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[Resource]

Station for structural studies on macro objects: beamline 6C Bio Medical Imaging at the Pohang Light Source-II

Jae-Hong Lim 1, *, Seung-Jun Seo 1,† , Hyo-Yun Kim 1 , Chun Kil Ryu 1 , Seungyu Rah 1 , Jung Yun Huang 1 , Chae-Soon Lee 1 , In Deuk Seo 1 , Seob-Gu Kim 1 , Dongyun Lee 2 and Moo-Hyun Cho /p>

1 Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang, Gyeongbuk 37673, Korea, 2 Division of Advanced Nuclear Engineering, Pohang University of Science and Technology, Pohang, Gyeongbuk 37673, Korea. *Correspondence: limjh@postech.ac.kr † Current address: Department of Biomedical Engineering and Radiology, School of Medicine, Catholic University of Daegu, Daegu 42472, Korea
*Correspondence: limjh@postech.ac.kr

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  • [Resource] Station for structural studies on macro objects: beamline 6C Bio Medical Imaging at the Pohang Light Source-II

    Similar to macromolecular crystallography (MX), radiography uses X-rays to reveal structures, however, of macro objects, which can be several orders of magnitude larger in size than the subjects of MX. Beamline 6C Bio Medical Imaging of the Pohang Light Source-II (PLS-II) utilizes synchrotron radiation X-rays to implement parallel beam projection radiography. Major applications are virtual anatomy of small animals and plants, non-invasive physiological monitoring and 3D histology in biology and medicine. Uses in material science and defect analysis of industrial products are also becoming popular. In this review, the use of the beamline is introduced including sample preparation, data acquisition and a workflow for 3D image reconstruction, visualization and analysis. For biologists who are familiar with MX, a brief comparison has been made between the two techniques. The mechanisms for spatial resolution determination and contrast formation in the parallel beam projection radiography are also presented.

  • [Crystallization] Purification, crystallization and X-ray crystallographic analysis of Csm5 in Type III-A Crispr-Cas system

    The CRISPR-Cas system is a microbial adaptive and heritable immune system, of which mechanism relies on the effector ribonucleoprotein (RNP) complex to degrade foreign genetic elements. The effector surveillance complex of the Type III-A CRISPR-Cas system has five Csm components, Csm1~Csm5. The Csm5 protein is placed in the crRNA 3' ends in the effector RNP complex, leading to speculations that crRNA maturation may be catalyzed by the Csm5 subunit. However, the crystal structure and the detailed function of Csm5 still remain elusive. In this study, the Csm5 from Thermococcus onnurineus NA1 was purified and crystallized by the sitting drop method in the condition of 20% w/v PEG 8000 and 100 mM CHES/sodium hydroxide pH 9.5 at 291K. The diffraction data were collected to a resolution of 3.5 Å. The crystal belonged to space group P4 3 2 1 2, with unit cell parameters a=81.0 Å, b=81.0 Å, c=169.1 Å. One protomer was presented in the asymmetric unit with a corresponding V M of 2.10 Å 3 Da -1 and solvent content of 50%.

  • [Crystallization] Expression, purification, crystallization, and preliminary crystallographic analysis of the Fab fragment of golimumab, a therapeutic antibody against TNFα

    TNFα is an inflammatory cytokine required for immune responses, playing a crucial role in the pathogenesis of inflammatory autoimmune diseases via interactions with its cognate receptors, TNFR1 and TNFR2. Therapeutic antibodies targeting TNFα, including infliximab, adalimumab, certolizumab pegol, and golimumab, are clinically used for the treatment of the inflammatory diseases such as rheumatoid arthritis, psoriatic arthritis, Crohn’s disease, and inflammatory bowel disease. Here, the Fab fragment of golimumab was overexpressed in the periplasmic region of Escherichia coli and purified by affinity and gel-filtration chromatography. The purified protein was crystallized by hanging-drop vapor diffusion in the presence of 100 mM Tris, pH 8.5, 0.1 M ammonium phosphate, and 12% (w/v) PEG 6,000 at 20°C. The crystal diffracted X-ray to 2.60 Å resolution and belonged to the orthogonal space group P2 1 2 1 2, with unit cell parameters a = 72.74, b = 130.29, and c = 93.35 Å. An asymmetric unit of the crystal contains two molecules of the Fab fragment of golimumab with a V M of 2.14 Å 3 Da -1 and a solvent content of 42.48%.

  • [Crystallization] Purification, crystallization and preliminary X-ray crystallographic analysis of VCA0593 with a c-di-GMP binding activity in Vibrio cholerae

    Many kinds of nucleotides are differentially synthesized and degraded in response to external and internal signaling in bacterial cells. Cyclic di-GMP (c-di-GMP) is ubiquitous bacterial nucleotide secondary signaling molecule, and is involved in virulence, antibiotic resistance, biofilm formation and cell division. VCA0593 was predicted or screened as a c-di-GMP or 5’-phosphguanylyl-(3’,5’)-guanosine binding protein in Vibrio cholerae genome by genome-wide system approaches, suggesting that VCA0593 might be involved in a c-di-GMP-mediated signaling pathway. In this study, the full-length VCA0593 was overexpressed in the E. coli expression system, and successfully crystallized. X-rays were diffracted by the crystals to 1.60 Å resolution, revealing that the crystals belong to space group P2 1 2 1 2, with unit cell parameters a = 68.9, b = 149.0, and c = 58.9 Å. According to cell content analysis, the asymmetric unit was expected to contain two molecules of VCA0593. The molecular replacement was not available due to lack of the homologous models. We are now under way to solve the crystal structure using the anomalous signals from Zn-soaked crystals. The crystal structure would help reveal the function of the protein and its mechanism of action, which could be associated with the pathogenesis of V. cholerae.

  • [Crystallization] Overexpression, crystallization, and preliminary X-ray crystallographic analysis of a putative Diguanylate cyclase from Thermotoga maritima by the Tm0107 gene

    Bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP), a bacterial secondary messenger, is a crucial regulator of collective versus individual behavior in bacteria. Diguanylate cyclases (DGCs) are essential for the synthesis of c-di-GMP. To elucidate the structural features of DGCs, we initiated the crystallization of a putative DGC (Tm0107) from Thermotoga maritima. Tm0107 protein was overexpressed in Escherichia coli and crystallized at 295 K using PEG 3000 as a precipitant. Crystals of Tm0107 protein diffracted to 2.1 Å resolution and belong to a Tetragonal space group P4 3 2 1 2, with unit cell parameters of a = 61.3 Å, b = 61.3 Å and c = 101.1 Å. The crystallization of selenomethionine-substituted protein is in progress to solve the crystal structure of Tm0107 protein.

  • [Crystallization] Purification, crystallization and X-ray crystallographic analysis of meso-diaminopimelic acid decarboxylase from Corynebacterium glutamicum

    meso-Diaminopimelic acid decarboxylase from Corynebacterium glutamicum (CgDAPDC) is the key enzyme for the production of L -lysine and it catalyzes meso-DAP to produce the final product, L -lysine. The CgDAPDC was overexpressed and purified to homogeneity by Ni-NTA affinity and size-exclusion chromatography. The CgDAPDC protein was crystallized using sitting-drop vapor-diffusion method in the presence of 0.8 M sodium citrate tribasic and 0.1 M sodium cacodylate, pH 6.5 at 293 K. X-ray diffraction data were collected to a maximum resolution of 2.4 Å. The crystal belonged to space group P2 1 2 1 2, with unit cell parameters a = 114.54 Å, b = 91.702 Å, c = 95.161, α = β = γ = 90°. With one molecules per asymmetric unit, the crystal volume per unit protein mass was 2.64 Å 3 Da -1 , which correspond to a solvent content of approximately 53.37%.

  • [Crystallization] Purification, crystallization and X-ray crystallographic analysis of RabA1a from Arabidopsis thaliana

    RabA1a is a small GTPase that contain a conserved GTP binding pocket to promote GTP hydrolysis. Fifty-seven isotypes of Rab GTPase have been found in Arabidopsis thaliana, and the twenty-seven RabA proteins are classified into six subgroups, from RabA1 to RabA6. RabA members play crucial roles in controlling docking and fusion during vesicle transport, yet their molecular and physiological functions remain unknown. Moreover, no RabA group protein structures have yet been determined. In this study, in order to understand the structural features and regulatory mechanisms of RabA from the crystal structures, an engineered N-terminal His-tagged RabA1a protein was overexpressed and purified. The RabA1a protein was crystallized with either GDP for an inactive form or GppNHp as a non-hydrolyzable GTP analog for an active form. X-ray diffraction data from crystals of RabA1a-GDP and RabA1a-GppNHp complexes were collected to resolutions of 2.8 Å and 2.6 Å, respectively. The crystal of the RabA1a-GDP complex belonged to the space group P2 1 , with unit cell parameters a = 36.7, b = 70.4, c = 68.0 Å, and β = 97.2°. The crystal of the RabA1a-GppNHp complex belonged to the space group P1, with unit cell parameters, a = 37.6, b = 48.4, c = 68.5 Å, α = 88.5, β = 74.1, and γ = 84.4°. Both crystals contained two macromolecules each containing GDP or GppNHp in the asymmetric units.