Transcription with a laser: Radiation-damage-free diffraction of RNA Polymerase II crystals.

Well-diffracting crystals are essential to obtain relevant structural data that will lead to understanding of RNA Polymerase II (Pol II) transcriptional processes at a molecular level. Here we present a strategy to study Pol II crystals using negative stain transmission electron microscopy (TEM) and a methodology to optimize radiation damage free data collection using free electron laser (FEL) at the Linac Coherent Light Source (LCLS). The use of negative stain TEM allowed visualization and optimization of crystal diffraction by monitoring the lattice quality of crystallization conditions. Nano crystals bearing perfect lattices were seeded and used to grow larger crystals for FEL data collection. Moreover, the use of in house designed crystal loops together with ultra-violet (UV) microscopy for crystal detection facilitated data collection. Such strategy permitted collection of multiple crystals of radiation-free-damage data, resulting in the highest resolution of wild type (WT) Pol II crystals ever observed.

MicroED Structure of Au146(p-MBA)57 at Subatomic Resolution Reveals a Twinned FCC Cluster.

Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. Herein we present the structure of the largest aqueous gold cluster, Au146(p-MBA)57 (p-MBA: para-mercaptobenzoic acid), solved by electron micro-diffraction (MicroED) to subatomic resolution (0.85 Å) and by X-ray diffraction at atomic resolution (1.3 Å). The 146 gold atoms may be decomposed into two constituent sets consisting of 119 core and 27 peripheral atoms. The core atoms are organized in a twinned FCC structure, whereas the surface gold atoms follow a C2 rotational symmetry about an axis bisecting the twinning plane. The protective layer of 57 p-MBAs fully encloses the cluster and comprises bridging, monomeric, and dimeric staple motifs. Au146(p-MBA)57 is the largest cluster observed exhibiting a bulk-like FCC structure as well as the smallest gold particle exhibiting a stacking fault.

Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.

Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.

Transmission electron microscopy for the evaluation and optimization of crystal growth.

The crystallization of protein samples remains the most significant challenge in structure determination by X-ray crystallography. Here, the effectiveness of transmission electron microscopy (TEM) analysis to aid in the crystallization of biological macromolecules is demonstrated. It was found that the presence of well ordered lattices with higher order Bragg spots, revealed by Fourier analysis of TEM images, is a good predictor of diffraction-quality crystals. Moreover, the use of TEM allowed (i) comparison of lattice quality among crystals from different conditions in crystallization screens; (ii) the detection of crystal pathologies that could contribute to poor X-ray diffraction, including crystal lattice defects, anisotropic diffraction and crystal contamination by heavy protein aggregates and nanocrystal nuclei; (iii) the qualitative estimation of crystal solvent content to explore the effect of lattice dehydration on diffraction and (iv) the selection of high-quality crystal fragments for microseeding experiments to generate reproducibly larger sized crystals. Applications to X-ray free-electron laser (XFEL) and micro-electron diffraction (microED) experiments are also discussed.

Structural Basis for the Interconversion of Maltodextrins by MalQ, the Amylomaltase of Escherichia coli.

Amylomaltase MalQ is essential for the metabolism of maltose and maltodextrins in Escherichia coli. It catalyzes transglycosylation/disproportionation reactions in which glycosyl or dextrinyl units are transferred among linear maltodextrins of various lengths. To elucidate the molecular basis of transglycosylation by MalQ, we have determined three crystal structures of this enzyme, i.e. the apo-form, its complex with maltose, and an inhibitor complex with the transition state analog acarviosine-glucose-acarbose, at resolutions down to 2.1 Å. MalQ represents the first example of a mesophilic bacterial amylomaltase with known structure and exhibits an N-terminal extension of about 140 residues, in contrast with previously described thermophilic enzymes. This moiety seems unique to amylomaltases from Enterobacteriaceae and folds into two distinct subdomains that associate with different parts of the catalytic core. Intriguingly, the three MalQ crystal structures appear to correspond to distinct states of this enzyme, revealing considerable conformational changes during the catalytic cycle. In particular, the inhibitor complex highlights the requirement of both a 3-OH group and a 4-OH group (or α1-4-glycosidic bond) at the acceptor subsite +1 for the catalytically competent orientation of the acid/base catalyst Glu-496. Using an HPLC-based MalQ enzyme assay, we could demonstrate that the equilibrium concentration of maltodextrin products depends on the length of the initial substrate; with increasing numbers of glycosidic bonds, less glucose is formed. Thus, both structural and enzymatic data are consistent with the extremely low hydrolysis rates observed for amylomaltases and underline the importance of MalQ for the metabolism of maltodextrins in E. coli.

[Diagnosis of space-occupying renal processes: sonography and aimed fine-needle biopsy]. / Diagnostik raumfordernder renaler Prozesse: Sonographie und gezielte Feinnadel-Biopsie.

It is reported on 3 cases in which by sonographic examination a tumorous process could be established. In all cases the additional investigation by fine needle biopsy and cytological examination proved the presence of malignant cells. This local renal process proved by sonography could be verified neither by i.v. pyelographic nor arteriographic methods. For two patients the diagnosis based on sonography, fine needle puncture and cytological test could be verified by operation. The third patient was not operated in spite of malignant cells in the cytogram because of negative computer tomography provided by an external hospital.

[The normal pancreas. Demonstration in the sonogram in relation to age]. / Das normale Pankreas. Darstellung im Sonogramm in Abhängigkeit zum Lebensalter.

For 191 patients without any clinically or anamnestically traceable disease of pancreas sonographic inspection was performed, a real time-equipment type Aloka, SSD 240 was used. The age of the patients, divided into 5 groups ranged from 15 to 90 years. The measurements of the pancreas sonographic inspection was performed, a real time-equipment type Aloka, SSD 240 was used. The age of the patients, divided into 5 groups ranged from 15 to 90 years. The measurements of the pancreas were established for head, neck, corpus and tail respectively. The following mean values for the thickness were found: Head: 2,41 +/- 0,41 cm; neck: 1,53 +/- 0,34 cm; corpus: 1,90 +/- 0,37 cm; tail: 1,67 +/- 0,37 cm. The width of the organ was in the area of the head: 1,3 to 3,5 cm, neck 1,0 to 2,7 cm, corpus 1,1 to 3,0 cm, tail 0,8 to 2,6 cm. A comparison between normal weight- and overweight-patients showed no significant differences at all. The echostructure of the pancreas showed a clear increase of intensity with progress in age. The reason for this more detailed echostructure is seen in an infiltration of fat as well as collagenous and fibrous elements. No change in organ size was found in this connection.

[Unexplained accelerated blood cells sedimentation rate in the aged (author's transl)]. / Unklare Blutsenkungsbeschleunigung im höheren Lebensalter.

There is one reason for unexplained accelerated blood cells sedimentation rate in the aged, which is often neglected:Polymyalgia arteriitica or Arteriitis temporalis. Long-term therapy with cortison normalizes pains and sedimentation rate. Nevertheless, short-term treatment is a way to diagnose the disease "ex juvantibus".