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Abstract: Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning. We explored the possibility of employing more than one PCR fragment to introduce various mutations in a single step, and found that when up to five PCR fragments were co-transformed into yeast, the recombination frequency was maintained as the number of fragments was increased. All transformants expressed the model membrane protein, while the resulting plasmid from each clone contained the designed mutations only. Thus, we have demonstrated a technique allowing the expression of mutant membrane proteins within 5 days, combining a GFP-fusion expression system and yeast homologous recombination.

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Abstract: We expand the previous theoretical treatment for the strong anisotropy of the x-ray magnetic linear dichroism
XMLD in a crystal field of cubic point-group symmetry to the more general case of tetragonal pointgroup symmetry. For the cubic symmetry, there are only two fundamental spectra, which have the same shape for rotation of either linear light polarization E or magnetization direction H. For the tetragonal symmetry, the XMLD is a linear combination of four fundamental spectra, with a different shape for linear dichroism
rotation of E and magnetic dichroism
rotation of H. However, only one extra spectrum is required to relate the linear and magnetic dichroism. The validity of the theory is demonstrated using a CoFe2O4
011 thin film on SrTiO3, which has both tetrahedrally distorted symmetry and large magnetic anisotropy. The XMLD at the Co L2,3 edges was found to exhibit a strong dependence on the relative orientation of external magnetic field, x-ray polarization, and crystalline axes. The large vaiations in the peak structure as a function of angle are not caused by the spin-orbit-induced magnetocrystall ne anisotropy but arise from the symmetry of the measurement geometry. The results are compared with calculated spectra using atomic multiplet theory for Co2+ d7 ?2p5d8 in octahedral and tetragonal crystal field symmetry. Although the magnitude of the dichroism is strongly influenced by the temperature, its spectral shape remains largely unaffected. The measured fundamental spectra are also robust against incomplete magnetization. The influence of the tetragonal distortion is revealed by small differences between the linear and magnetic dichroism. It is shown that the magnetic dichroism spectra can be transferred from CoFe2O4 to CoO. Therefore, the rich structure in the Co2+ L3 XMLDprovides a sensitive probe to determine the orientation of the spin axis with respect to the crystalline axes, hence offering a valuable tool for experimentalists for the study of exchange bias in Co oxides. In contrast, the Co2+ L2 edge, where the fundamental spectra have similar spectral shape but with opposite sign, does not al w an unambiguous determination.

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Abstract: Coherent X-ray Diffraction Imaging (CXDI) is a powerful method of imaging single crystalline grains within a powder. Direct Fourier transformation of the oversampled diffraction pattern surrounding a Bragg peak is possible once the phases have been obtained using a 'support' constraint. The image is in general complex with the phase representing a projection of the atomic displacements allowing access to the internal strains inside the crystal. CXDI relies crucially on the production of a coherent beam of X-rays, which is one of tire technical advances of the latest 3rd generation Synchroton Radiation (SR) sources. It is shown here how the use of a secondary Source in the design of the X-ray beamline allows the coherence to be controlled continuously over a finite range without any loss of flux. The conclusions will have significant impact on instruments currently being designed at tire Diamond Light Source and the new National Synchrotron Light Source (NSLS-2).

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Abstract: Outer membrane proteins are structurally distinct from those that reside in the inner membrane and play important roles in bacterial pathogenicity and human metabolism. X-ray crystallography studies on 40 different outer membrane proteins have revealed that the transmembrane portion of these proteins can be constructed from either -sheets or less commonly from -helices. The most common architecture is the -barrel, which can be formed from either a single anti-parallel sheet, fused at both ends to form a barrel or from multiple peptide chains. Outer membrane proteins exhibit considerable rigidity and stability, making their study through x-ray crystallography particularly tractable. As the number of structures of outer membrane proteins increases a more rational approach to their crystallization can be made. Herein we analyse the crystallization data from 53 outer membrane proteins and compare the results to those obtained for inner membrane proteins. A targeted sparse matrix screen for outer membrane protein crystallization is presented based on the present analysis.

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Abstract: The orientational ordering of the nematic phase of a polyethylene glycol (PEG)-peptide block copolymer in aqueous solution is probed by small-angle neutron scattering (SANS), with the sample subjected to steady shear in a Couette cell. The PEG-peptide conjugate forms fibrils that behave as semiflexible rodlike chains. The orientational order parameters P2 and P4 are obtained by modeling the data using a series expansion approach to the form factor of uniform cylinders. The method used is independent of assumptions on the form of the singlet orientational distribution function. Good agreement with the anisotropic two-dimensional SANS patterns is obtained. The results show shear alignment starting at very low shear rates, and the orientational order parameters reach a plateau at higher shear rates with a pseudologarithmic dependence on shear rate. The most probable distribution functions correspond to fibrils parallel to the flow direction under shear, but a sample at rest shows a bimodal distribution with some of the rodlike peptide fibrils oriented perpendicular to the flow direction.