Practical Image Restoration of Thick Biological Specimens Using Multiple Focus Levels in Transmission Electron Microscopy

Three-dimensional electron tomographic studies of thick specimens such as cellular organelles or supramolecular structures require accurate interpretations of transmission electron micrograph intensities. In addition to microscope lens aberrations, thick specimen imaging is complicated by additional distortions resulting from multiple elastic and inelastic scattering. Extensive analysis of the mechanism of image formation using electron energy-loss spectroscopy and imaging as well as exit wavefront reconstruction demonstrated that multiple scattering does not contribute to the coherent component of the exit wave (Hanet al.,1996, 1995). Although exit wavefront restored images showed enhanced contrast and resolution, that technique, which requires the collection of more than 30 images at different focus levels, is not practical for routine data collection in 3D electron tomography, where usually over 100 projection views are required for each reconstruction. Using a 0.7-μm-thick specimen imaged at 200 keV, the accuracy of reconstructions using small numbers of defocused images and a simple linear filter (Schiske, 1968) was assessed by comparison to the complete exit wave restoration. We demonstrate that only four optimal focus levels are required to effectively restore the coherent component (deviation 5.1%). By contrast, the optimal single image (zero defocus) shows a 25.5% deviation to the exit wave restoration. Two pairs of under- and over-defocus images should be taken: one pair at quite high defocus (>10 μm) to differentiate the coherent (single elastic scattering) from the incoherent (multiple elastic and inelastic scattering) components, and the second pair to optimize information content at the highest desired resolution (e.g., 5 μm for (2.5 nm)⁻¹resolution). We also propose a new interpretation of the restored amplitude and phase components where the specimen mass-density is proportional to the logarithm of the amplitude component and linearly related to the phase component. This approach should greatly facilitate the collection of high resolution tomographic data from thick samples.     

Effect of Oxidation on the Structure of Human Low- and High-Density Lipoproteins

This work presents a controlled study of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) structural changes due to in vitro oxidation with copper ions. The changes were studied by small-angle x-ray scattering (SAXS) and dynamic light scattering (DLS) techniques in the case of LDL and by SAXS, DLS, and Z-scan (ZS) techniques in the case of HDL. SAXS data were analyzed with a to our knowledge new deconvolution method. This method provides the electron density profile of the samples directly from the intensity scattering of the monomers. Results show that LDL particles oxidized for 18 h show significant structural changes when compared to nonoxidized particles. Changes were observed in the electrical density profile, in size polydispersity, and in the degree of flexibility of the APO-B protein on the particle. HDL optical results obtained with the ZS technique showed a decrease of the amplitude of the nonlinear optical signal as a function of oxidation time. In contrast to LDL results reported in the literature, the HDL ZS signal does not lead to a complete loss of nonlinear optical signal after 18 h of copper oxidation. Also, the SAXS results did not indicate significant structural changes due to oxidation of HDL particles, and DLS results showed that a small number of oligomers formed in the sample oxidized for 18 h. All experimental results for the HDL samples indicate that this lipoprotein is more resistant to the oxidation process than are LDL particles.     

The effect of model approximations on single-collision distributions of low-energy electrons in liquid water

The development of cross sections for the inelastic interaction of low-energy electrons with condensed tissue-like media is best accomplished within the framework of the dielectric theory. In this work we investigate the degree to which various model approximations, used in the above methodology, influence electron single-collision distributions. These distributions are of major importance to Monte Carlo track structure codes, namely, the energy-loss spectrum, the inelastic inverse mean free path, and the ionization efficiency. In particular, we make quantitative assessment of the influence of (1) the optical data set, (2) the dispersion algorithm, and (3) the perturbation and exchange Born corrections. It is shown that, although the shape and position of the energy-loss spectrum remains almost fixed, its peak height may vary by up to a factor of 1.5. Discrepancies in the calculated inelastic inverse mean free path are largely within 20-30% above 100 eV; they increase drastically, though, at lower energies. Exchange and perturbation Born corrections increase gradually below 1 keV leading to a approximately 30 to 40% reduction of the inverse mean free path at 100 eV. The perturbation effect contributes more than the exchange effect to this reduction. Similar to the dispersion situation, the effect of Born corrections at lower energies is also unclear since the models examined disagree strongly below 100 eV. In comparison, the vapor data are higher than the liquid calculations by 20 to 50% as the energy decreases from 1 to 0.1 keV, respectively. The excitation contribution is the main cause of this difference, since the ionization efficiency in the liquid levels off at approximately 90%, whereas the plateau value for the vapor is approximately 70%. It is concluded that electron inelastic distributions for liquid water, although in some respects distinctively different from the vapor phase, have associated uncertainties that are comparable in magnitude to the phase differences. The situation below 100 eV is uncertain.     

3.9 ? structure of the nucleosome core particle determined by phase-plate cryo-EM

The Volta phase plate is a recently developed electron cryo-microscopy (cryo-EM) device that enables contrast enhancement of biological samples. Here we have evaluated the potential of combining phase-plate imaging and single particle analysis to determine the structure of a small protein–DNA complex. To test the method, we made use of a 200 kDa Nucleosome Core Particle (NCP) reconstituted with 601 DNA for which a high-resolution X-ray crystal structure is known. We find that the phase plate provides a significant contrast enhancement that permits individual NCPs and DNA to be clearly identified in amorphous ice. The refined structure from 26,060 particles has an overall resolution of 3.9 Å and the density map exhibits structural features consistent with the estimated resolution, including clear density for amino acid side chains and DNA features such as the phosphate backbone. Our results demonstrate that phase-plate cryo-EM promises to become an important method to determine novel near-atomic resolution structures of small and challenging samples, such as nucleosomes in complex with nucleosome-binding factors.     

Quantitative evaluation of neurons in the mucosal plexus of adult human intestines

Scanning transmission electron tomography offers enhanced contrast compared to regular transmission electron microscopy, and thicker samples, up to 1 μm or more, can be analyzed, since the depth of focus and inelastic scattering are not limitations. In this study, we combine this novel imaging approach with state of the art specimen preparation by using novel light transparent sapphire specimen carrier for high-pressure freezing and a freeze substitution protocol for better contrast of membranes. This combination allows for imaging membranes and other subcellular structures with unsurpassed quality. This is demonstrated with mitochondria, where the inner and outer mitochondrial membranes as well as the membranes in the cristae appear in very close apposition with a minimal intermembrane space. These findings correspond well with old observations using freeze fracturing. In 880-nm thick sections of hemophagocytes, the three-dimensional structure of membrane sheets could be observed in the virtual sections of the tomogram. Microtubules, actin and intermediate filaments could be visualized within one sample. Intermediate filaments, however, could even be better observed in 3D using surface scanning electron tomography.     

Solution x-ray scattering-based estimation of electron cryomicroscopy imaging parameters for reconstruction of virus particles

Structure factor amplitudes and phases can be computed directly from electron cryomicroscopy images. Inherent aberrations of the electromagnetic lenses and other instrumental factors affect the structure factors, however, resulting in decreased accuracy in the determined three-dimensional reconstruction. In contrast, solution x-ray scattering provides absolute and accurate measurement of spherically averaged structure factor amplitudes of particles in solution but does not provide information on the phases. In the present study, we explore the merits of using solution x-ray scattering data to estimate the imaging parameters necessary to make corrections to the structure factor amplitudes derived from electron cryomicroscopic images of icosahedral virus particles. Using 400-kV spot-scan images of the bacteriophage P22 procapsid, we have calculated an amplitude contrast of 8.0 +/- 5.2%. The amplitude decay parameter has been estimated to be 523 +/- 188 A2 with image noise compensation and 44 +/- 66 A2 without it. These results can also be used to estimate the minimum number of virus particles needed for reconstruction at different resolutions.     

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 µm tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells – on the level of a few protein molecules in germinal centers.     

An atypical BRCT–BRCT interaction with the XRCC1 scaffold protein compacts human DNA Ligase IIIα within a flexible DNA repair complex

The XRCC1–DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT–BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS–MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT–BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.     

Electron stopping power and inelastic mean free path in amino acids and protein over the energy range of 20–20,000 eV

Systematic calculations of stopping power (SPs) and inelastic mean free path (IMFP) values for 20–20,000 eV electrons in a group of 15 amino acids and a simple protein have been performed. The calculations are based on the dielectric response model and take into account the exchange effect between the incident electron and target electrons. The optical energy-loss functions for the 15 investigated amino acids and the protein are evaluated by using an empirical approach, because of the lack of experimental optical data. For all the considered materials, the calculated mean ionization potentials are in good agreement with those given by Bragg’s rule, and the evaluated SP values at 20 keV converge well to the Bethe–Bloch predictions. The data shown represent the first results of SP and IMFP, for these 15 amino acids and the protein in the energy range below 20 keV, and might be useful for studies of various radiation effects in these materials. In addition, the average energy deposited by inelastic scattering of the electrons on this group of 15 amino acids, on the protein, on Formvar and on DNA, respectively, has been estimated for energies below 20 keV. The dependences of the average energy deposition on the electron energy are given. These results are important for any detailed studies of radiation-induced inactivation of proteins and the DNA.     

Zero-loss energy filtering as improved imaging mode in cryoelectronmicroscopy of frozen-hydrated specimens

One of the remaining problems in attaining higher structural resolution with cryoelectronmicroscopy of frozen-hydrated specimens is the low contrast of micrographs taken close to the electron optical focus. By measuring electron energy loss spectra (EELS) of ice layers we show that a large fraction of incident electrons undergoes an inelastic electron-plasmon scattering process. Thus these electrons do not carry structural information of the protein but increase the background of the electron image and therefore reduce the contrast of the negative. Here we report the improvement in contrast gained by filtering out inelastically scattered electrons using an energy-filtered transmission electron microscope (EFTEM). This gain in contrast permits a dramatic decrease in defocusing values, resulting in improved structural resolution. In addition, the increased signal to noise ratio allows the recording of micrographs at a reduced electron dose. This should result in less damage to vitrified and unstained proteins and other beam-sensitive specimens.     

Effect of Oxidation on the Structure of Human Low- and High-Density Lipoproteins

This work presents a controlled study of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) structural changes due to in vitro oxidation with copper ions. The changes were studied by small-angle x-ray scattering (SAXS) and dynamic light scattering (DLS) techniques in the case of LDL and by SAXS, DLS, and Z-scan (ZS) techniques in the case of HDL. SAXS data were analyzed with a to our knowledge new deconvolution method. This method provides the electron density profile of the samples directly from the intensity scattering of the monomers. Results show that LDL particles oxidized for 18 h show significant structural changes when compared to nonoxidized particles. Changes were observed in the electrical density profile, in size polydispersity, and in the degree of flexibility of the APO-B protein on the particle. HDL optical results obtained with the ZS technique showed a decrease of the amplitude of the nonlinear optical signal as a function of oxidation time. In contrast to LDL results reported in the literature, the HDL ZS signal does not lead to a complete loss of nonlinear optical signal after 18 h of copper oxidation. Also, the SAXS results did not indicate significant structural changes due to oxidation of HDL particles, and DLS results showed that a small number of oligomers formed in the sample oxidized for 18 h. All experimental results for the HDL samples indicate that this lipoprotein is more resistant to the oxidation process than are LDL particles.     

Inhaled steroid/tobacco smoke particle interactions: a new light on steroid resistance

Background

Inhaled steroid resistance is an obstacle to asthma control in asthmatic smokers. The reasons of this phenomenon are not yet entirely understood. Interaction of drug particles with environmental tobacco smoke (ETS) could change the aerodynamic profile of the drug through the particle coagulation phenomenon. Aim of the present study was to examine whether steroid particles interact with smoke when delivered in the presence of ETS.

Methods

Beclomethasone-hydrofluoralkane (BDP-HFA) pMDI particle profile was studied after a single actuation delivered in ambient air or in the presence of ETS in an experimental chamber using a light scattering Optical Particle Counter capable of measuring the concentrations of particle sized 0.3–1.0, 1.1–2.0, 2.1–3.0, 3.1–4.0, 4.1–5.0, and > 5.1 μm in diameter with a sampling time of one second. The number of drug particles delivered after a single actuation was measured as the difference between total particle number after drug delivery and background particle number. Two groups of experiments were carried out at different ambient background particle concentrations. Two-tail Student''s t-test was used for statistical analysis.

Results

When delivered in ambient air, over 90% of BDP-HFA particles were found in the 0.3–1.0 μm size class, while particles sized 1.1–2.0 μm and 2.1–3.0 represented less than 6.6% and 2.8% of total particles, respectively. However, when delivered in the presence of ETS, drug particle profile was modified, with an impressive decrease of 0.3–1.0 μm particles, the most represented particles resulting those sized 1.1–2.0 μm (over 66.6% of total particles), and 2.1–3.0 μm particles accounting up to 31% of total particles.

Conclusion

Our data suggest that particle interaction between inhaled BDP-HFA pMDI and ETS takes place in the first few seconds after drug delivery, with a decrease in smaller particles and a concurrent increase of larger particles. The resulting changes in aerosol particle profile might modify regional drug deposition with potential detriment to drug efficacy, and represent a new element of steroid resistance in smokers. Although the present study does not provide any functional or clinical assessment, it might be useful to advise smokers and non smokers with obstructive lung disease such as asthma or COPD, to avoid to act inhaled drugs in the presence of ETS in order to obtain the best therapeutic effect.     

Considerations of coherent scattering and electron binding in incoherent scattering, in computation of dose deposition in tissue from low-energy photon beams

Two different sets of Monte Carlo computations were carried out for the study of dose penetration of monoenergetic, low-energy (10 to 100 keV) photon beams incident on slabs of tissue. One program took into account coherent scattering and considered electron binding when finding the angle of scattering during incoherent scattering; the other simpler program, customarily used at higher energies, largely ignored these effects. For calculations at the source photon energy of 100 keV, it was found that there was negligible difference in dose distribution in the slab between the more and less complex type of calculations. The same thing was found to be true for the 30 and 10-keV source photon energies only for shallow penetration distances; and at deeper penetrations the simple approach tended to overestimate the dose appreciably. It is concluded that for penetration of low-energy photon beams into tissue, accurate calculational results cannot be assured with the neglect of coherent scattering effects and electron binding considerations in determining the scattering angles except for shallow depths of penetration.     

Dynamics of Protein and its Hydration Water: Neutron Scattering Studies on Fully Deuterated GFP

We present a detailed analysis of the picosecond-to-nanosecond motions of green fluorescent protein (GFP) and its hydration water using neutron scattering spectroscopy and hydrogen/deuterium contrast. The analysis reveals that hydration water suppresses protein motions at lower temperatures (<∼200 K), and facilitates protein dynamics at high temperatures. Experimental data demonstrate that the hydration water is harmonic at temperatures <∼180–190 K and is not affected by the proteins’ methyl group rotations. The dynamics of the hydration water exhibits changes at ∼180–190 K that we ascribe to the glass transition in the hydrated protein. Our results confirm significant differences in the dynamics of protein and its hydration water at high temperatures: on the picosecond-to-nanosecond timescale, the hydration water exhibits diffusive dynamics, while the protein motions are localized to <∼3 Å. The diffusion of the GFP hydration water is similar to the behavior of hydration water previously observed for other proteins. Comparison with other globular proteins (e.g., lysozyme) reveals that on the timescale of 1 ns and at equivalent hydration level, GFP dynamics (mean-square displacements and quasielastic intensity) are of much smaller amplitude. Moreover, the suppression of the protein dynamics by the hydration water at low temperatures appears to be stronger in GFP than in other globular proteins. We ascribe this observation to the barrellike structure of GFP.     

A complete dielectric response model for liquid water: a solution of the Bethe ridge problem

We present a complete yet computationally simple model for the dielectric response function of liquid water over the energy-momentum plane, which, in contrast to earlier models, is consistent with the recent inelastic X-ray scattering spectroscopy data at both zero and finite momentum transfer values. The model follows Ritchie's extended-Drude algorithm and is particularly effective at the region of the Bethe ridge, substantially improving previous models. The present development allows for a more accurate simulation of the inelastic scattering and energy deposition process of low-energy electrons in liquid water and other biomaterials. As an example, we calculate the stopping power of liquid water for electrons over the 0.1-10 keV range where direct experimental measurements are still impractical and the Bethe stopping formula is inaccurate. The new stopping power values are up to 30-40% lower than previous calculations. Within the range of validity of the first Born approximation, the new values are accurate to within the experimental uncertainties (a few percent). At the low end, the introduction of Born corrections raises the uncertainty to perhaps approximately 10%. Thus the present model helps extend the ICRU electron stopping power database for liquid water down to about two orders of magnitude with a comparable level of uncertainty.     

Label-Free Single-Particle Imaging of the Influenza Virus by Objective-Type Total Internal Reflection Dark-Field Microscopy

Here we report label-free optical imaging of single particles of the influenza virus attached on a glass surface with a simple objective-type total internal reflection dark-field microscopy (TIRDFM). The capability of TIRDFM for the imaging of single viral particles was confirmed from fine correlation of the TIRDFM images with fluorescent immunostaining image and scanning electron microscopy image. The density of scattering spots in the TIRDFM images showed a good linearity against the virus concentration, giving the limit of detection as 1.2×10⁴ plaque-forming units per milliliter. Our label-free optical imaging method does require neither elaborated sample preparation nor complex optical systems, offering a good platform for rapid and sensitive counting of viral particles.     

Dual Energy CT Angiography of Peripheral Arterial Disease: Feasibility of Using Lower Contrast Medium Volume

ObjectiveOne of the main drawbacks associated with Dual Energy Computed Tomography Angiography (DECTA) is the risk of developing contrast medium-induced nephropathy (CIN). The aim of the present study was firstly, to design an optimal CT imaging protocol by determining the feasibility of using a reduced contrast medium volume in peripheral arterial DECTA, and secondly, to compare the results with those obtained from using routine contrast medium volume.MethodsThirty four patients underwent DECTA for the diagnosis of peripheral arterial disease. They were randomly divided into two groups: Group 1 (routine contrast volume group) with n = 17, injection rate 4–5 ml/s, and 1.5 ml/kg of contrast medium, and Group 2 ((low contrast volume group), with n = 17, injection rate 4–5ml/s, and contrast medium volume 0.75 ml/kg. A fast kilovoltage—switching 64-slice CT scanner in the dual-energy mode was employed for the study. A total of 6 datasets of monochromatic images at 50, 55, 60, 65, 70 and 75 keV levels were reconstructed with adaptive statistical iterative reconstruction (ASIR) at 50%. A 4-point scale was the tool for qualitative analysis of results. The two groups were compared and assessed quantitatively for image quality on the basis of signal-to-noise ratio (SNR) and contrast-to-noise-ratio (CNR). Radiation and contrast medium doses were also compared.ResultsThe overall mean CT attenuation and mean noise for all lower extremity body parts was significantly lower for the low volume contrast group (p<0.001), and varied significantly between groups (p = 0.001), body parts (p<0.001) and keVs (p<0.001). The interaction between group body parts was significant with CT attenuation and CNR (p = 0.002 and 0.003 respectively), and marginally significant with SNR (p = 0.047), with minimal changes noticed between the two groups. Group 2 (low contrast volume group) displayed the lowest image noise between 65 and 70 keV, recorded the highest SNR and CNR at 65 keV, and produced significantly lower results with respect to contrast medium volume and duration of contrast injection (p<0.001). The effect of radiation dose was not statistically significant between the two groups.ConclusionsDECTA images created at 65 keV and 50% ASIR with low contrast medium volume protocol, yielded results that were comparable to routine contrast medium volume, with acceptable diagnostic images produced during the evaluation of peripheral arteries.     

Low-energy electron penetration range in liquid water

Monte Carlo simulations of electron tracks in liquid water are performed to calculate the energy dependence of the electron penetration range at initial electron energies between 0.2 eV and 150 keV, including the subexcitation electron region (<7.3 eV). Our calculated electron penetration distances are compared with available experimental data and earlier calculations as well as with the results of simulations using newly reported amorphous ice electron scattering cross sections in the range approximately 1-100 eV.     

The beamlines of ELETTRA and their application to structural biology

Protein crystallographers are nowadays regular users of synchrotron radiation (SR) facilities for several applications. The goal of majority of users is simply to extract more accurate, higher resolution data from existing crystals; they use monochromatic radiation and the rotation method, in order to get a complete survey of the reciprocal space in a short time. In fact the brilliance of SR is essential, due to the weak scattering power of the samples, and because of their sensibility to radiation damage. Over the last few years, however, a general increase of interest for measurements at multiple wavelengths, which exploit the anomalous dispersion for the phase problem (multiwavelength anomalous diffraction — MAD), has generated the need of intense tuneable sources. For these applications, the emphasis is on accurate measurements of the small differences between the intensities of Bragg reflections at various energies across the absorption edge of an element present in the sample. The macromolecular diffraction beamline at ELETTRA, which is now running routinely since spring 1995, has been designed to provide a high flux — highly collimated tuneable X-rays source in the spectral range between 4 and 25 keV. The radiation source is the 57-pole wiggler, which delivers a very intense radiation up to 25 keV, and is shared and used simultaneously with the small angle X-ray scattering (SAXS) beamline. The front-end filter system has a cut-off energy at about 4 keV. The beamline optics consists of a pseudo-channel-cut double-crystal monochromator followed by a double focusing toroidal mirror. The tunability and the stability of the monochromator allows the user to perform MAD experiments, and for this purpose, a fluorescence probe for the exact calibration of the absorption edge is available on-line. The experimental station is based on an imaging plate area detector from MarResearch, with a sensible area of 345 mm in diameter. A cooled N₂-stream is available to cool the sample crystal in order to reduce the radiation damage. SAXS is an experimental technique used to derive structural information about supra-molecular assemblies, amorphous materials and partly ordered systems (e.g. size and shape of large molecules). The high-flux SAXS beamline at ELETTRA is mainly intended for time-resolved studies on fast structural transitions in the sub-millisecond time region in solutions and in partly ordered systems, triggered by external or process parameters, with a SAXS resolution between 10 and 1400 Å in real space. The source is the already mentioned 57-pole and the SAXS beamline accepts three discrete energies of its spectrum, namely 5.4, 8 and 16 keV. The beamline optics consists of a flat double-crystal monochromator and a double focusing toroidal mirror. A multi-purpose sample stage, movable along an optical table in order to optimise the sample to detect distance, allows to perform fast time-resolved relaxation studies based on temperature- or pressure-jumps as well as stopped flow experiments. Moreover, the users have option to install their own specialised sample surrounding equipment. The optimisation of the beamline with respect to high-flux and consequently high-flux density, allows to perform the following experiments: low contrast solution scattering, grazing incidence surface diffraction, micro-spot scanning, X-ray fluorescence analysis, time-resolved studies 11 s, simultaneous small- and wide-angle measurements on gels, liquid crystals, biopolymers, amorphous materials, muscles.This revised version was published online in October 2005 with corrections to the Cover Date.     

Quantitative analysis of the formation of nucleoprotein complexes between HIV-1 Gag protein and genomic RNA using transmission electron microscopy

In HIV, the polyprotein precursor Gag orchestrates the formation of the viral capsid. In the current view of this viral assembly, Gag forms low-order oligomers that bind to the viral genomic RNA triggering the formation of high-ordered ribonucleoprotein complexes. However, this assembly model was established using biochemical or imaging methods that do not describe the cellular location hosting Gag–gRNA complex nor distinguish gRNA packaging in single particles. Here, we studied the intracellular localization of these complexes by electron microscopy and monitored the distances between the two partners by morphometric analysis of gold beads specifically labeling Gag and gRNA. We found that formation of these viral clusters occurred shortly after the nuclear export of the gRNA. During their transport to the plasma membrane, the distance between Gag and gRNA decreases together with an increase of gRNA packaging. Point mutations in the zinc finger patterns of the nucleocapsid domain of Gag caused an increase in the distance between Gag and gRNA as well as a sharp decrease of gRNA packaged into virions. Finally, we show that removal of stem loop 1 of the 5′-untranslated region does not interfere with gRNA packaging, whereas combined with the removal of stem loop 3 is sufficient to decrease but not abolish Gag-gRNA cluster formation and gRNA packaging. In conclusion, this morphometric analysis of Gag-gRNA cluster formation sheds new light on HIV-1 assembly that can be used to describe at nanoscale resolution other viral assembly steps involving RNA or protein–protein interactions.