Photon Factory Activity Report 2022 #40 (2023)

BL-20B/ 2021G037

Control of strain in subgrains of protein crystals by the introductionof grown-in dislocations
Haruhiko KOIZUMI1,*, Satoshi UDA2, Ryo SUZUKI3 and Masaru TACHIBANA3
1
Graduate School of Integrated Sciences for Life, Hiroshima University,
1-4-4 Kagamiyama, Higashi-Hiroshima, 739-8528, Japan2
New Industry Creation Hatchery Center, Tohoku University,
6-6-10 Aramaki Aza Aoba, Aoba-ku, Sendai, 980-8579, Japan
3
Graduate School of Nanobioscience, Yokohama City University,
22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan
It is important to reveal the exact cause of poor diffractivity in protein crystals in order to determine the accurate structureof protein molecules. It is shown that there is a large amount of local strain in subgrains of glucose isomerase crystals eventhough the overall crystal quality is rather high, as shown by clear equal-thickness fringes in X-ray topography. Thus, alarge stress is exerted on the subgrains of protein crystals, which could significantly lower the resistance of the crystals toradiation damage. It is also demonstrated that this local strain can be reduced through the introduction of dislocations in
the crystal. This suggests that the introduction of dislocations in protein crystals can be effective in enhancing the crystal
quality of subgrains of protein crystals. By exploiting this effect, the radiation damage in subgrains could be decreased,leading to the collection of X-ray diffraction data sets with high diffractivity.
1 Introduction
The three-dimensional (3D) structures of protein
molecules are intimately related to the proteins found inliving organisms. Therefore, it is important to determine
the 3D structures of protein molecules from the points ofview of both fundamentals and applications in medicalscience. The determination of the 3D structures of protein
molecules has typically been carried out at synchrotronradiation facilities with high brilliance. A structure
determined using data at a resolution of better than 1.5 Å,corresponding to the length of a covalent carbon–carbonbond, is necessary to achieve structure-guided drug designand controlled drug delivery. This means that thecollection of many high-order reflections from protein
crystals, i.e. high diffraction efficiency (diffractivity), is
desirable to obtain accurate 3D structures of protein
molecules. As such, many researchers have concentrated
on developing high-brilliance sources and/or improving
the sensitivity of the detector systems [1]. However,protein molecules useful in structural analysis (<1.5 Åresolution) for structure-guided drug design and controlled
drug delivery represent only 9% of all protein molecules
deposited in the Protein Data Bank, even when usingsynchrotron-radiation facilities with high brilliance such asSPring-8. This suggests the importance of the growth ofhigh-quality protein crystals that allow the collection ofmany high-order reflections. In fact, it has been reported
that the improvement of crystal quality leads to an increasein the integrated intensities of diffraction spots [2].
Therefore, it is important to enhance the crystal quality ofprotein crystals.
In this report, we observed a large amount of local strain
in the subgrains of the present glucose isomerase crystals,
even though the overall quality of the crystal as a whole isnear-perfect as verified by microscopic XRD rockingcurve measurements. We believe that this points to ainherent crystal imperfection in protein crystals thatprevents the achievement of high diffractivity, which isassociated with radiation damage due to synchrotron
radiation. Moreover, we demonstrate control over the local
strain in subgrains of glucose isomerase crystals byintroducing dislocations.
2 Experiment
Glucose isomerase was purchased from Hampton
Research and was used without further purification. The
crystallization conditions were the same as those usedpreviously [3, 4]. Glucose isomerase crystals were grownfrom seed crystals that were either cross-linked using aglutaraldehyde solution or left non-cross-linked. Thecrystals grown under these conditions were approximately
2.5 mm in size. The crystals were orthorhombic, with spacegroup I222 and unit-cell parameters a = 93.88, b = 99.64,
c = 102.90 Å, and contained two molecules per unit cell[5].
Microscopic XRD rocking-curve measurements wereperformed at room temperature using synchrotron
radiation on the BL20B beamline at the Photon Factory,
High Energy Accelerator Research Organization (KEK),
Japan. A monochromatic beam with a wavelength of 1.2 Å
was selected. Instrument parameter details are provided ina previous report [4]. The glucose isomerase crystals weresealed in an acrylic cell as reported previously [3, 4]. A
high spatial resolution, two-dimensional digital CCD
camera (Photonic Science X-RAY FDI 1.00:1, 6.45 × 6.45μm effective pixels) was employed to measure themicroscopic XRD rocking curves for the 011 family ofreflections obtained from glucose isomerase crystals. XRD
rocking curves were reconstructed from reflected
intensities outside the crystals using various beam-spot
sizes ranging from 64.5 μm (ten pixels) to 1096.5 μm (170pixels).

Photon Factory Activity Report 2022 #40 (2023)

3 Results and Discussion
Fig. 1 shows a digital X-ray topograph of a glucose
isomerase crystal taken using the 011 reflection. As seen inFig. 1, no contrast attributable to the seed crystal or
dislocations was observed because the seed crystal was notcross-linked using glutaraldehyde solution. XRD rocking
curves for each beam-spot size were reconstructed fromthe region within the white circle. Fig. 2 shows theestimated dependence of local strain and misorientation on
beam-spot size in dislocation-free glucose isomerase
crystals. The filled and open symbols represent the data for
type A1 and type A2 crystals, respectively. As shown in Fig.
2, the local strain increases with decreasing beam-spot sizefor beam-spot sizes below 451.5 μm (70 pixels), levelingoff at around 28.2 με. This means that a large amount oflocal strain is accumulated in subgrains, because the beamspot size (64.5 μm) is smaller than the previously estimatedsubgrain size of tetragonal henn-egg-white (HEW)
lysozyme crystals (200–600 μm) [6].

Fig. 1: Digital X-ray topograph of a glucose isomerase
crystal taken using the 011 reflection. The white circle
is the region from which the XRD rocking-curves foreach beam-spot size were reconstructed.

Fig. 3: Dependence of the local strain on beam-spotsize estimated using a glucose isomerase crystal witha few dislocations. The filled and open symbols
represent data obtained from type B1 and type B2
crystals, respectively. The analyzed region enclosedby the white circle does not contain any dislocations.
Next, we demonstrate that the local strain in subgrains
can potentially be controlled by introducing grown-in
dislocations in the crystal. However, no dislocations wereincluded in the analyzed region in the present case. Fig. 3
shows the dependence of local strain on beam-spot size forglucose isomerase crystals containing a few dislocations.
The filled and open symbols represent data obtained fromtype B1 and type B2 crystals, respectively. As clearly seenin the digital X-ray topograph in Fig. 3, dislocations weregenerated in the crystal. In the case of glucose isomerase
crystals with a few dislocations (types B1 and B2), it isobserved that the local strain in the subgrains decreases asa whole relative to that for dislocation-free crystals (typeA1 and type A2), and the beam-spot size at which the local
strain in the subgrains becomes detectable also decreases.
This means that dislocations have a significant effect on
the local strain in subgrains, even if the analyzed region,
which is free of dislocations, is far removed from thegrown-in dislocations. Thus, the influence of the strain
field of each dislocation could extend over a distance of theorder of a millimetre.
References[1] N.E. Chayen et al., Macromolecular Crystallization
and Crystal Perfection. (2010).
[2] E.H. Snell et al., Acta Cryst. D51, 1099 (1995).
[3] H. Koizumi et al., Cryst. Growth Des. 14, 5111 (2014).
[4] H. Koizumi et al., Cryst. Growth Des. 16, 4905 (2016).
[5] H. Carrell et al., PNAS 86, 4440 (1989).
[6] H. Koizumi et al., Cryst. Growth Des. 14, 5662 (2014).
* h-koizumi@hiroshima-u.ac.jp

Fig. 2: The dependence of local strain andmisorientation on beam-spot size estimated using a
glucose isomerase crystal grown from a non-crosslinked seed crystal. The filled and open symbols
represent data obtained from type A1 and A2 crystals,respectively.