LSCALE – the new normalization, scaling and absorption correction program in the Daresbury Laue software suite

MTAN (5′-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine moiety is mediated almost exclusively by hydrogen bonds, and the proposed catalytic mechanism requires multiple protontransfer events. Of particular interest is the protonation state of residue D198, which possesses a pK a above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wildtype HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA) as well as one neutron/X-ray crystal structure of an inactive variant (HpMTAN-D198N) cocrystallized with SAH. These results support a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs.neutron diffraction | enzyme mechanism | proton transfer | nucleosidase | Helicobacter

Section: Methodsmentioning

confidence: 99%

Neutron structures of the Helicobacter pylori 5′-methylthioadenosine nucleosidase highlight proton sharing and protonation states

Banco

Mishra

Ostermann

et al. 2016

“…Using the data from all crystal settings, six iterations of a combined five cycles of wavelength normalization and four cycles of intercycle scaling were performed. The wavelength normalization curve was modeled by using nine Chebyshev polynomials (28). To obtain reasonable values for R merge , the wavelength range was restricted to 0.9-6.5 Å, and only reflections with I Ͼ 3 were used in determining the wavelength normalization polynomial.…”

Section: Methodsmentioning

confidence: 99%

Locating active-site hydrogen atoms in d -xylose isomerase: Time-of-flight neutron diffraction

Katz

Carrell

et al. 2006

Time-of-flight neutron diffraction has been used to locate hydrogen atoms that define the ionization states of amino acids in crystals of D-xylose isomerase. This enzyme, from Streptomyces rubiginosus, is one of the largest enzymes studied to date at high resolution (1.8 Å) by this method. We have determined the position and orientation of a metal ion-bound water molecule that is located in the active site of the enzyme; this water has been thought to be involved in the isomerization step in which D-xylose is converted to D-xylulose or D-glucose to D-fructose. It is shown to be water (rather than a hydroxyl group) under the conditions of measurement (pH 8.0). Our analyses also reveal that one lysine probably has an ؊NH2-terminal group (rather than NH 3 ؉ ). The ionization state of each histidine residue also was determined. High-resolution x-ray studies (at 0.94 Å) indicate disorder in some side chains when a truncated substrate is bound and suggest how some side chains might move during catalysis. This combination of time-of-flight neutron diffraction and x-ray diffraction can contribute greatly to the elucidation of enzyme mechanisms.amino acid ionization states ͉ enzyme mechanism ͉ x-ray diffraction ͉ deuterium͞hydrogen in proteins ͉ proton transfer T he enzyme D-xylose isomerase (XI) from the bacterium Streptomyces rubiginosus, a homotetramer of molecular mass 173 kDa, is one of a large class of aldose-ketose isomerases that require two divalent metal ions for function. The folding of its backbone, that of a (␤␣) 8 barrel, was first determined by us in 1984 (1). We report here our structural studies by time-of-flight neutron diffraction; XI is among the largest enzymes studied by this method. One metal ion (M1) binds four carboxylate groups (Glu-181, Glu-217, Asp-245, and Asp-287) and two water molecules (W1116 and W1218 in Fig. 1). The substrate binds at this site, displacing the two metal-bound water molecules. The other metal site (M2) binds three carboxylate groups (Glu-217, Asp-257, and bidentate Asp-255), one water molecule, and a His residue (His-220). The carboxylate group of Glu-217 is shared by both metal ions.XI catalyzes the interconversion of the aldo-sugars D-xylose or D-glucose to the keto-sugars D-xylulose and D-fructose, respectively. There are two sites in XI where hydrogen transfer is involved in a catalytic mechanism. One involves the opening of the ring of the cyclic sugar substrate to give a straight-chain sugar that the enzyme then can isomerize. This ring-opening takes place near one of the metal ions, M1 (in the upper region of Fig.

“…Integration of each Laue reflection was performed. The program LSCALE (18) was used to derive the wavelength-normalization curve by using the intensities of symmetry-equivalent reflections measured at different wavelengths. No account was made for crystal damage since neutrons do not induce detectable radiation damage and no explicit absorption corrections were applied.…”

Section: Methodsmentioning

confidence: 99%

The 15-K neutron structure of saccharide-free concanavalin A

Blakeley

Kalb

Helliwell

et al. 2004

The positions of the ordered hydrogen isotopes of a protein and its bound solvent can be determined by using neutron crystallography. Furthermore, by collecting neutron data at cryo temperatures, the dynamic disorder within a protein crystal is reduced, which may lead to improved definition of the nuclear density. It has proved possible to cryo-cool very large Con A protein crystals (>1.5 mm 3 ) suitable for high-resolution neutron and x-ray structure analysis. We can thereby report the neutron crystal structure of the saccharide-free form of Con A and its bound water, including 167 intact D2O molecules and 60 oxygen atoms at 15 K to 2.5-Å resolution, along with the 1.65-Å x-ray structure of an identical crystal at 100 K. Comparison with the 293-K neutron structure shows that the bound water molecules are better ordered and have lower average B factors than those at room temperature. Overall, twice as many bound waters (as D2O) are identified at 15 K than at 293 K. We note that alteration of bound water orientations occurs between 293 and 15 K; such changes, as illustrated here with this example, could be important more generally in protein crystal structure analysis and ligand design. Methodologically, this successful neutron cryo protein structure refinement opens up categories of neutron protein crystallography, including freeze-trapped structures and cryo to room temperature comparisons. W ater molecules located in both the interior and on the surface of a protein have been shown to play diverse and important roles for the efficient functioning of proteins. Vital information on their hydrogen-bonding interactions can be gained from neutron data. Neutron crystallography can be used to directly assign the positions of hydrogen isotopes in a protein and its bound solvent, which can be done more effectively and at lower resolutions than required with x-rays, thereby providing more information from a diffraction experiment (1). In x-ray protein crystallography, x-rays are scattered in proportion to the number of electrons. Therefore, the ability to locate the hydrogen isotopes of a protein and its bound solvent is only possible if data are available at ultra-high resolution (Ͼ1 Å) and critically where the relevant atoms are ordered sufficiently well; an examplar of this approach is the 0.66-Å x-ray study of aldose reductase (2). For neutrons, however, the scattering centers are the atomic nuclei, and each nucleus has a characteristic strong force interaction with a neutron. There is considerably less variation between the elements, and furthermore the interaction can be different for different isotopes of the same element. Hydrogen atoms have a negative neutron scattering length (b H ϭ Ϫ0.374), which is of similar magnitude but opposite in sign to C (b C ϭ 0.665), N (b N ϭ 0.936), O (b O ϭ 0.580), and D (b D ϭ 0.667). The practical effect of this sign difference is that hydrogen atoms appear as negative peaks in neutron Fourier maps, in contrast to the majority of atoms, which appear as positive peaks (3) and can...