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Ahn, Jinsook,Lee, Dukwon,Jo, Inseong,Jeong, Hyeongseop,Hyun, Jae-Kyung,Woo, Jae-Sung,Choi, Sang-Ho,Ha, Nam-Chul Korean Society for Molecular and Cellular Biology 2020 Molecules and cells Vol.43 No.3
Cryo-electron microscopy (cryo-EM) is now the first choice to determine the high-resolution structures of huge protein complexes. Grids with two-dimensional arrays of holes covered with a carbon film are typically used in cryo-EM. Although semi-automatic plungers are available, notable trial-and-error is still required to obtain a suitable grid specimen. Herein, we introduce a new method to obtain thin ice specimens using real-time measurement of the liquid amounts in cryo-EM grids. The grids for cryo-EM strongly diffracted laser light, and the diffraction intensity of each spot was measurable in real-time. The measured diffraction patterns represented the states of the liquid in the holes due to the curvature of the liquid around them. Using the diffraction patterns, the optimal time point for freezing the grids for cryo-EM was obtained in real-time. This development will help researchers rapidly determine high-resolution protein structures using the limited resource of cryo-EM instrument access.
Lamin Filament Assembly Derived from the Atomic Structure of the Antiparallel Four-Helix Bundle
하남출,Jinsook Ahn,Inseong Jo,Soyeon Jeong,Jinwook Lee 한국분자세포생물학회 2023 Molecules and cells Vol.46 No.5
The nucleoskeletal protein lamin is primarily responsible for the mechanical stability of the nucleus. The lamin assembly process requires the A11, A22, and ACN binding modes of the coiled-coil dimers. Although X-ray crystallography and chemical cross-linking analysis of lamin A/C have provided snapshots of A11 and ACN binding modes, the assembly mechanism of the entire filament remains to be explained. Here, we report a crystal structure of a coil 2 fragment, revealing the A22 interaction at the atomic resolution. The structure showed detailed structural features, indicating that two coiled-coil dimers of the coil 2 subdomain are separated and then re-organized into the antiparallel-four-helix bundle. Furthermore, our findings suggest that the ACN binding mode between coil 1a and the C-terminal part of coil 2 when the A11 tetramers are arranged by the A22 interactions. We propose a full assembly model of lamin A/C with the curvature around the linkers, reconciling the discrepancy between the in situ and in vitro observations. Our model accounts for the balanced elasticity and stiffness of the nuclear envelopes, which is essential in protecting the cellular nucleus from external pressure.
Yongseong Hyun,Jinsook Ahn,Yeongjin Baek,Yongbin Xu,Nam-Chul Ha 한국구조생물학회 2020 Biodesign Vol.8 No.3
Brucella abortus is an intracellular bacterial pathogen that causes brucellosis in humans and livestock. The genome of B. abortus encodes the VirB type IV secretion system (T4SS), which is essential to its virulence. B. abortus produces and secretes effector proteins through the T4SS to survive in the intracellular environment and manipulate host immunity. The T4SS spans the peptidoglycan layer through vacancies in the peptidoglycan chain. Recently, secretion activator gene A (SagA) from B. abortus was identified as a lysozyme-like enzyme that creates holes in the peptidoglycan layer. In this study, SagA from B. abortus was overexpressed, purified, and crystallized. Crystal diffraction data were acquired at 2.0 Å resolution, a P213 space group with a unit cell parameter of 79.04 Å. We are currently exploring the crystal structure of SagA using the anomalous signal from selenomethionine-substituted crystals and an X-ray free-electron laser.
Crystal structure of the nuclease and capping domain of SbcD from Staphylococcus aureus
Lee Jinwook,Jo Inseong,Ahn Jinsook,Hong Seokho,Jeong Soyeon,Kwon Aeran,Ha Nam-Chul 한국미생물학회 2021 The journal of microbiology Vol.59 No.6
The SbcCD complex is an essential component of the DNA double-strand break (DSB) repair system in bacteria. The bacterial SbcCD complex recognizes and cleaves the DNA ends in DSBs by ATP-dependent endo- and exonuclease activities as an early step of the DNA repair process. SbcD consists of nuclease, capping, and helix-loop-helix domains. Here, we present the crystal structure of a SbcD fragment from Staphylococcus aureus, which contained nuclease and capping domains, at a resolution of 2.9 Å. This structure shows a dimeric assembly similar to that of the corresponding domains of SbcD from Escherichia coli. The S. aureus SbcD fragment exhibited endonuclease activities on supercoiled DNA and exonuclease activity on linear and nicked DNA. This study contributes to the understanding of the molecular basis for how bacteria can resist sterilizing treatment, causing DNA damage.
Nam-Chul Ha,김수현,김송희,Jinsook Ahn,조인성,이지원,Sang Ho CHOI 한국분자세포생물학회 2019 Molecules and cells Vol.42 No.12
The Gram-negative opportunistic pathogen, Pseudomonas aeruginosa, has multiple multidrug efflux pumps. MexT, a LysR-type transcriptional regulator, functions as a transcriptional activator of the MexEF-OprN efflux system. MexT consists of an N-terminal DNA‑binding domain and a C‑terminal regulatory domain (RD). Little is known regarding MexT ligands and its mechanism of activation. We elucidated the crystal structure of the MexT RD at 2.0 Å resolution. The structure comprised two protomer chains in a dimeric arrangement. MexT possessed an arginine-rich region and a hydrophobic patch lined by a variable loop, both of which are putative ligand‑binding sites. The three-dimensional structure of MexT provided clues to the interacting ligand structure. A DNase I footprinting assay of full-length MexT identified two MexT-binding sequence in the mexEF-oprN promoter. Our findings enhance the understanding of the regulation of MexT-dependent activation of efflux pumps.
Balanced Scorecard Based Performance Analysis of Accreditation for Engineering Education
Ju, Yonghan,Sohn, So Young,Ahn, Jinsook,Choi, Jin Young Korean Institute of Industrial Engineers 2014 Industrial Engineeering & Management Systems Vol.13 No.1
The number of students graduating from accredited programs has been increasing annually since the first students graduated from accredited engineering programs in Korean universities in 2004. In this paper, we evaluate the effect of engineering education accreditation by the Accreditation Board for Engineering Education of Korea (ABEEK). We developed performance evaluation indices based on the balanced scorecard concept and applied the proposed indicators to graduates, faculty, and industry employers to see if there are significant differences between accredited and non-accredited groups. Overall, regardless of survey object, engineering education accreditation was perceived to contribute to the elevation of engineering and science and the level of national growth. However, the differences between accredited and non-accredited groups for some key performance indicators were statistically insignificant. The results of this paper are expected to provide crucial feedback information for the improvement of engineering education accreditation in Korea.
Balanced Scorecard Based Performance Analysis of Accreditation for Engineering Education
Yonghan Ju,So Young Sohn,Jinsook Ahn,Jin Young Choi 대한산업공학회 2014 Industrial Engineeering & Management Systems Vol.13 No.1
The number of students graduating from accredited programs has been increasing annually since the first students graduated from accredited engineering programs in Korean universities in 2004. In this paper, we evaluate the effect of engineering education accreditation by the Accreditation Board for Engineering Education of Korea (ABEEK). We developed performance evaluation indices based on the balanced scorecard concept and applied the proposed indicators to graduates, faculty, and industry employers to see if there are significant differences between accredited and non-accredited groups. Overall, regardless of survey object, engineering education accreditation was perceived to contribute to the elevation of engineering and science and the level of national growth. However, the differences between accredited and non-accredited groups for some key performance indicators were statistically insignificant. The results of this paper are expected to provide crucial feedback information for the improvement of engineering education accreditation in Korea.
Kim, Suhyeon,Kim, Songhee H.,Ahn, Jinsook,Jo, Inseong,Lee, Zee-Won,Choi, Sang Ho,Ha, Nam-Chul Korean Society for Molecular and Cellular Biology 2019 Molecules and cells Vol.42 No.12
The Gram-negative opportunistic pathogen, Pseudomonas aeruginosa, has multiple multidrug efflux pumps. MexT, a LysR-type transcriptional regulator, functions as a transcriptional activator of the MexEF-OprN efflux system. MexT consists of an N-terminal DNA-binding domain and a C-terminal regulatory domain (RD). Little is known regarding MexT ligands and its mechanism of activation. We elucidated the crystal structure of the MexT RD at 2.0 Å resolution. The structure comprised two protomer chains in a dimeric arrangement. MexT possessed an arginine-rich region and a hydrophobic patch lined by a variable loop, both of which are putative ligand-binding sites. The three-dimensional structure of MexT provided clues to the interacting ligand structure. A DNase I footprinting assay of full-length MexT identified two MexT-binding sequence in the mexEF-oprN promoter. Our findings enhance the understanding of the regulation of MexT-dependent activation of efflux pumps.
Jo, Inseong,Kim, Dukyun,Bang, Ye-Ji,Ahn, Jinsook,Choi, Sang Ho,Ha, Nam-Chul American Society for Biochemistry and Molecular Bi 2017 The Journal of biological chemistry Vol.292 No.17
<P>Most Gram-negative bacteria respond to excessive levels of H2O2 using the peroxide-sensing transcriptional regulator OxyR, which can induce the expression of antioxidant genes to restore normality. Vibrio vulnificus has two distinct OxyRs (OxyR1 and OxyR2), which are sensitive to different levels of H2O2 and induce expression of two different peroxidases, Prx1 and Prx2. Although OxyR1 has both high sequence similarity and H2O2 sensitivity comparable with that of other OxyR proteins, OxyR2 exhibits limited sequence similarity and is more sensitive to H2O2. To investigate the basis for this difference, we determined crystal structures and carried out biochemical analyses of OxyR2. The determined structure of OxyR2 revealed a flipped conformation of the peptide bond before Glu-204, a position occupied by glycine in other OxyR proteins. Activity assays showed that the sensitivity to H2O2 was reduced to the level of other OxyR proteins by the E204G mutation. We solved the structure of the OxyR2-E204G mutant with the same packing environment. The structure of the mutant revealed a dual conformation of the peptide bond before Gly-204, indicating the structural flexibility of the region. This structural duality extended to the backbone atoms of Gly-204 and the imidazole ring of His-205, which interact with H2O2 and invariant water molecules near the peroxidatic cysteine, respectively. Structural comparison suggests that Glu-204 in OxyR2 provides rigidity to the region that is important in H2O2 sensing, compared with the E204G structure or other OxyR proteins. Our findings provide a structural basis for the higher sensitivity of OxyR2 to H2O2 and also suggest a molecular mechanism for bacterial regulation of expression of antioxidant genes at divergent concentrations of cellular H2O2.</P>