Zhening Zhang

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.

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].

Deep sequencing with longitudinal sampling of a VRC01-like-antibody response in a chronically infected individual

Background VRC01-like antibodies use heavy chain mimicry of the CD4-receptor to achieve effective neutralization of HIV-1. The VRC01-like antibodies that have been observed in a number of HIV-1-infected individuals (i) display extensive somatic changes (70-100 nucleotide changes in VH-gene), (ii) can be detected only after several years of infection, (iii) derive from VH1-2, and (iv) are compatible with several different heavy J chains and different light chains. Methods To understand the persistence, evolution, and lineage of VRC01-like antibodies, we sampled PBMCs from donor 45, the source of VRC01 and VRC03 antibodies, at approximately yearly intervals over a 15-year period, and performed deep sequencing on the heavy and light chain variable portions of expressed antibodies. Anti-idiotypic antibodies were used to correlate mRNA levels of antibodies identified by the deep sequencing with expressed levels of these antibodies in serum. Results High expression levels of VRC01-like antibody sequences persisted over the entire 15-year period. Multiple lineages of VRC01-like antibodies were detected at each time point, and some of these, in particular the lineages that include VRC01 and VRC03, persisted over multiple time points, and displayed extensive branching in their evolution. Conclusion Deep sequencing provides a means to define the genetic record of the lineage and maturation of antibodies effective at neutralizing HIV-1. Precise definition of the natural ontogeny of broadly neutralizing antibodies may be essential in defining appropriate strategies to elicit such antibodies in vaccine settings.

Self-Blotting Nanowire Grids for Cryo-EM Sample Preparation

Almost every aspect of cryo electron microscopy (CryoEM) has been automated over the last few decades. One of the challenges that remains to be addressed is the robust and reliable preparation of vitrified specimens of suitable ice thickness. The development of a new self-blotting nanowire (Zhang et al., 2013 ) grid in conjunction with a piezo electric dispensing robot called Spotiton (Jain et al., 2012 ) enables spreading a sample to a thin film without the use of externally applied filter paper. This new approach has the advantage of using small amounts of protein material, resulting in large areas of ice of a well- defined thickness containing evenly distributed particles (Razinkov et al., 2016 ).

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.