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In the framework of expanding the range of copper‐based compounds in the pyroxene family, we have synthesized at high pressure and high temperature a powder containing a mixture of a new phase with stoichiometry Sr5CuGe9O24 having two identified impurity phases. Electron crystallography showed that the new phase crystallizes in the monoclinic space group P2/c, with unit‐cell parameters a = 11.8 Å, b = 8.1 Å, c = 10.3 Å and β = 101.3°. We applied the recently developed low‐dose electron diffraction tomography method to solve the structure by direct methods. The obtained structure model contains all 9 independent cation positions and all 13 oxygen positions. A subsequent refinement against powder X‐ray diffraction data ascertained the high quality of the structure solution, in particular, the unusual structural arrangement that there are three different environments for Ge in this phase. [ABSTRACT FROM AUTHOR]

Synthetic riboswitches mediating ligand-dependent RNA cleavage or splicing-modulation represent elegant tools to control gene expression in various applications, including next-generation gene therapy. However, due to the limited understanding of context-dependent structure–function relationships, the identification of functional riboswitches requires large-scale-screening of aptamer-effector-domain designs, which is hampered by the lack of suitable cellular high-throughput methods. Here we describe a fast and broadly applicable method to functionally screen complex riboswitch libraries (~1.8 × 104 constructs) by cDNA-amplicon-sequencing in transiently transfected and stimulated human cells. The self-barcoding nature of each construct enables quantification of differential mRNA levels without additional pre-selection or cDNA-manipulation steps. We apply this method to engineer tetracycline- and guanine-responsive ON- and OFF-switches based on hammerhead, hepatitis-delta-virus and Twister ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices. In summary, our method enables fast and efficient high-throughput riboswitch identification, thereby overcoming a major hurdle in the development cascade for therapeutically applicable gene switches. Riboswitches can mediate ligand-dependent RNA cleavage and splicing to control gene expression. Here the authors present a method to functionally screen large libraries and identify functional variants. [ABSTRACT FROM AUTHOR]

• LD-EDT allows obtaining complete electron diffraction data sets in an automated way. • LD-EDT is optimized for dose efficiency on beam sensitive samples. • The high data quality allows solving structures even if the completeness is low. • No special equipment in needed for this method, its implementation is easy. • LD-EDT is fast and can be a routine method for screening inhomogeneous samples. A new electron diffraction tomography method is presented that is aimed to enable high performance electron crystallography experiments on beam sensitive materials using a standard TEM without any special equipment. Low-dose electron diffraction tomography minimizes the electron dose necessary for obtaining data sets suitable for structure solution by irradiating the crystal exclusively during the acquisition of the diffraction patterns. The performance of the method is successfully tested on two model structures, a complex oxide Sr 5 CuGe 9 O 24 and the beam sensitive metal-organic framework (MOF) of manganese formiate. Even when the limited lifetime of a beam sensitive material only allows obtaining a data set of rather low completeness, the data quality is high enough for the structures to be solved to a high precision. Low-dose electron diffraction tomography is easy to implement and doesn't require any special equipment or lengthy calibration processes, making it accessible to a large number of scientists. [ABSTRACT FROM AUTHOR]

• Beam damage in 3D ED is reduced with a data acquisition scheme adapted from cryoET. • Damage-free data from multiple particles is merged to improve data completeness. • Effective electron dose can be lowered up to an order of magnitude. Beam sensitive nanomaterials such as zeolites or metal-organic frameworks (MOF) represent a great challenge for crystallographic structure determination and refinement. The strong electron-matter interaction and the high spatial resolution achievable make electron diffraction the technique of choice for particles of sizes below a micrometer and many different 3-dimensional electron diffraction (3D ED) techniques have been developed in recent years. Nevertheless, beam sensitivity of the samples can lead to the crystal structure being damaged during the data acquisition impeding the determination of its structure. A simple way to reduce beam damage is to lower the dose during the experiment. However, this implies weaker diffraction intensities which can become problematic for the exploitation of the data. In order to obtain complete data sets with strong intensities without damaging the crystals, we developed the dose symmetric electron diffraction tomography (DS-EDT) method, combining the low-dose electron diffraction tomography (LD-EDT) technique with the dose-symmetric tomography scheme known from cryo-EM. In order to reduce the dose on an individual crystal and still obtain enough data for a structure solution and refinement, we partition the dose over several crystals. The individual datasets are then merged in order to achieve the necessary completeness. On two test structures we first show that merging of data from small domains of the reciprocal space is indeed sufficient to obtain reliable data for structure solution and refinement. Second, we show on the beam sensitive manganese formate that high-quality data can be obtained on a few frames while the frames that have suffered from beam damage can still be used to determine the orientation matrix and the unit cell of the crystals. The results from the dynamical refinement on the obtained data show a high accuracy of the atom positions. In this way, DS-EDT can reduce the total dose on an individual crystal by an order of magnitude with respect to the already very dose-efficient LD-EDT. [ABSTRACT FROM AUTHOR]