X-ray phase-contrast tomography of cells manipulated with an optical stretcher.

X-rays can penetrate deeply into biological cells and thus allow for examination of their internal structures with high spatial resolution. In this study, X-ray phase-contrast imaging and tomography is combined with an X-ray-compatible optical stretcher and microfluidic sample delivery. Using this setup, individual cells can be kept in suspension while they are examined with the X-ray beam at a synchrotron. From the recorded holograms, 2D phase shift images that are proportional to the projected local electron density of the investigated cell can be calculated. From the tomographic reconstruction of multiple such projections the 3D electron density can be obtained. The cells can thus be studied in a hydrated or even living state, thus avoiding artifacts from freezing, drying or embedding, and can in principle also be subjected to different sample environments or mechanical strains. This combination of techniques is applied to living as well as fixed and stained NIH3T3 mouse fibroblasts and the effect of the beam energy on the phase shifts is investigated. Furthermore, a 3D algebraic reconstruction scheme and a dedicated mathematical description is used to follow the motion of the trapped cells in the optical stretcher for multiple rotations.

A small-angle X-ray scattering study of red blood cells in continuous flow.

Owing to their large penetration depth and high resolution, X-rays are ideally suited to study structures and structural changes within intact biological cells. For this reason, X-ray-based techniques have been used to investigate adhesive cells on solid supports. However, these techniques cannot easily be transferred to the investigation of suspended cells in flow. Here, an X-ray compatible microfluidic device that serves as a sample delivery system and measurement environment for such studies is presented. As a proof of concept, the microfluidic device is applied to investigate chemically fixed bovine red blood cells by small-angle X-ray scattering (SAXS). A very good agreement is found between in-flow and static SAXS data. Moreover, the data are fitted with a hard-sphere model and screened Coulomb interactions to obtain the radius of the protein hemoglobin within the cells. Thus, the utility of this device for studying suspended cells with SAXS in continuous flow is demonstrated.

Structural model of the M7G46 Methyltransferase TrmB in complex with tRNA.

TrmB belongs to the class I S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) and introduces a methyl group to guanine at position 7 (m7G) in tRNA. In tRNAs m7G is most frequently found at position 46 in the variable loop and forms a tertiary base pair with C13 and U22, introducing a positive charge at G46. The TrmB/Trm8 enzyme family is structurally diverse, as TrmB proteins exist in a monomeric, homodimeric, and heterodimeric form. So far, the exact enzymatic mechanism, as well as the tRNA-TrmB crystal structure is not known. Here we present the first crystal structures of B. subtilis TrmB in complex with SAM and SAH. The crystal structures of TrmB apo and in complex with SAM and SAH have been determined by X-ray crystallography to 1.9 Å (apo), 2.5 Å (SAM), and 3.1 Å (SAH). The obtained crystal structures revealed Tyr193 to be important during SAM binding and MTase activity. Applying fluorescence polarization, the dissociation constant Kd of TrmB and tRNAPhe was determined to be 0.12 µM ± 0.002 µM. Luminescence-based methyltransferase activity assays revealed cooperative effects during TrmB catalysis with half-of-the-site reactivity at physiological SAM concentrations. Structural data retrieved from small-angle x-ray scattering (SAXS), mass-spectrometry of cross-linked complexes, and molecular docking experiments led to the determination of the TrmB-tRNAPhe complex structure.