Structure of a canonical RNase YoeB bound to the ribosome

As a typical endoribonuclease, YoeB mediates cellular adaptation in diverse bacteria by degrading mRNAs on its activation. Although the catalytic core of YoeB is thought to be identical to well-studied nucleases, this enzyme specifically targets mRNA substrates that are associated with ribosomes in vivo. However, the molecular mechanism of mRNA recognition and cleavage by YoeB, and the requirement of ribosome for its optimal activity, largely remain elusive. Here, we report the structure of YoeB bound to 70S ribosome in pre-cleavage state, revealing that both the 30S and 50S subunits participate in YoeB binding. The mRNA is recognized by the catalytic core of YoeB, of which the general base/acid (Glu46/His83) are within hydrogen-bonding distance to their reaction atoms, demonstrating an active conformation of YoeB on ribosome. Also, the mRNA orientation involves the universally conserved A1493 and G530 of 16S rRNA. In addition, mass spectrometry data indicated that YoeB cleaves mRNA following the second position at the A-site codon, resulting in a final product with a 3'-phosphate at the newly formed 3' end. Our results demonstrate a classical acid-base catalysis for YoeB-mediated RNA hydrolysis and provide insight into how the ribosome is essential for its specific activity.

Room Temperature Serial Crystallography at Synchrotrons

Serial Femtosecond Crystallography (SFX) is the most commonly used method for the emerging structure determination at X-ray free-electron lasers (FELs). The high peak brilliance of the FEL and the possibility of using femtosecond pulses afford use of nano-to-micron sized crystals in a diffraction-before-destruction approach for the acquisition of high-resolution undamaged diffraction data [1]. The crystals are obliterated upon exposure to an FEL X-ray pulse so only a single snapshot can be collected per crystal, necessitating a constant supply of fresh crystals. The crystals are therefore injected in a liquid microjet [2], [3]. We show that this serial method of data collection and the associated data analysis can be successfully adapted to serial crystallography (SX) measurements at synchrotrons, enabling room temperature studies using the unattenuated beam. Given the continuous supply of fresh crystals, the full tolerable dose can be used for each single crystal exposure, permitting analysis of small or weakly scattering crystals. FEL X-ray pulses are much shorter than the fraction of a second exposure time at a synchrotron, so SFX injection conditions are modified in SX such as to slow down the typically fast travelling crystals. By embedding the crystals in a viscous material the crystals remain in the beam long enough to yield measurable diffraction and smearing out of the diffraction peaks due to crystal tumbling is avoided. We demonstrate the successful application of room temperature SX at the Swiss Light Source at ambient pressure. Our experimental setup allows collection of both still and rotation data. Recent progress using model systems will be presented, establishing this high throughput, high dose rate approach as a new route to structure determination of macromolecules in their native environment and at room temperature.

Optimal data collection using photon-counting Hybrid Pixel Detectors

The data collection parameters used in a diffraction experiment have a strong impact on the quality of the acquired data. A careful choice of parameters leads to better data and can make the difference between success and failure in phasing attempts and better data will also result in a more accurate atomic model. The selection of data acquisition parameters has to account for the application of the data in various phasing methods or high-resolution refinement. Furthermore, experimental factors like crystal characteristics and the properties of X-ray source and detector have to be considered. Hybrid Pixel Detectors are now for several years in use in macromolecular crystallography and an increasing number of synchrotron beamlines as well as laboratory instruments are equipped with such detectors. Photon-counting Hybrid Pixel Detectors have fundamentally different characteristics and offer various advantages over other detector technologies. To fully exploit the advantages of Hybrid Pixel Detectors, different data collection strategies than those established for other detector types have to be applied. Fine φ-slicing is a strategy particularly well suited because of the fast readout time and the absence of readout noise. This strategy was systematically investigated collecting a large number of data sets from crystals of four different proteins to investigate the benefit of fine φ-slicing on data quality with a noise-free detector in practice. The results show that fine φ-slicing can substantially improve scaling statistics and anomalous signal. Furthermore, when collecting data in continuous rotation at high frame rates up to hundreds of images per second, quality might be impaired by detector readout. Results on the influence of readout time on data quality will be presented and strategies to easily avoid detrimental effects of detector readout will be discussed.

Advancing Native-SAD Phasing at Synchrotron with 13 Real-life Case Studies

The key step in elucidating de novo 3D X-ray structures relies on the incorporation of heavy elements into proteins or crystals. Selenomethionine incorporation or heavy metal derivatization are however not always possible and require additional efforts. Exploiting anomalous signals from intrinsically present elements like S, P, and Ca2+ from proteins and nucleic acids, as well as Cl-, SO42-, and PO42- from crystallization solutions, is therefore an appealing alternative. Such a method has been shown to be valid by collecting data from several crystals and combining them(1). Recent developments at macromolecular crystallography beamlines are however pushing the limits of what could be obtained out of a single crystal. Here we introduce a novel data collection routine for native-SAD phasing, which distributes tolerable X-ray life-doses to very high multiplicity X-ray diffraction data sets measured at 6 keV energy and at different crystal orientations on a single crystal. This allows the extraction of weak anomalous signals reliably by reducing both systematic and random measurement errors. The data collection method has been applied successfully to thirteen real-life examples including membrane proteins, a protein/DNA complex, and a large protein complex. In addition to de novo structure determination, we advocate such a data collection protocol for molecular replacement solvable structures where unbiased phase information is crucial in objective map interpretation and model building, especially for medium and low-resolution cases.

Native SAD Structure Solution from Merohedrally Twinned Data

Up until now, comparatively few structures were solved by native SAD. Recent advances in multi crystal averaging [1] have shown that native SAD can be applied to an increasing number of cases. Though theoretically possible [2], successful structure solutions from twinned data have not been reported yet. Here, we report the structure solution of the human Centromere protein M from a merohedrally twinned crystal with a twinning fraction of 0.45 in the space group P3. The data were collected at the bending magnet beamline X06DA at the Swiss Light Source, which is equipped with the in-house developed multi-axis goniometer PRIGo and the PILATUS 2M detector. A highly redundant 2.2 Å dataset was collected in a number of different crystal orientations. A substructure solution could only be obtained after 50000 SHELXD [3] tries. Automatic model building after phasing and density modification resulted in a model with the majority of residues built correctly. We will present this particularly difficult case together with other more routine cases, all solved with the same experimental setup and at the beamline X06DA.

Recent developments at the MX beamline X10SA at the SLS

The three macromolecular crystallography (MX) beamlines at the Swiss Light Source (SLS) rank among the most productive in Europe. The very successful design of the first beamline, X06SA, inaugurated in 2001, was the basis for the second beamline, X10SA, operated by the Paul Scherrer Institut and financed by the partners Max Planck Society, Novartis and Hofmann-La Roche. To keep up with the increasing demand for high throughput crystallographic experiments, especially in an industrial environment, as well as the rising interest in more challenging targets, the beamline is under constant development. Here we will present the recent advances in usability and performance, including software integration and automation with the completely new data acquisition software DA+, in-situ screening for diffraction candidates, (serial) micro-crystallography with micro-beam, and beamline hardware.

Structural basis for hydroxymethylcytosine recognition by the SRA domain of UHRF2

Methylated cytosine of CpG dinucleotides in vertebrates may be oxidized by Tet proteins, a process that can lead to DNA demethylation. The predominant oxidation product, 5-hydroxymethylcytosine (5hmC), has been implicated in embryogenesis, cell differentiation and human diseases. Recently, the SRA domain of UHRF2 (UHRF2-SRA) has been reported to specifically recognize 5hmC, but how UHRF2 recognizes this modification is unclear. Here we report the structure of UHRF2-SRA in complex with a 5hmC-containing DNA. The structure reveals that the conformation of a phenylalanine allows the formation of an optimal 5hmC-binding pocket, and a hydrogen bond between the hydroxyl group of 5hmC and UHRF2-SRA is critical for their preferential binding. Further structural and biochemical analyses unveiled the role of SRA domains as a versatile reader of modified DNA, and the knowledge should facilitate further understanding of the biological function of UHRF2, and the comprehension of DNA hydroxymethylation in general.