Three macromolecular crystallography (MX) beamlines at the HelmholtzZentrum Berlin (HZB) are available for the regional, national and international structural biology user community. The state-of-the-art synchrotron beamlines for MX BL14.1, BL14.2 and BL14.3 are located within the low-section of the BESSY II electron storage ring. All beamlines are fed from a superconducting 7 T wavelength-shifter insertion device. BL14.1 and BL14.2 are energy tunable in the range 5-16 keV, while BL14.3 is a fixed-energy side station operated at 13.8 keV. All three beamlines are equipped with CCD detectors. BL14.1 and BL14.2 are in regular user operation providing about 200 beam days per year and about 600 user shifts to approximately 50 research groups across Europe. BL14.3 has initially been used as a test facility and was brought into regular user mode operation during the year 2010. BL14.1 has recently been upgraded with a microdiffractometer including a mini-goniometer and an automated sample changer. Additional user facilities include office space adjacent to the beamlines, a sample preparation laboratory, a biology laboratory (safety level 1) and highend computing resources. In this article the instrumentation of the beamlines is described, and a summary of the experimental possibilities of the beamlines and the provided ancillary equipment for the user community is given.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate–ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.
The flavin cofactor of Escherichia coli DNA photolyase in its neutral radical form, FADH • , was investigated by high-frequency/high-field continuous-wave electron paramagnetic resonance at 360 GHz. The data presented are the first flavin radical spectra where the full rhombic symmetry of the g-tensor is resolved. A fit of the spectrum yields accurate principal values of g, which show only a small anisotropy: g X ) 2.004 31(5), g Y ) 2.003 60(5) and g Z ) 2.002 17(7). The hyperfine splitting observed in the g Y region could be assigned to a hyperfine tensor component of the H(5) proton in the 7,8-dimethyl isoalloxazine moiety of FADH • . From a comparison of this effective hyperfine coupling with the principal value obtained from pulsed (Davies) electron-nuclear double resonance, the orientation of the g-tensor principal axes with respect to the H(5) hyperfine principal axes could be derived. Remaining ambiguities in the sign of the angle between the principal axes of g and the molecular axes are discussed by taking into account results from g-tensor calculations using density functional theory and semiempirical AM1 based methods.
A new chip-based crystal-mounting approach for rapid room-temperature data collection from numerous crystals is described. This work was motivated by the recent development of X-ray free-electron lasers. These novel sources deliver very intense femtosecond X-ray pulses that promise to yield high-resolution diffraction data of nanocrystals before their destruction by radiation damage. Thus, the concept of `diffraction before destruction' requires rapid replenishment of the sample for each exposure. The chip promotes the self-assembly of an array of protein crystals on a surface. Rough features on the surface cause the crystals to adopt random orientations, allowing efficient sampling of reciprocal space
The last decade witnessed a tremendous growth in combined efforts of biologists, chemists and physicists to understand the dominant factors determining the specificity and directionality of transmembrane transfer processes in proteins. A large variety of experimental techniques is being used including X-ray and neutron diffraction, but also time-resolved optical, infrared and magnetic resonance spectroscopy. This is done in conjunction with genetic engineering strategies to construct site-specific mutants for controlled modification of the proteins. As a general perception of these efforts, the substantial influence of weak interactions within the protein and its membrane interfaces is recognized. The weak interactions are subject to subtle changes during the reaction cycle owing to the inherent flexibility of the protein-membrane complex. Specific conformational changes accomplish molecular-switch functions for the transfer process to proceed with optimum efficiency. Characteristic examples of time varying non-bonded interactions are specific H-patterns and/or polarity effects of the microenvironment. The present perception has emerged from the coupling of newly developed spectroscopic techniques - and advanced EPR certainly deserves credit in this respect - with newly developed computational strategies to interpret the experimental data in terms of protein structure and dynamics. By now, the partners of this coupling, particularly high-field EPR spectroscopy and DFT-based quantum theory, have reached a level of sophistication that applications to large biocomplexes are within reach. In this review, a few large paradigm biosystems are surveyed which were explored lately in our laboratory. Taking advantage of the improved spectral and temporal resolution of high-frequency/high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), three biosystems are characterized with respect to structure and dynamics: (1) Light-induced electron-transfer intermediates in wild-type and mutant reaction-centre proteins from the photosynthetic bacterium Rhodobacter sphaeroides, (2) light-driven proton-transfer intermediates of site-specifically nitroxide spin-labelled mutants of bacteriorhodopsin proteins from Halobacterium salinarium, (3) refolding intermediates of site-specifically nitroxide spin-labelled mutants of the channel-forming protein domain of Colicin A bacterial toxin produced in Escherichia coli. The detailed information obtained is complementary to that of protein crystallography, solid-state NMR, infrared and optical spectroscopy techniques. A unique strength of high-field EPR is particularly noteworthy: it can provide highly desired detailed information on transient intermediates of proteins in biological action. They can be observed and characterized while staying in their working states on biologically relevant time scales. The review introduces the audience to origins and basic experiments of EPR in relation to NMR, describes the underlying strategies for extending...
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