Research Project: Time-Resolved Serial Femtosecond X-Ray Kinetocrystallography and CryoEM Studies of Biomacromolecules
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Contributors
Funders
ID
TB.00460
Authors
Demirci, Hasan
Faculty Member
Publications
An advanced workflow for single-particle imaging with the limited data at an X-ray free-electron laser
(International Union of Crystallography, 2020)
Assalauova, Dameli
Kim, Young Yong
Bobkov, Sergey
Khubbutdinov, Ruslan
Rose, Max
An improved analysis for single-particle imaging (SPI) experiments, using the limited data, is presented here. Results are based on a study of bacteriophage PR772 performed at the Atomic, Molecular and Optical Science instrument at the Linac Coherent Light Source as part of the SPI initiative. Existing methods were modified to cope with the shortcomings of the experimental data: inaccessibility of information from half of the detector and a small fraction of single hits. The general SPI analysis workflow was upgraded with the expectation-maximization based classification of diffraction patterns and mode decomposition on the final virus-structure determination step. The presented processing pipeline allowed us to determine the 3D structure of bacteriophage PR772 without symmetry constraints with a spatial resolution of 6.9 nm. The obtained resolution was limited by the scattering intensity during the experiment and the relatively small number of single hits.
Intersubunit coupling enables fast CO2-fixation by reductive carboxylases
(American Chemical Society (ACS), 2022)
Rao, Y.
Stoffel, G.M.
Vögeli, B.
Schell, K.
Gomez, A.
Enoyl-CoA carboxylases/reductases (ECRs) are some of the most efficient CO2-fixing enzymes described to date. However, the molecular mechanisms underlying the extraordinary catalytic activity of ECRs on the level of the protein assembly remain elusive. Here we used a combination of ambient-temperature X-ray free electron laser (XFEL) and cryogenic synchrotron experiments to study the structural organization of the ECR from Kitasatospora setae. The K. setae ECR is a homotetramer that differentiates into a pair of dimers of open- and dosed-form subunits in the catalytically active state. Using molecular dynamics simulations and structure-based mutagenesis, we show that catalysis is synchronized in the K. setae ECR across the pair of dimers. This conformational coupling of catalytic domains is conferred by individual amino acids to achieve high CO2-fixation rates. Our results provide unprecedented insights into the dynamic organization and synchronized inter- and intrasubunit communications of this remarkably efficient CO2-fixing enzyme during catalysis.
Structural characterization of TRAF6 N-terminal for therapeutic uses and computational studies on new derivatives
(Multidisciplinary Digital Publishing Institute (MDPI), 2023)
Sever, Belgin
Basoglu-Unal, Faika
Ece, Abdulilah
Tateishi, Hiroshi
Koga, Ryoko
Tumor necrosis factor receptor-associated factors (TRAFs) are a protein family with a wide variety of roles and binding partners. Among them, TRAF6, a ubiquitin ligase, possesses unique receptor binding specificity and shows diverse functions in immune system regulation, cellular signaling, central nervous system, and tumor formation. TRAF6 consists of an N-terminal Really Interesting New Gene (RING) domain, multiple zinc fingers, and a C-terminal TRAF domain. TRAF6 is an important therapeutic target for various disorders and structural studies of this protein are crucial for the development of next-generation therapeutics. Here, we presented a TRAF6 N-terminal structure determined at the Turkish light source "Turkish DeLight" to be 3.2 angstrom resolution at cryogenic temperature (PDB ID: 8HZ2). This structure offers insight into the domain organization and zinc-binding, which are critical for protein function. Since the RING domain and the zinc fingers are key targets for TRAF6 therapeutics, structural insights are crucial for future research. Separately, we rationally designed numerous new compounds and performed molecular docking studies using this template (PDB ID:8HZ2). According to the results, 10 new compounds formed key interactions with essential residues and zinc ion in the N-terminal region of TRAF6. Molecular dynamic (MD) simulations were performed for 300 ns to evaluate the stability of three docked complexes (compounds 256, 322, and 489). Compounds 256 and 489 was found to possess favorable bindings with TRAF6. These new compounds also showed moderate to good pharmacokinetic profiles, making them potential future drug candidates as TRAF6 inhibitors.
Rapid and efficient ambient temperature X-ray crystal structure determination at Turkish Light Source
(Nature Portfolio, 2023)
KUTTAM (Koç University Research Center for Translational Medicine)
Department of Molecular Biology and Genetics
KUYTAM (Koç University Surface Science and Technology Center)
Graduate School of Sciences and Engineering
School of Medicine
High-resolution biomacromolecular structure determination is essential to better understand protein function and dynamics. Serial crystallography is an emerging structural biology technique which has fundamental limitations due to either sample volume requirements or immediate access to the competitive X-ray beamtime. Obtaining a high volume of well-diffracting, sufficient-size crystals while mitigating radiation damage remains a critical bottleneck of serial crystallography. As an alternative, we introduce the plate-reader module adapted for using a 72-well Terasaki plate for biomacromolecule structure determination at a convenience of a home X-ray source. We also present the first ambient temperature lysozyme structure determined at the Turkish light source (Turkish DeLight). The complete dataset was collected in 18.5 min with resolution extending to 2.39 angstrom and 100% completeness. Combined with our previous cryogenic structure (PDB ID: 7Y6A), the ambient temperature structure provides invaluable information about the structural dynamics of the lysozyme. Turkish DeLight provides robust and rapid ambient temperature biomacromolecular structure determination with limited radiation damage.
A brief atlas of insulin
(Bentham Science Publishers, 2023)
KUISCID (Koç University İşbank Center for Infectious Diseases)
Department of Molecular Biology and Genetics
Yes
Demirci, Hasan
Ayan, Esra
Insulin is an essential factor for mammalian organisms: a regulator of glucose metabolism and other key signaling pathways. Insulin is also a multifunctional hormone whose absence can cause many diseases. Recombinant insulin is widely used in the treatment of diabetes. Understanding insulin, biosimilars, and biobetters from a holistic perspective will help pharmacologically user-friendly molecules design and develop personalized medicine-oriented therapeutic strategies for diabetes. Additionally, it helps to understand the underlying mechanism of other insulin-dependent metabolic disorders. The purpose of this atlas is to review insulin from a biotechnological, basic science, and clinical perspective, explain nearly all insulin-related disorders and their underlying molecular mechanisms, explore exogenous/recombinant production strategies of patented and research-level insulin/analogs, and highlight their mechanism of action from a structural perspective. Combined with computational analysis, comparisons of insulin and analogs also provide novel information about the structural dynamics of insulin.