample and grid characterization by tomography

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

Reducing effects of particle adsorption to the air–water interface in cryo-EM

Most protein particles prepared in vitreous ice for single-particle cryo-electron microscopy (cryo-EM) are adsorbed to air–water or substrate–water interfaces, which can cause the particles to adopt preferred orientations. By using a rapid plunge-freezing robot and nanowire grids, we were able to reduce some of the deleterious effects of the air–water interface by decreasing the dwell time of particles in thin liquid films. We demonstrated this by using single-particle cryo-EM and cryo-electron tomography (cryo-ET) to examine hemagglutinin, insulin receptor complex, and apoferritin.Reducing the length of time that protein particles spend on a sample grid prior to freezing mitigates deleterious effects caused by particle adsorption to the air–water interface in single-particle cryo-EM.

Structure of the Insulin Receptor in Complex with Insulin using Single Particle CryoEM Analysis

Insulin Receptor (IR) mediated signaling is crucial in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels [1, 2]. Aberrations in Insulin signaling have been associated with a variety of disease states, including diabetes, cancer and Alzheimer’s [1, 3, 4]. IR is composed of two heterodimers ( and  chains), each containing an extracellular portion (ectodomain), a single transmembrane helix (TM), and a cytoplasmic tyrosine kinase domain (TK) (Figure 1). One single disulfide bond links the  and  chains in the monomer, while the dimer is stabilized by two interchain disulfide bonds (Figure 1). Insulin is thought to bind to two distinct sites (per monomer), in a complex process that exhibits negative cooperativity [5]. Insulin binding site 1 was mapped by alanine scanning to portion of the L1 domain (Asp12-Asn15, Leu37, Phe39, Phe64 and Arg65) and to the CT helix (Gln692-Pro718) located at the C-terminal end of the ID domain. Site 2 was mapped to loop regions near the junction between the FNIII-1 and FNIII-2 domains [5 and references therein].

Strategies for Automated CryoEM Data Collection Using Direct Detectors

The new generation of direct electron detectors has been a major contributor to the recent resolution revolution in cryo-electron microscopy. Optimal use of these new cameras using automated data collection software is critical for high-throughput near-atomic resolution cryo-electron microscopy research. We present an overview of the practical aspects of automated data collection in the context of this new generation of direct detectors, highlighting the differences, challenges, and opportunities the new detectors provide compared to the previous generation of data acquisition media.

Spotiton: a new method for vitrifying samples for cryo-EM

Almost every aspect of cryo-electron microscopy (cryoEM) has been automated over the last few decades. One of the challenges that remain to be addressed is the robust and reliable preparation of vitrified specimens[2] of suitable ice thickness. We will present results from the next generation of a new device for preparing vitrified samples. The device combines a picoliter dispensing system with a new ‘‘self-blotting”[3] grid that we have developed to provide a method for spreading a sample to a thin film without the use of externally applied filter paper. This new method[1] consumes very small amounts of protein material and results in large areas of vitrified ice of a well-defined thickness and single particles that are evenly and well distributed within the ice. We will discuss the current state of the development of Spotiton and future possibilities for further improvements. We also show here the high-resolution structure of Proteasome at 2.9 Angstrom achieved in 2days from making grid to structure with only 8hr of data collection on Spotiton grid.