Maxim E. Kuil

Cryo-electron microscopy of extracellular vesicles in fresh plasma

Introduction Extracellular vesicles (EV) are phospholipid bilayer-enclosed vesicles recognized as new mediators in intercellular communication and potential biomarkers of disease. They are found in many body fluids and mainly studied in fractions isolated from blood plasma in view of their potential in medicine. Due to the limitations of available analytical methods, morphological information on EV in fresh plasma is still rather limited. Objectives To image EV and determine the morphology, structure and size distribution in fresh plasma by cryo-electron microscopy (cryo-EM). Methods Fresh citrate- and ethylenediaminetetraacetic acid (EDTA)-anticoagulated plasma or EV isolated from these plasmas were rapidly cryo-immobilized by vitrification and visualized by cryo-EM. Results EV isolated from fresh plasma were highly heterogeneous in morphology and size and mostly contain a discernible lipid bilayer (lipid vesicles). In fresh plasma there were 2 types of particles with a median diameter of 30 nm (25–260 nm). The majority of these particles are electron dense particles which most likely represent lipoproteins. The minority are lipid vesicles, either electron dense or electron lucent, which most likely represent EV. Lipid vesicles were occasionally observed in close proximity of platelets in citrate and EDTA-anticoagulated platelet-rich plasma. Cryo-electron tomography (cryo-ET) was employed to determine the 3D structure of platelet secretory granules. Conclusions Cryo-EM is a powerful technique that enables the characterization of EV in fresh plasma revealing structural details and considerable morphological heterogeneity. Only a small proportion of the submicron structures in fresh plasma are lipid vesicles representing EV.

Heterogeneous nucleation of three-dimensional protein nanocrystals.

Nucleation is the rate-limiting step in protein crystallization. Introducing heterogeneous substrates may in some cases lower the energy barrier for nucleation and thereby facilitate crystal growth. To date, the mechanism of heterogeneous protein nucleation remains poorly understood. In this study, the nucleating properties of fragments of human hair in crystallization experiments have been investigated. The four proteins that were tested, lysozyme, glucose isomerase, a polysaccharide-specific Fab fragment and potato serine protease inhibitor, nucleated preferentially on the hair surface. Macrocrystals and showers of tiny crystals of a few hundred nanometres thickness were obtained also under conditions that did not produce crystals in the absence of the nucleating agent. Cryo-electron diffraction showed that the nanocrystals diffracted to at least 4 A resolution. The mechanism of heterogeneous nucleation was studied using confocal fluorescent microscopy which demonstrated that the protein is concentrated on the nucleating surface. A substantial accumulation of protein was observed on the sharp edges of the hairs cuticles, explaining the strong nucleating activity of the surface.

The prospects of protein nanocrystallography

The miniaturization of protein crystallographys experimental method has several advantages. Firstly, it reduces the amount of protein required for identifying crystallization conditions, allowing crystallographic studies of rare natural proteins and complexes. Secondly, higher levels of supersaturation can be obtained in very small volumes, allowing the exploration of additional crystallization conditions. Thirdly, there are indications that protein crystals grown in very small volumes may be better ordered. Fourthly, miniaturization and automation go hand in hand, opening the prospects of easier and more reproducible experimentation. Progress in the development of nanocrystallography is discussed and the remaining bottlenecks are highlighted.

A novel approximation method of CTF amplitude correction for 3D single particle reconstruction

The typical resolution of three-dimensional reconstruction by cryo-EM single particle analysis is now being pushed up to and beyond the nanometer scale. Correction of the contrast transfer function (CTF) of electron microscopic images is essential for achieving such a high resolution. Various correction methods exist and are employed in popular reconstruction software packages. Here, we present a novel approximation method that corrects the amplitude modulation introduced by the contrast transfer function by convoluting the images with a piecewise continuous function. Our new approach can easily be implemented and incorporated into other packages. The implemented method yielded higher resolution reconstructions with data sets from both highly symmetric and asymmetric structures. It is an efficient alternative correction method that allows quick convergence of the 3D reconstruction and has a high tolerance for noisy images, thus easing a bottleneck in practical reconstruction of macromolecules.

Area detectors in structural biology

Abstract An overview of area detectors in structural biology is presented. Development of these detectors is one of the main reasons for the exponential rise in the number of structure determinations of large biological complexes. The different techniques used for structure determination put different demands on area detectors. The techniques used in structural biology, X-ray and electron diffraction and electron imaging are discussed and the requirements for a good detector are highlighted. Furthermore, an overview is given of the current state of the art of high-resolution area detectors.

Protein nano-crystallogenesis

We demonstrate the feasibility of growing crystals of protein in volumes as small as 1 nanoliter. Advances in the handling of very small volumes (i.e. through inkjet and other technologies) open the way towards fully automated systems. The rationale for these experiments is the desire to develop a system that speeds up the structure determination of proteins by crystallographic techniques, where most of the precious protein sample is wasted for the identification of the ideal crystallisation conditions. An additional potential benefit of crystallisation in very small volumes is the potential improvement of the crystal quality through reduced convection during crystal growth. Furthermore, in such small volumes even very highly supersaturated conditions can be stable for prolonged periods, allowing additional regions of phase-space to be prospected for elusive crystallisation conditions. A massive improvement in the efficiency of protein crystallogenesis will cause a paradigm shift in the biomolecular sciences and will have a major impact in product development in (for example) the pharmaceutical industry.

Neutron scattering experiments on magnetically aligned liquid crystalline DNA fragment solutions

Abstract Small angle neutron scattering experiments have been performed on liquid crystalline 163 basepair NaDNA fragments in aqueous solution in the concentration range 190–285 mg ml−1. To induce a macroscopic alignment, a magnetic field was applied either parallel (B ∥ configuration) or perpendicular (B ⊊ configuration) to the incoming neutron beam. The isotropic scattering pattern in the B ∥ configuration and the anisotropic scattering in the B ⊊ configuration agree with the cholesteric structure of the liquid crystalline solutions. From the anisotropic scattering in the B ⊊ configuration, information on the orientation ordering can be derived. For this purpose, the experimental data are compared to the form function of a uniform rod including a gaussian orientation distribution. The standard deviation of this distribution is approximately 20° for all concentrations investigated here.

Pesticide monitoring in the Netherlands: can it be improved?

In the Netherlands, pesticide monitoring of the surface water is separately managed by different water authorities. These water authorities can decide when, where, and what pesticide will be monitored and at what frequency. To help make the decisions more reasonable and make the monitoring system more efficient and systematic, three new types of monitoring maps were developed. The maps are based on actual pesticide measurement from current monitoring system and crop data. Combined temporal and spatial distribution maps give an overview of the pesticide problem in the whole Netherlands. It can be used to develop a strategy in which season and where the pesticide should be intensively monitored. Temporal distribution map, together with the pesticide usage prediction map, provide information of individual pesticide. They help to find out when and at what frequency and which pesticide be monitored, based on which monitoring facilities can be applied more efficiently.

Screening crystallisation conditions using fluorescence correlation spectroscopy

We investigate the potential of fluorescence correlation spectroscopy (FCS) in screening for crystallisation conditions. Solutions that nucleate protein crystals must have different interactions than solutions that do not give rise to crystals. Due to these different interactions the average mean squared displacement of the individual proteins changes. By monitoring protein self-diffusion, we can distinguish crystallizing from non-crystallizing solutions. The method introduced can be applied at extremely low concentrations in femtoliter volumes as an early diagnostic for molecular association. Based on our preliminary findings FCS has the potential to become a routine screening method for crystallography.

The thermodynamic characteristics of the conformational transitions of native xanthan

Abstract Xanthan shows a well-known conformational transition in salt free solution at slightly elevated temperature, upon charging. Recently, a second transition was reported at higher temperature, when potentiometric results were analyzed using a Henderson-Hasselbalch representation of the titration curves. The molar fractions of the xanthan monomers in the different conformational states A, B, and C could then be determined as a function of the degree of proton dissociation, θ. Here it is shown that a simple linear dependence of the standard free enthalpy changes (Δ G °) of the transitions on θ adequately describes the changing molar fractions throughout the titration curves. From a temperature series of the first transition ( A → B ), the dependence of the changes of standard enthalpy and entropy on θ Δ H °(θ), and Δ S °(θ) are inferred. Surprisingly, irrespective of the degree of dissociation, the A → B transition is dominated by the change of standard entropy of the transition. Beyond θ ≈ 0.3 the B state is entropy stabilized relative to the A state. The dependence of the thermally induced transitions on the polymer charge is illustrated with viscosity data.