Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus

Bae, Jong Hoa; Hwang, Kwang Yeon; Nam, Ki Hyun

doi:10.1016/j.bbrc.2018.06.074

Cited by 20 publications

(14 citation statements)

References 27 publications

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“…The active site of glucose isomerase contains two metal binding sites for substrate binding and catalytic function. 29,30 These metal binding sites of glucose isomerase are well-defined in the electron density map, and there was no negative fo-fc electron density map counted at 3 σ, indicating there was no significant radiation damage (Supplementary Figure S5). The glucose isomerase structures delivered in shortenings A and B had high similarity with previously reported glucose isomerase structures delivered using grease (PDB code 4W4Q) 31 and nylon mesh-based fixed target scanning (6IRK) 12 with a root mean squared deviation of 0.1594–0.2549 for Cα atoms (Supplementary Figure S6).…”

Section: Resultsmentioning

confidence: 99%

“…34 The hit images were indexed and merged using the CrystFEL program. 35 The phase problem of the lysozyme and glucose isomerase was solved by molecule replacement using the Phaser-MR in PHENIX 36 , with the crystal structures of lysozyme (PDB code 6IG6) 21 and glucose isomerase (PDB code 5ZYD) 30 used as the search models. The model building and refinement were conducted using Coot 37 and Phenix.refinement in PHENIX, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Shortening injection matrix for serial crystallography

Nam

2019

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Serial crystallography (SX) allows crystal structures to be observed at room temperature through the steady delivery of crystals to the X-ray interaction point. Viscous delivery media are advantageous because they afford efficient sample delivery from an injector or syringe at a low flow rate. Hydrophobic delivery media, such as lipidic cubic phase (LCP) or grease, provide a very stable injection stream and are widely used. The development of new hydrophobic delivery materials can expand opportunities for future SX studies with various samples. Here, I introduce fat-based shortening as a delivery medium for SX experiments.This material is commercially available at low cost and is straightforward to handle because its phase (i.e., solid or liquid) can be controlled by temperature. Shortening was extruded from a syringe needle in a very stable injection stream even below 200 nl/min. X-ray exposed shortening produced several background scattering rings, which have similar or lower intensities than those of LCP and contribute negligibly to data processing. Serial millisecond crystallography was performed using two shortening delivery media, and the room temperature crystal structures of lysozyme and glucose isomerase were successfully determined at resolutions of 1.5-2.0 Å. Therefore, shortening can be used as a sample delivery medium in SX experiments. more biologically reliable crystal structure of macromolecules beyond conventional X-ray crystallography using single crystal diffraction at cryogenic temperatures. In SX experiments, it is important to deliver the crystal sample serially and stably to the X-ray interaction point. 5Delivering the sample reliably not only reduces sample consumption but also enables data collection for accurate responses in time-resolved studies. 6 Among the various sample delivery methods, including liquid jet injector, 7 high viscosity injector, 8,9 fixed-target scanning, 10-12 and microfluidics, 13,14 sample delivery using a viscous medium with an injector or syringe is widely applied to serial femtosecond crystallography (SFX) or serial millisecond crystallography (SMX), which can successfully determine structure using an XFEL or synchrotron, respectivley. [15][16][17][18][19][20][21][22] The advantage of crystal delivery with a viscous delivery medium is the drastic reduction of sample consumption by lowering the flow rate of the sample loading from the injector or syringe. 22Based on their physical and chemical properties, sample delivery media can be categorized as either lipidic cubic phase (LCP), 15 oil-based, [16][17][18] or hydrogel-based. 17-21 Among them, LCP and grease, which have hydrophobic characteristics, provide the most stable injection streams and are the most widely used delivery media in SX experiments. 15-18 LCP, which is usually used with monoolein, is widely applied in the process of membrane protein crystallization, and its crystallized membrane protein embedded in LCP can be used as a sample delivery medium. 15,23 The LCP delivery medium enables the sample t...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Shortening injection matrix for serial crystallography

Nam

2019

Preprint

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“…4a). The active site of glucose isomerase contains two magnesium binding sites for substrate binding and catalytic function 34,35 . These magnesium binding sites of glucose isomerase are well-defined in the electron density map, and there was no negative fo-fc electron density map counted at 3 σ, indicating there was no significant radiation damage ( Supplementary Fig.…”

Section: Smx Using the Shortening Injection Matrixmentioning

confidence: 99%

“…The hit images were indexed and merged using the CrystFEL program 39 . The phase problem of the lysozyme and glucose isomerase was solved by molecule replacement using the Phaser-MR in PHENIX 40 , with the crystal structures of lysozyme (PDB code 6IG6) 24 and glucose isomerase (PDB code 5ZYD) 35 used as the search models. The model building and refinement were conducted using Coot 41 and Phenix.refinement in PHENIX, respectively 40 .…”

Section: Characterization Of Shorteningmentioning

confidence: 99%

Shortening injection matrix for serial crystallography

Nam

2020

Sci Rep

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Serial crystallography allows crystal structures to be determined at room temperature through the steady delivery of crystals to the X-ray interaction point. Viscous delivery media are advantageous because they afford efficient sample delivery from an injector or syringe at a low flow rate. Hydrophobic delivery media, such as lipidic cubic phase (LCP) or grease, provide a stable injection stream and are widely used. the development of new hydrophobic delivery materials can expand opportunities for future SX studies with various samples. Here, I introduce fat-based shortening as a delivery medium for SX experiments. this material is commercially available at low cost and is straightforward to handle because its phase (i.e., solid or liquid) can be controlled by temperature. Shortening was extruded from a syringe needle in a stable injection stream even below 200 nl/min. X-ray exposed shortening produced several background scattering rings, which have similar or lower intensities than those of LCP and contribute negligibly to data processing. Serial millisecond crystallography was performed using two shortening delivery media, and the room temperature crystal structures of lysozyme and glucose isomerase were successfully determined at resolutions of 1.5-2.0 Å. Therefore, shortening can be used as a sample delivery medium in SX experiments.

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“…The diffraction patterns were filtered and processed using the Cheetah 27 and CrystFEL 28 programs, respectively. Molecular replacement was performed using Phaser-MR 29 with the crystal structure of lysozyme (PDB code 6IG6) 21 or glucose isomerase (5ZYE) 30 as the search modes. The model was established and its structure was refined using the COOT 31 and PHENIX 32 programs, respectively.…”

Section: Structure Determinationmentioning

confidence: 99%

Sample delivery using capillary and delivery medium for serial crystallography

Nam¹

2019

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Serial crystallography (SX) is an innovative technology in structural biology that enables the visualization of molecular dynamics of macromolecules at room temperature. SX experiments always require a considerable amount of effort to deliver a crystal sample to the X-ray interaction point continuously and reliably. Here, a sample delivery method using a capillary and a delivery medium is introduced. The crystals embedded in the delivery medium can pass through the capillary tube, which is aligned with the X-ray beam, at very low flow rates without requiring elaborate delivery techniques and drastically reducing sample consumption. This simple but highly efficient sample delivery method can allow researchers to deliver crystals precisely to X-rays in SX experiments.Keyword: serial crystallography, sample delivery, capillary, viscus medium I thank the beamline staff at 11C beamline at Pohang Accelerator Laboratory for their assistance with data collection. The authors thank Global Science experimental Data hub Center (GSDC) at Korea Institute of Science and Technology Information (KISTI) for computational support.

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Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus

Cited by 20 publications

References 27 publications

Shortening injection matrix for serial crystallography

Shortening injection matrix for serial crystallography

Shortening injection matrix for serial crystallography

Sample delivery using capillary and delivery medium for serial crystallography

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