Structure of the native chemotaxis core signaling unit from phage E-protein lysed E. coli cells.

IMPORTANCE: Bacterial chemotaxis is a ubiquitous behavior that enables cell movement toward or away from specific chemicals. It serves as an important model for understanding cell sensory signal transduction and motility. Characterization of the molecular mechanisms underlying chemotaxis is of fundamental interest and requires a high-resolution structural picture of the sensing machinery, the chemosensory array. In this study, we combine cryo-electron tomography and molecular simulation to present the complete structure of the core signaling unit, the basic building block of chemosensory arrays, from Escherichia coli. Our results provide new insight into previously poorly-resolved regions of the complex and offer a structural basis for designing new experiments to test mechanistic hypotheses.

Application of super-resolution and correlative double sampling in cryo-electron microscopy.

Developments in cryo-EM have allowed atomic or near-atomic resolution structure determination to become routine in single particle analysis (SPA). However, near-atomic resolution structures determined using cryo-electron tomography and sub-tomogram averaging (cryo-ET STA) are much less routine. In this paper, we show that collecting cryo-ET STA data using the same conditions as SPA, with both correlated double sampling (CDS) and the super-resolution mode, allowed apoferritin to be reconstructed out to the physical Nyquist frequency of the images. Even with just two tilt series, STA yields an apoferritin map at 2.9 Å resolution. These results highlight the exciting potential of cryo-ET STA in the future of protein structure determination. While processing SPA data recorded in super-resolution mode may yield structures surpassing the physical Nyquist limit, processing cryo-ET STA data in the super-resolution mode gave no additional resolution benefit. We further show that collecting SPA data in the super-resolution mode, with CDS activated, reduces the estimated B-factor, leading to a reduction in the number of particles required to reach a target resolution without compromising the data size on disk and the area imaged in SerialEM. However, collecting SPA data in CDS does reduce throughput, given that a similar resolution structure, with a slightly larger B-factor, is achievable with optimised parameters for speed in EPU (without CDS).

High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity.

Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography STA in in situ structural cell biology, we previously developed emClarity, a graphics processing unit-accelerated image-processing software that offers STA and classification of macromolecular complexes at high resolution. However, the workflow remains challenging, especially for newcomers to the field. In this protocol, we describe a detailed workflow, processing and parameters associated with each step, from initial tomography tilt-series data to the final 3D density map, with several features unique to emClarity. We use four different samples, including human immunodeficiency virus type 1 Gag assemblies, ribosome and apoferritin, to illustrate the procedure and results of STA and classification. Following the processing steps described in this protocol, along with a comprehensive tutorial and guidelines for troubleshooting and parameter optimization, one can obtain density maps up to 2.8 Å resolution from six tilt series by cryo-electron tomography STA.

Skin dose contamination conversion coefficients. Benchmark with three simulation codes.

Handling of radioactive material by operators can lead to contamination at the surface of the skin in case of an accident. The quantification of the dose received by the skin due to a contamination scenario is performed by means of dedicated dose coefficients as it is the case for other radiation protection dose quantities described in the literature. However, most available coefficients do not match realistic scenarios according to state-of-the-art of science and technology. Therefore, this work deals with dedicated dose conversion factors for skin contamination. Since there is an increasing demand on dose coefficients in general, these specific coefficients can be used for various calculations in radiation protection. In this work a method to evaluate such coefficients for the skin contamination dose related to photons, electrons, positrons, alpha and neutron particles is proposed. The coefficients are generated using Monte-Carlo simulations with three well established calculation codes (FLUKA, MCNP, and GEANT4). The results of the various codes are compared against each other for benchmarking purposes. The new dose coefficients allow the computation of the skin received dose, in the case of skin contamination scenario of an individual, taking into account the decay radiation of the radionuclides of interest. To benchmark the quantity derived here, comparisons of radionuclide contamination doses to the skin using the VARSKIN code available in the literature are performed with the results of this work.

Photons fluence to local skin Dose coefficients and benchmark with three Monte-Carlo codes. Application to the computation of radioactive material transport limits.

Fluence to Local Skin Dose Conversion Coefficients (LSD-CC) are radiological protection quantities used for external radiation exposures which allow the conversion of particle fluences into local skin equivalent dose. The International Commission on Radiological Protection published LSD-CC for electrons with an energy range from 10 keV to 10 MeV. However, the literature does not address these radiation protection quantities for all particle types, in particular for photons. In this article, computed LSD-CC values for photons are presented which enrich the literature and are of interest for the radiation protection community. As an example for an application of the use of the computed LSD-CC values, the IAEA A1/A2 working group, which supports the review of the international regulation related to the transport of radioactive material, has decided to estimate the dose to the skin using such coefficients. In this publication, LSD-CC for photons are computed and benchmarked using GEANT4, FLUKA and MCNP. In addition, the FLUKA Monte-Carlo calculation code is used to compute the LSD-CC values for electrons and positrons to compare with existing data in the literature and validate the presented models. As one application of these LSD-CC values, the transfer functions for calculating the IAEA A-values are determined using the LSD-CC and are compared to a one-step direct calculation method.

Calculation and benchmark of fluence-to-local skin equivalent dose coefficients for neutrons with FLUKA, MCNP, and GEANT4 Monte-Carlo codes.

Dose equivalent limits for single organs are recommended by the ICRP (International Commission for the Radiological Protection publication 103). These limits do not lend themselves to be measured. They are assessed by convoluting conversion factors with particle fluences. The Fluence-to-Dose conversion factors are tabulated in the ICRP literature. They allow assessing the organ dose of interest using numerical simulations. In particular, the literature lacks the knowledge of local skin equivalent dose (LSD) coefficients for neutrons. In this article, we compute such values for neutron energies ranging from 1 meV to 15 MeV. We use FLUKA, MCNP and GEANT4 Radiation transport Monte-Carlo simulation codes to perform the calculations. A comparison between these three codes is performed. These calculated values are important for radiation protection studies and radiotherapy applications.

CryoET structures of immature HIV Gag reveal six-helix bundle.

Gag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection.

Qualification of the activities measured by gamma spectrometry on unitary items of intermediate-level radioactive waste from particle accelerators.

In the frame of maintenance, upgrade and dismantling activities, activated equipment are removed from the accelerator complex and require characterization in view of their disposal as radioactive waste. The characterization process consists of a series of radiation measurements, complemented by analytical studies, which quantify the activity of radionuclides inside an object. A fraction of the radioactive waste produced at CERN presents contact dose-rates higher than 100 µSv/h, and can therefore be classified as LILW Waste ("Low and intermediate level radioactive waste"). These objects, due to the activation mechanisms, are often subject to large activity heterogeneities. The quantification of gamma-emitting radionuclides is typically performed by gamma spectrometry, under the assumption of homogeneous distributions of activity within an object. However, this assumption can lead to underestimating the activity value of such radionuclides. In this article we perform a gamma spectrometry qualification in order to quantify the impact of assuming homogenous distribution.

Qualification of gamma spectrometry measurement for the radiological characterization of mixed VLLW cables in particle accelerators.

In the framework of maintenance activities in particle accelerators, such as upgrades and dismantling, a large number of activated equipment are removed from the accelerator complex and require characterization in view of their disposal as radioactive waste. In particular, cables can be of different types. This feature induces variations of the efficiency calibration curves due to the variation of the material composition, source distribution and density. Hence, quantifying the activities of the gamma-emitting radionuclides can be quite challenging for mixed cables. In this article, we propose a new qualification methodology, based on gamma spectrometry, in order to assess the activity results uncertainties of gamma-emitting radionuclides. This new methodology is developed to define the envelop efficiency calibration curves and allows for the establishment of more accurate activity values with their corresponding uncertainties.

An enhanced characterization process for the elimination of very low level radioactive waste in particle accelerators.

The elimination of very low level waste towards the French national repository requires their radiological characterization to estimate the radionuclide inventory and the associated activities within a waste package. Such characterization is performed by means of activation calculations and measurements. Two elimination projects have been identified at CERN, to dispose of bulk metallic waste and cables activated in the CERN accelerator complex. Based on the experience gained over the last 4 years, we develop a large scale elimination process to dispose of such types of activated equipment. A program for quality controls has therefore been developed through a novel software tool whose purpose is to compute the radiological data required by the repository for the acceptance of the waste as well as performing quality controls.

Classification of radiological objects at the exit of accelerators with a dose-rate constraint.

Maintenance activities and operations of high-energy particle accelerators can lead to the collection of radioactive equipment as well as waste materials. In order to ensure their proper classification as radioactive or non-radioactive, one has to quantify the activities of radionuclides produced. According to the regulatory requirements in Switzerland, these activities need to be compared with nuclide-specific clearance limits. In particular, a new set of clearance limits was introduced by the Swiss authorities in January 2018, leading to more conservative values for a number of relevant radionuclides. We describe in this paper a new methodology based on dose-rate measurements to classify potentially radioactive objects at the exit of the CERN accelerator complex. This methodology concerns the specific material compositions typically found at CERN and takes into account the latest clearance limits introduced by the Swiss authorities.

Generation of low-energy neutrons cross-sections for the Monte Carlo code FLUKA and the deterministic code ActiWiz.

Activation of material is of interest for waste treatment and hazard assessment. In particular, activation of printed circuit can lead to the production of radionuclides at an isomeric state, for example, coming from silver. In particle accelerators, the production of silver isomeric states mainly come from low energy neutrons, below 20 MeV. The quantification of activation and associated doses at CERN is based on the FLUKA and ActiWiz codes. In the FLUKA release 2011.2c, all branching ratios for isomer production were set at 50% by default. The present work provides a set of nuclide- and energy-dependent branching ratios, extracted from the library EAF-2010. In the ActiWiz release 3.3, the library JEFF3.1.1 was used for low energy neutron cross-sections. This study provides a new set of neutron cross-sections extracted from JEFF3.3, ENDFB/VIII.0 and EAF-2010 for future update of ActiWiz.

A new gamma spectroscopy methodology based on probabilistic uncertainty estimation and conservative approach.

The gamma spectroscopy technique is commonly used in many applications to evaluate the activity of gamma emitters in a given sample. This assessment of activity is of particular interest for the disposal of radioactive waste or for clearance purposes. However, for these specific applications, one needs to show that the evaluated activities are reasonably conservative. This paper shows an application of a methodology developed to quantify the efficiency calibration curve uncertainties originating from a test case sample and its associated geometry modelling. Therefore, the effects of enclosing geometries on the activity measurement results are discussed. The purpose is to provide an example of uncertainty analysis for an approach that could be applied to other studies in which a conservative estimation of the activity is required.

A novel technique for the optimization and reduction of gamma spectroscopy geometry uncertainties.

Material activation can sometimes cause large heterogeneities in the distribution of radioactivity (hotspots). Moreover, the sample geometry parameters are not always well known. When performing gamma-spectroscopy to quantify the radionuclide inventory in activated materials, often predefined models are used to represent the sample geometry (dimensions, source-to-detector distance, material type) and their activity distribution, for efficiency calibration. This simplification causes uncertainties of the efficiency curves associated with the model and consequently, to the activity results. In this paper, we develop a new approach, based on ISOCS/LabSOCS to quantify and reduce uncertainties originating from the geometry model. The theory is described in this document and an experimental case is discussed.

Spectrum and Yield to Dose Conversion Coefficients for Beta Skin Doses Linked to the Q System.

Monte Carlo simulations are a state-of-the-art method to calculate dose coefficients and could be used with the Q system for radioactive material packaging. These simulations often take a long time to converge with sufficient precision. Furthermore, if multiple sources have to be taken into account, many weeks of calculations may be needed. In order to reduce the calculation time, this paper proposes a new method based on a transfer function to instantly compute Q values associated with beta skin doses. The method developed in this paper can be applied to compute beta skin dose and easily could be extended to other particles and different depths in organs with various kinds of shielding configurations between source and target.