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

Abstract

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...

Temperature-dependent macromolecular X-ray crystallography

“…Another beneficial effect of cryocooling originates from reduced dynamic disorder. For example, about twice as many water molecules are detected at cryo-temperature compared with room temperature in protein structures determined using X-ray (Nakasako, 1999) or neutron crystallography (Blakeley et al, 2004). Today, more than 90% of all macromolecular X-ray crystal structures are determined from data collected at 100 K (Garman & Owen, 2006).…”

Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase

“…Neutron diffraction offers a more reliable solution to the problem, because hydrogen and, particularly, its isotope deuterium (D) are visible at much lower resolutions than required with X-rays (14,15). The approach does present a number of challenges, particularly the need to cool the large crystals required for neutron crystallography down to the cryogenic temperatures required for the study of enzyme intermediates (16). But the fact that neutrons are non-ionizing and are scattered by the atomic nuclei (rather than by the electrons, as in X-ray crystallography) means that photoreductionwhich has been the source of most of the confusion in previous X-ray work -does not occur at all.…”

Protein crystal perfection and its application

“…However, we have recently shown that it is possible to freeze and collect high resolution X-ray (1.65 Å on a rotating anode) and neutron data (2.5 Å on LADI; Fig. 7) from large concanavalin A protein crystals ($2 and 5 mm 3 ) as examples (Blakeley, Kalb et al, 2004). These data have allowed a combined 'X + n' protein structure analysis to be undertaken as performed previously with room temperature X-ray and neutron data sets (Habash et al, 2000).…”

“…Bigger crystals for neutron structural studies are also needed and where they are reasonably perfect. In an interesting twist of this story, freezing of very large crystals of proteins suitable for high resolution neutron data collection is a recent breakthrough (Blakeley, Kalb et al, 2004). Neutron cryo-crystallography benefits the clarity of bound water structure, including the water deuteriums, and also opens up freeze-trap neutron protein structural studies.…”