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Softer X-rays (softer + x-ray)
Selected AbstractsOverview and new developments in softer X-ray (2Å < , < 5Å) protein crystallographyJOURNAL OF SYNCHROTRON RADIATION, Issue 1 2004John R. Helliwell New methodologies with synchrotron radiation and X-ray free electron lasers (XFELs) in structural biology are being developed. Recent trends in harnessing softer X-rays in protein crystallography for phase determination are described. These include reference to a data-collection test at 2.6 Å wavelength with a lysozyme crystal on SRS station 7.2 (Helliwell, 1983) and also use of softer X-rays (2,Å wavelength) to optimise f," at the xenon L1 absorption edge in the Single Isomorphous Replacement Optimised Anomalous Scattering ('SIROAS') structure determination of apocrustacyanin A1 with four, partially occupied, xenon atoms (Cianci et al., 2001; Chayen et al., 2000). The hand of the protein was determined using the f," enhanced sulphur anomalous signal from six disulphides in the protein dimer of 40,kDa. In a follow-up study the single wavelength xenon L1 -edge f," optimised data set alone was used for phase determination and phase improvement by solvent flattening etc. (CCP4 DM) (Olczak et al., 2003). Auto-tracing of the protein was feasible but required additional diffraction data at higher resolution. This latter could be avoided in future by using improved tilted detector settings during use of softer X-rays, i.e. towards back-scattering recording (Helliwell, 2002). The Olczak et al. study has already led to optimisation of the new SRS beamline MPW,MAD,10 (see www.nwsgc.ac.uk) firstly involving the thinning of the beryllium windows as much as possible and planning for a MAR Research tilted detector `desk top beamline' geometry. Thus the use of softer, i.e. 2 to 3,Å wavelength range, X-rays will allow optimisation of xenon and iodine L -edge f," and enhancing of sulphur f," signals for higher throughput protein crystallography. Softer X-rays utilisation in protein crystallography includes work done on SRS bending-magnet station 7.2 in the early 1980s by the author as station scientist (Helliwell, 1984). In the future development of XFELs these softer X-ray wavelengths could also be harnessed and relax the demands to some extent on the complexity and cost of an XFEL. Thus, by use of say 4,Å XFEL radiation and use of a back-scattering geometry area detector the single molecule molecular transform could be sampled to a spatial resolution of 2,Å, sufficient, in principle, for protein model refinement (Miao et al., 1999). Meanwhile, Miao et al. (2003) report the first experimental recording of the diffraction pattern from intact Escherichia coli bacteria using coherent X-rays, with a wavelength of 2,Å, at a resolution of 30,nm and a real-space image constructed. The new single-particle X-ray diffraction-imaging era has commenced. [source] Going soft and SAD with manganeseACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2005Paula S. Salgado SAD phasing has been revisited recently, with experiments being carried out using previously unconventional sources of anomalous signal, particularly lighter atoms and softer X-rays. A case study is reported using the 75,kDa RNA-dependent RNA polymerase of the bacteriophase ,6, which binds a Mn atom and crystallizes with three molecules in the asymmetric unit. X-ray diffraction data were collected at a wavelength of 1.89,Å and although the calculated anomalous signal from the three Mn atoms was only 1.2%, SHELXD and SOLVE were able to locate these atoms. SOLVE/RESOLVE used this information to obtain SAD phases and automatically build a model for the core region of the protein, which possessed the characteristic features of the right-hand polymerase motif. These results demonstrate that with modern synchrotron beamlines and software, manganese phasing is a practical tool for solving the structure of large proteins. [source] Crystallization and phasing of focal adhesion protein 52 from Gallus gallusACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2004Imre Tör Focal adhesion protein 52 (FAP52) is a multidomain adaptor protein of 448 amino acids characterized as an abundant component of focal adhesions. FAP52 binds to filamin via its N-terminal ,-helical domain, suggesting a role in linking focal adhesions to the actin-based cytoskeleton. The recombinant protein was crystallized using the hanging-drop vapour-diffusion method, which yielded two crystal forms. Native data were collected from both crystal forms to 2.8 and 2.1,Å resolution, respectively. For one of the crystal forms, initial MAD phasing was successfully performed using two data sets from xenon-derivatized crystals. The derivative data sets were collected using softer X-rays of 1.5 and 1.9,Å wavelength. Preliminary structural analysis reveals the presence of a dimer in the asymmetric unit. [source] Structure of lobster apocrustacyanin A1 using softer X-raysACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2001M. Cianci The molecular basis of the camouflage colouration of marine crustacea is often provided by carotenoproteins. The blue colour of the lobster carapace, for example, is intricately associated with a multimacromolecular 16-mer complex of protein subunits each with a bound astaxanthin molecule. The protein subunits of crustacyanin fall into two distinct subfamilies, CRTC and CRTA. Here, the crystal structure solution of the A1 protein of the CRTC subfamily is reported. The problematic nature of the structure solution of the CRTC proteins (both C1 and A1) warranted consideration and the development of new approaches. Three putative disulfides per protein subunit were likely to exist based on molecular-homology modelling against known lipocalin protein structures. With two such subunits per crystallographic asymmetric unit, this direct approach was still difficult as it involved detecting a weak signal from these sulfurs and suggested the use of softer X-rays, combined with high data multiplicity, as reported previously [Chayen et al. (2000), Acta Cryst. D56, 1064,1066]. This paper now describes the structure solution of CRTC in the form of the A1 dimer based on use of softer X-rays (2,Å wavelength). The structure solution involved a xenon derivative with an optimized xenon LI edge signal and a native data set. The hand of the xenon SIROAS phases was determined by using the sulfur anomalous signal from a high-multiplicity native data set also recorded at 2,Å wavelength. For refinement, a high-resolution data set was measured at short wavelength. All four data sets were collected at 100,K. The refined structure to 1.4,Å resolution based on 60,276 reflections has an R factor of 17.7% and an Rfree of 22.9% (3137 reflections). The structure is that of a typical lipocalin, being closely related to insecticyanin, to bilin-binding protein and to retinol-binding protein. This A1 monomer or dimer can now be used as a search motif in the structural studies of the oligomeric forms ,- and ,-crustacyanins, which contain bound astaxanthin molecules. [source] Apocrustacyanin A1 from the lobster carotenoprotein ,-crustacyanin: crystallization and initial X-ray analysis involving softer X-raysACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2000N. E. Chayen The A1 subunit of the carotenoprotein ,-crustacyanin, isolated from lobster carapace, has been crystallized using the vapour-diffusion method. The crystals, grown in solutions of ammonium sulfate containing methylpentanediol (MPD), diffracted to 2.0,Å. The crystals are stable to radiation. The space group of the crystals is P212121. The unit-cell parameters are a = 41.9, b = 80.7, c = 110.8,Å. `Standard structure determination' has been unsuccessful within this crustacyanin family. Instead, an approach based on the S atoms is being undertaken involving softer X-rays at the SRS, Daresbury. [source] | ||||||||||