The microbial resistance to antibiotics is a genuine global threat. Consequently, a search of new inhibitors remains of acute importance due to the increasing spread of multidrug resistance. Here we present a new type of non-β-lactam β-lactamase inhibitor PA-34 based on natural phenoxyaniline, identified using computer-assisted screening of scaffolds related to those of known low-affinity inhibitors. The compound displays reversible competitive inhibition of bacterial β-lactamase TEM-171, with a Ki of 88 μM. Using enzyme kinetics, infra-red spectroscopy, fluorescence quenching and computer docking, we propose that the inhibitor binds at the entrance to the enzyme active site. This is a novel inhibition mechanism compared to binding covalently to the catalytic serine in the active site or non-covalently to the allosteric site. The residues involved in binding the inhibitor are conserved among molecular class A β-lactamases. The identified compound and its proposed binding mode may have a potential for a regulation of the catalytic activity of a wide range of class A β-lactamases. We also hypothesise that the presented route for finding non-β-lactam compounds may be an effective and durable approach for combating bacterial antibiotic resistance.

The P13 macromolecular crystallography beamline, based on the low-emittance source PETRA III, enables X-ray diffraction experiments on macromolecular crystals over a wide wavelength range (0.7–3.1 Å). The beam has a variable focus size and a small divergence enabling data collection on micrometre-sized crystals.

Telomeres are regions of non-coding DNA that cap the chromosomes, preventing the loss of coding DNA during cell division and contributing to chromosomal stability. In actively dividing cells, such as embryonic stem cells, the telomeres need to elongated by telomerase. The telomerase complex consist of the enzyme telomerase reverse transcriptase (TERT), telomerase RNA (TR) and additional proteins. TERT and TR are required for the telomerase activity in vitro. Telomerase is active in vast majority of the cancer cells ensuring continuous cell division and tumor growth. Syndromes leading to premature aging are often associated with short telomeres. Finding ways to regulate the telomerase activity would help to advance therapies for these conditions. However, the structural information available of the telomerase complex is very limited. We have chosen thermophilic yeast Hansenula polymorpha as a model system due to the stability of its proteins. The N-terminal domain of the TERT is essential for telomerase activity and possibly is involved in binding of TR, telomeric DNA and additional protein components of the telomerase complex. We have crystallised the N-terminal domain of H. polymorpha TERT and, in lack of a homologious structure, produced a seleno-methionine derivative of the protein. MAD data on N-terminal domain has been collected to resolution of 2.0 Å at the PETRA-III beamline P13 (EMBL/DESY) in Hamburg. We will discuss the structure-function relationship of the N-domain and the whole TERT component.

A couple of years ago a study was presented that set the record for the highest X-ray crystallographic resolution for a biological macromolecule [1]. The structure of the small protein crambin was determined to 0.48 Å resolution - this almost doubled the amount of available experimental data. Crambin is a small protein consisting of 46 amino acids belonging to the thionin family. Although the protein and its structure has long been known, it lacks any obvious enzymatic activity and has a hard-to-guess biological function. The protein crystallizes readily and serves as an excellent specimen for exploring the limits of resolution of the diffraction. The results demonstrated the possibilities that can be offered by a high-energy synchrotron source. The structure refined with Refmac, Shelxl and Mopro revealed a wealth of details. Bonding electron density became visible along the main chain. However, no fundamental additional structural features could be detected in comparison to the previously collected data set to 0.54 Å resolution. The availability of extremely high-resolution data is certainly of great help to drive further software development and methods for data interpretation. The question will always remain as to what the true limits are in terms of what can be seen in a biological macromolecule. Here we will present the results of our recent efforts in interpretation of such ultra-high resolution data.

Since 2012, EMBL Hamburg operates two new beamlines for macromolecular crystallography - P13 and P14 - at PETRA III at DESY (Hamburg, Germany). We exploit the high brilliance and the wide energy range offered by PETRA III to offer a wide range of conditions to fit the experimental conditions to the challenges posed by the samples. P13 provides high photon flux down to 4 keV. With a helium cone and a kappa goniostat, this allows optimized data collection for SAD phasing. Using adaptive mirrors, the focus size (H x V) can be adjusted between 30 x 20 μm^2 and 150 x 100 μm^2 to match the size of the sample. A MARVIN sample changer is in operation for rapid loading and unloading of samples. P14 offers a high photon flux (>10^12 ph/sec at 12 keV into 5 x 5 µm^2). The beamsize can be varied between 1 x 1.5 mm^2 (unfocused) and 5 x 5 µm^2 (fully focused) in less than a minute by moving the KB mirrors in and out of the beam. For small crystals, an MD3 vertical diffractometer with a sphere of confusion smaller than 100 nm offers excellent conditions. Both beamlines are equipped with PILATUS 6M-F detectors for shutter-less data collection and dedicated data processing computers. The beamlines are embedded into the 'Integrated Facility for Structural Biology' offering facilities for sample preparation and characterization, a laboratory specifically equipped for the preparation of heavy atom derivatives, and downstream facilities for data evaluation We will report about the status of the beamlines and describe typical experimental situations (small crystals, large unit cells, serial crystallography, low-energy phasing, small molecules and others).