Large crystal growth for neutron protein crystallography

Budayova‐Spano, Monika; Koruza, Katarina; Fisher, Zoë

doi:10.1016/bs.mie.2019.11.015

Cited by 8 publications

(7 citation statements)

References 51 publications

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“…Detailed procedures and practical examples of crystallization for neutron crystallography have been described in, for example, Ref. [3]. An anaerobic environment is desirable for growing high-quality crystals because it prevents the oxidation of protein solution during crystallization [4].…”

Section: Sample Preparation For Neutron Crystallographymentioning

confidence: 99%

Current status of neutron crystallography in structural biology

2022

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Hydrogen atoms and hydration water molecules in proteins are essential for many biochemical processes, especially enzyme catalysis. Neutron crystallography enables direct observation of hydrogen atoms, and reveals molecular recognition through hydrogen bonding and catalytic reactions involving proton-coupled electron transfer. The use of neutron crystallography is still limited for proteins, but its popularity is increasing owing to an increase in the number of diffractometers for structural biology at neutron facilities and advances in sample preparation. According to the characteristics of the neutrons, monochromatic or quasi-Laue methods and the time-of-flight method are used in nuclear reactors and pulsed spallation sources, respectively, to collect diffraction data. Growing large crystals is an inevitable problem in neutron crystallography for structural biology, but sample deuteration, especially protein perdeuteration, is effective in reducing background levels, which shortens data collection time and decreases the crystal size required. This review also introduces our recent neutron structure analyses of copper amine oxidase and coppercontaining nitrite reductase. The neutron structure of copper amine oxidase gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions. Electron transfer via a hydrogen-bond jump and a hydroxide ion ligation in copper-containing nitrite reductase are clarified, and these observations are consistent with the results from the quantum chemical calculations. This review article is an extended version of the Japanese article, Elucidation of

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Section: Sample Preparation For Neutron Crystallographymentioning

confidence: 99%

Current status of neutron crystallography in structural biology

2022

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“…2) were harvested to generate microseeds with Hampton Research Seed Beads. The microseeding technique provides the reliable reproduction of single crystals, allowing optimization trials for precipitant concentration and pH using grid searching to be carried out (Budayova-Spano et al, 2020). It is also important to determine whether the freeze-thaw process of concentrated protein samples and microseed stocks has deleterious effects on crystallization.…”

Section: CL M Pro Crystallizationmentioning

confidence: 99%

Room-temperature neutron and X-ray data collection of 3CL M^profrom SARS-CoV-2

Kneller

Phillips

Kovalevsky

et al. 2020

Acta Crystallogr F Struct Biol Commun

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The replication of SARS-CoV-2 produces two large polyproteins, pp1a and pp1ab, that are inactive until cleavage by the viral chymotrypsin-like cysteine protease enzyme (3CL Mpro) into a series of smaller functional proteins. At the heart of 3CL Mpro is an unusual catalytic dyad formed by the side chains of His41 and Cys145 and a coordinated water molecule. The catalytic mechanism by which the enzyme operates is still unknown, as crucial information on the protonation states within the active site is unclear. To experimentally determine the protonation states of the catalytic site and of the other residues in the substrate-binding cavity, and to visualize the hydrogen-bonding networks throughout the enzyme, room-temperature neutron and X-ray data were collected from a large H/D-exchanged crystal of ligand-free (apo) 3CL Mpro.

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“…Significant effort is put into obtaining large crystals to compensate for the low flux of neutron beams. Although crystals of 0.1-0.3 mm 3 are acceptable for current neutron protein crystallography (Blakeley & Podjarny, 2018;Budayova-Spano et al, 2020), these are still larger than the crystals used in X-ray protein crystallography. It is difficult to obtain high-resolution diffraction data in neutron crystallography.…”

Section: Introductionmentioning

confidence: 99%

Protonation states of hen egg-white lysozyme observed using D/H contrast neutron crystallography

Chatake¹,

Tanaka²,

Kusaka³

et al. 2022

Acta Cryst Sect D Struct Biol

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Hen egg-white lysozyme (HEWL) is an enzymatic protein with two acidic amino acids, Glu35 and Asp52, in its active site. Glu35 acts as a proton donor to the substrate and Asp52 interacts with the positively charged substrate, suggesting different protonation states of these residues. However, neutron crystallographic studies thus far have not provided a consistent picture of the protonation states of these residues. Only one study succeeded in observing the active protonation states of Glu35 and Asp52 in the triclinic crystal system. However, their active states in the most widely studied tetragonal crystal system are still unknown. The application of the D/H contrast technique in neutron crystallography improves the ability to locate exchangeable D/H atoms in proteins. In the present study, D2O and H2O solvent crystals were prepared. Each neutron data set was collected for only five days by combining a time-of-flight diffractometer (iBIX) and the spallation neutron source at the Japan Proton Accelerator Research Complex. The D/H contrast map provided better visualization of the D/H atoms in HEWL than the conventional neutron scattering length density map. The neutron D/H contrast map demonstrated the alternative protonation of the OE1 and OE2 atoms in the carboxyl group of Glu35. This alternative protonation occurs in the absence of a substrate, where high selectivity of the protonation site does not occur. In this case, only the OE1—HE1 bond attacks the substrate in an equilibrium between OE1—HE1 and OE2—HE2, or the H+ ion of the OE2—HE2 bond moves to the OE1 atom just before or after substrate binding to initiate the catalytic reaction. In contrast, the carboxyl group of Asp52 is not protonated. Protonation of the carboxyl group was not observed for other Asp and Glu residues. These results are consistent with results from NMR spectroscopy and explain the protonation states at the active site in the apo form of HEWL.

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Large crystal growth for neutron protein crystallography

Cited by 8 publications

References 51 publications

Current status of neutron crystallography in structural biology

Current status of neutron crystallography in structural biology

Room-temperature neutron and X-ray data collection of 3CL M^profrom SARS-CoV-2

Protonation states of hen egg-white lysozyme observed using D/H contrast neutron crystallography

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Large crystal growth for neutron protein crystallography

Cited by 8 publications

References 51 publications

Current status of neutron crystallography in structural biology

Current status of neutron crystallography in structural biology

Room-temperature neutron and X-ray data collection of 3CL Mprofrom SARS-CoV-2

Protonation states of hen egg-white lysozyme observed using D/H contrast neutron crystallography

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Product

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Room-temperature neutron and X-ray data collection of 3CL M^profrom SARS-CoV-2