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.
Fatty acid synthases (FASs) and polyketide synthases (PKSs) condense acyl compounds to fatty acids and polyketides, respectively. Both, FASs and PKSs, harbor acyltransferases (ATs), which select substrates for condensation by β-ketoacyl synthases (KSs). Here, we present the structural and functional characterization of the polyspecific malonyl/acetyltransferase (MAT) of murine FAS. We assign kinetic constants for the transacylation of the native substrates, acetyl- and malonyl-CoA, and demonstrate the promiscuity of FAS to accept structurally and chemically diverse CoA-esters. X-ray structural data of the KS-MAT didomain in a malonyl-loaded state suggests a MAT-specific role of an active site arginine in transacylation. Owing to its enzymatic properties and its accessibility as a separate domain, MAT of murine FAS may serve as versatile tool for engineering PKSs to provide custom-tailored access to new polyketides that can be applied in antibiotic and antineoplastic therapy.
Radiation damage is an unwelcome and unavoidable aspect of macromolecular crystallography. In order to quantify the extent of X-ray-induced changes, knowledge of the dose (absorbed energy per unit mass) is necessary since it is the obvious metric against which to plot variables such as diffraction intensity loss and B factors. Significant improvements to the program RADDOSE for accurately calculating the dose absorbed by macromolecular crystals are presented here. Specifically, the probability of energy loss through the escape of fluorescent photons from de-excitation of an atom following photoelectric absorption is now included. For lighter elements, both the probability of fluorescence and of its subsequent escape from the crystal are negligible, but for heavier atoms the chance of fluorescence becomes significant (e.g. 30% as opposed to Auger electron decay from a K-shell excited iron atom), and this has the effect of reducing the absorbed dose. The effects of this phenomenon on dose calculations are presented for examples of crystals of an iron-containing protein, 2-selenomethionine proteins, a uranium derivatised protein, and for a nucleic acid sample. For instance, the inclusion of fluorescent escape results in up to a 27% decrease in the calculated absorbed dose for a typical selenomethionine protein crystal irradiated at the selenium K-edge.
De novo biosynthesis of fatty acids is an iterative process requiring strict regulation of the lengths of the produced fatty acids. In this review, we focus on the factors determining chain lengths in fatty acid biosynthesis. In a nutshell, the process of chain‐length regulation can be understood as the output of a chain‐elongating C−C bond forming reaction competing with a terminating fatty acid release function. At the end of each cycle in the iterative process, the synthesizing enzymes need to “decide” whether the growing chain is to be elongated through another cycle or released as the “mature” fatty acid. Recent research has shed light on the factors determining fatty acid chain length and has also achieved control over chain length for the production of the technologically interesting short‐chain (C4–C8) and medium‐chain (C10–C14) fatty acids.
Multigrain crystallography / 3DXRD microscopy / Crystal structure determination / Polycrystal diffraction / Crystal indexing Abstract. We summarize exploratory work on multigrain crystallography. The experimental arrangement comprises a monochromatic beam, a fully illuminated sample with up to several hundred grains in transmission geometry on a rotary table and a 2D detector. Novel algorithms are presented for indexing, integration and filtering with emphasis on handling the complications of spot overlap and the need for on-line analysis. The structure solution and refinement steps are performed by conventional single crystal programs. Simulations are used to verify the algorithms and to probe the overall limitations of the methodology in terms of number of grains, size of unit cell and direct space resolution. First experimental results in the fields of chemistry, structural biology and time-resolved studies in photochemistry are presented. As an outlook, the concept of TotalCrystallography is introduced, defined as the simultaneous characterization of the 3D atomic, and 3D grain-scale structure of polycrystalline specimens with phases of unknown composition and structure.
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