Radical doping and high-pressure freezing in an advanced neutron crystallography

The isotope effect in conventional neutron protein crystallography (NPC) can be eliminated by the proton polarization technique (ppt) as an advanced NPC. Furthermore, the ppt can improve detection sensitivity of hydrogen (relative neutron scattering length of polarized proton) by approximately eight times in comparison with conventional NPC. Several technical difficulties, however, should be overcome in order to perform the ppt. In this poster, two developing fundamental studies to realize ppt will be presented; 1) radical doping into protein crystals that facilitates sample electron polarization, which was analyzed by X-ray crystallography, liquid-chromatography/mass-spectrometry (LC/MS) and electron spin resonance (ESR) measurement, 2) high-pressure flash freezing performed especially using a new machine of HPC-201 (ADC Inc.), which has the advantage of making bulk water amorphous without destroying the single large crystal, may easily realize the low temperature environment of crystal at around 1K. The former results were that radical molecules distributed non-specifically around proteins, and that they were included in protein crystal to some extent [1]. These are a favorable tendency for better proton polarization.

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Cryoprotectant-free high-pressure cooling and dynamic nuclear polarization for more sensitive detection of hydrogen in neutron protein crystallography

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798318005028 ◽

2018 ◽

Vol 74 (8) ◽

pp. 787-791 ◽

Cited By ~ 3

Author(s):

Ichiro Tanaka ◽

Naoya Komatsuzaki ◽

Wen-Xue Yue ◽

Toshiyuki Chatake ◽

Katsuhiro Kusaka ◽

...

Keyword(s):

High Pressure ◽

Dynamic Nuclear Polarization ◽

Nuclear Polarization ◽

Protein Crystals ◽

Large Protein ◽

Proton Polarization ◽

Cooling Method ◽

Small Crystals ◽

Polarization Technique ◽

Neutron Protein Crystallography

To improve the sensitivity of hydrogen detection using neutrons, a proton-polarization technique together with a high-pressure cooling method is necessary. The highest pressure (200 MPa) used in the experiment described here enabled relatively large protein crystals to be cooled without any cryoprotectants while retaining the protein structure, and it was confirmed that high-pressure-cooled crystals diffracted to nearly the same resolution as flash-cooled small crystals soaked with cryoprotectants. Dynamic nuclear polarization was used as a proton-polarization technique for protein crystals, and ∼300 mg polycrystalline protein doped with TEMPOL gave a maximum proton polarization of 22.3% at a temperature of 0.5 K in a 2.5 T magnetic field.

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C−H- - -O Hydrogen Bonds in β-Sheetlike Networks: Combined X-ray Crystallography and High-Pressure Infrared Study

Journal of the American Chemical Society ◽

10.1021/ja036719z ◽

2003 ◽

Vol 125 (40) ◽

pp. 12358-12364 ◽

Cited By ~ 37

Author(s):

Kwang Ming Lee ◽

Hai-Chou Chang ◽

Jyh-Chiang Jiang ◽

Jack C. C. Chen ◽

Hsiang-En Kao ◽

...

Keyword(s):

High Pressure ◽

Hydrogen Bonds ◽

Infrared Study ◽

X Ray ◽

X Ray Crystallography

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Estimation of the Postmortem Duration of Mouse Tissue by Electron Spin Resonance Spectroscopy

Journal of Toxicology ◽

10.1155/2011/973172 ◽

2011 ◽

Vol 2011 ◽

pp. 1-11

Author(s):

Shinobu Ito ◽

Tomohisa Mori ◽

Hideko Kanazawa ◽

Toshiko Sawaguchi

Keyword(s):

Free Radicals ◽

Electron Spin Resonance ◽

Electron Spin ◽

Spin Trap ◽

Detection Sensitivity ◽

Mouse Tissue ◽

Spin Adduct ◽

Simple Method ◽

Oxidation Stress ◽

Spin Resonance

Electron spin resonance (ESR) method is a simple method for detecting various free radicals simultaneously and directly. However, ESR spin trap method is unsuited to analyze weak ESR signals in organs because of water-induced dielectric loss (WIDL). To minimize WIDL occurring in biotissues and to improve detection sensitivity to free radicals in tissues, ESR cuvette was modified and used with 5,5-dimethtyl-1-pyrroline N-oxide (DMPO). The tissue samples were mouse brain, hart, lung, liver, kidney, pancreas, muscle, skin, and whole blood, where various ESR spin adduct signals including DMPO-ascorbyl radical (AsA∗), DMPO-superoxide anion radical (OOH), and DMPO-hydrogen radical (H) signal were detected. Postmortem changes in DMPO-AsA∗and DMPO-OOH were observed in various tissues of mouse. The signal peak of spin adduct was monitored until the 205th day postmortem. DMPO-AsA∗in liver (y=113.8–40.7 log (day),R1=-0.779,R2=0.6,P<.001) was found to linearly decrease with the logarithm of postmortem duration days. Therefore, DMPO-AsA∗signal may be suitable for detecting an oxidation stress tracer from tissue in comparison with other spin adduct signal on ESR spin trap method.

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A high-pressure cryocooling method for protein crystals and biological samples with reduced background X-ray scatter

Journal of Applied Crystallography ◽

10.1107/s0021889812045013 ◽

2012 ◽

Vol 46 (1) ◽

pp. 234-241 ◽

Cited By ~ 11

Author(s):

Chae Un Kim ◽

Jennifer L. Wierman ◽

Richard Gillilan ◽

Enju Lima ◽

Sol M. Gruner

Keyword(s):

High Pressure ◽

Biological Samples ◽

Protein Crystal ◽

High Background ◽

X Ray ◽

Coherent Diffraction Imaging ◽

X Ray Scattering ◽

Alternative Method ◽

Helium Gas ◽

Diffraction Imaging

High-pressure cryocooling has been developed as an alternative method for cryopreservation of macromolecular crystals and successfully applied for various technical and scientific studies. The method requires the preservation of crystal hydration as the crystal is pressurized with dry helium gas. Previously, crystal hydration was maintained either by coating crystals with a mineral oil or by enclosing crystals in a capillary which was filled with crystallization mother liquor. These methods are not well suited to weakly diffracting crystals because of the relatively high background scattering from the hydrating materials. Here, an alternative method of crystal hydration, called capillary shielding, is described. The specimen is kept hydratedviavapor diffusion in a shielding capillary while it is being pressure cryocooled. After cryocooling, the shielding capillary is removed to reduce background X-ray scattering. It is shown that, compared to previous crystal-hydration methods, the new hydration method produces superior crystal diffraction with little sign of crystal damage. Using the new method, a weakly diffracting protein crystal may be properly pressure cryocooled with little or no addition of external cryoprotectants, and significantly reduced background scattering can be observed from the resulting sample. Beyond the applications for macromolecular crystallography, it is shown that the method has great potential for the preparation of noncrystalline hydrated biological samples for coherent diffraction imaging with future X-ray sources.

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Compressibility Studies of Zirconium Based Metal-Organic Frameworks

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314098428 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C157-C157

Author(s):

Claire Hobday ◽

Stephen Moggach ◽

Carole Morrison ◽

Tina Duren ◽

Ross Forgan

Keyword(s):

High Pressure ◽

Mechanical Stability ◽

Metal Organic Frameworks ◽

External Pressure ◽

X Ray Diffraction ◽

X Ray ◽

X Ray Crystallography ◽

Pressure Medium ◽

Metal Organic ◽

University Of Oslo

Metal-organic frameworks (MOFs) are a well-studied class of porous materials with the potential to be used in many applications such as gas storage and catalysis.[1] UiO-67 (UiO = University of Oslo), a MOF built from zirconium oxide units connected with 4,4-biphenyldicarboxylate (BDC) linkers, forms a face centred cubic structure. Zirconium has a high affinity towards oxygen ligands making these bridges very strong, resulting in UiO-based MOFs having high chemical and thermal stability compared to other MOF structures. Moreover, UiO-67 has become popular in engineering studies due to its high mechanical stability.[2] Using high pressure x-ray crystallography we can exert MOFs to GPa pressures, experimentally exploring the mechanical stability of MOFs to external pressure. By immersing the crystal in a hydrostatic medium, pressure is applied evenly to the crystal. On surrounding a porous MOF with a hydrostatic medium composed of small molecules (e.g. methanol), the medium can penetrate the MOF, resulting in medium-dependant compression. On compressing MOF-5 (Zn4O(BDC)3) using diethylformamide as a penetrating medium, the framework was shown to have an increased resistance to compression, becoming amorphous several orders of magnitude higher in pressure than observed on grinding the sample.[3] Here we present a high-pressure x-ray diffraction study on the UiO-based MOF UiO-67, and several new synthesised derivatives built from same metal node but with altered organic linkers, allowing us to study in a systematic way, the mechanical stability of the MOF, and its pressure dependence on both the linker, and pressure medium.

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Studies of the mechanism of Coal Hydrogenation by Electron Spin Resonance. Quarterly technical progress report, March 1-May 31, 1980. [For high-temperature, high pressure measurements]

10.2172/5175323 ◽

1980 ◽

Author(s):

Ira B. Goldberg

Keyword(s):

High Pressure ◽

Electron Spin Resonance ◽

High Temperature ◽

Electron Spin ◽

Technical Progress ◽

Progress Report ◽

Pressure Measurements ◽

Spin Resonance ◽

High Temperature High Pressure

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X-ray Crystallography at High Pressure to Probe Conformational Fluctuations in Biological Macromolecules

10.1063/1.2436439 ◽

2007 ◽

Author(s):

Eric Girard ◽

Richard Kahn ◽

Anne-Claire Dhaussy ◽

Isabella Ascone ◽

Mohamed Mezouar ◽

...

Keyword(s):

High Pressure ◽

Biological Macromolecules ◽

X Ray ◽

X Ray Crystallography

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MRPC (Missing Regions in Polypeptide Chains): a knowledgebase

Journal of Applied Crystallography ◽

10.1107/s1600576719012330 ◽

2019 ◽

Vol 52 (6) ◽

pp. 1422-1426

Author(s):

Rajendran Santhosh ◽

Namrata Bankoti ◽

Adgonda Malgonnavar Padmashri ◽

Daliah Michael ◽

Jeyaraman Jeyakanthan ◽

...

Keyword(s):

Protein Structures ◽

Three Dimensional ◽

Protein Molecule ◽

Data Bank ◽

Protein Crystal ◽

Dimensional Structure ◽

Protein Structure Analysis ◽

Three Dimensional Structure ◽

X Ray Crystallography ◽

Polypeptide Chains

Missing regions in protein crystal structures are those regions that cannot be resolved, mainly owing to poor electron density (if the three-dimensional structure was solved using X-ray crystallography). These missing regions are known to have high B factors and could represent loops with a possibility of being part of an active site of the protein molecule. Thus, they are likely to provide valuable information and play a crucial role in the design of inhibitors and drugs and in protein structure analysis. In view of this, an online database, Missing Regions in Polypeptide Chains (MRPC), has been developed which provides information about the missing regions in protein structures available in the Protein Data Bank. In addition, the new database has an option for users to obtain the above data for non-homologous protein structures (25 and 90%). A user-friendly graphical interface with various options has been incorporated, with a provision to view the three-dimensional structure of the protein along with the missing regions using JSmol. The MRPC database is updated regularly (currently once every three months) and can be accessed freely at the URL http://cluster.physics.iisc.ac.in/mrpc.

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