An Unconventional Mode of Protein Crystal Growth - American

Article pubs.acs.org/crystal

An Unconventional Mode of Protein Crystal Growth: Case Study Xylanase Mike Sleutel,*,† Alexander E. S. Van Driessche,*,‡ and Dominique Maes† †

Structural Biology Brussels, Flanders Interuniversity Institute for Biotechnology (VIB), Vrije Universiteit Brussel, Pleinlaan 2, 1050, Elsene, Belgium ‡ Laboratorio de Estudios Cristálograficos, IACT, CSIC - U. Granada, P.T. Ciencias de la Salud, Av. de las Palmeras 4, 18100 Armilla (Granada), Spain S Supporting Information *

ABSTRACT: We report on the violation of the succession of crystal growth modes as a function of supersaturation. Typically, a crystal transitions from spiral growth to two-dimensional (2D) nucleation mediated growth with increasing supersaturation. The defect-prone monoclinic xylanase crystals studied in this work constitute an exception to this well-maintained rule. This is the result of an unique interplay between the dominating layer generation mechanism and the subsequent occurrence and propagation of large numbers of lattice discontinuities. The defect density becomes so high that, given enough time, a fully developed, crystal-wide network of interlinked stacking faults is generated. This network effectively abolishes any advancement of steps emanating from the sole step source, being a spiral dislocation. The crystals manage to recover from this growth cessation by switching over to a secondary layer generating mechanism at lower supersaturation, that is, 2D nucleation. This work shows that by simply departing from well-established purified protein model systems, one can obtain unconventional and highly complex protein crystals with nontrivial growth mechanisms.

■

microscopy (AFM) 5 and more recently laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM).6 Early applications of AFM on protein crystal samples focused on the mechanisms and kinetics of growth and defect formation on a nano- and microscopic scale.7−10 Experimental results were interpreted within a theoretical framework that is heavily grafted onto the proven theories of small molecule crystallization.11−14 These initial in situ experiments mainly demonstrated that the mechanisms of protein crystal growth do not differ substantially from those for small molecules grown from solution, quantitative differences notwithstanding.15,16 Indeed, classical mechanisms of layer generation such as spiral growth and two-dimensional (2D) nucleation are also the dominating sources of steps on protein crystals.17 Likewise, conventional defects found on small molecule crystals, such as vacancies, interstitials, edge dislocations, stacking faults, striations, etc., are equally abundant in protein crystals. More recent efforts on protein samples, however, have uncovered new phenomena, previously undocumented for small molecules. For instance, mesoscopic observations on lysozyme crystals using LCM-DIM have identified a new

INTRODUCTION The field of protein crystallization has received considerable impetus the past three decades due to the convergence of two critical factors: (i) an increasing need to rationalize protein crystallization and (ii) the continuous development of highly performant in situ diagnostics methods. The exponential expanse of the field of molecular biology has generated a quasi unstoppable demand for detailed structural information on the biological constituents under study. As the field matures and the body of known structures grows, it is becoming increasingly more difficult to solve the structures of the remaining proteins; this class of proteins includes intrinsically disordered proteins, membrane proteins, quaternary complexes, etc. Notwithstanding recent developments in NMR1,2 and Xray free-electron lasers,3,4 protein crystallization remains a significant part of and in many cases bottleneck in the gene-tostructure pathway. Solving this bottleneck or at least broadening it can in part be achieved through fundamental research which provides insights into the mechanisms and fundamental parameters that govern the output of a protein crystallization experiment. Such research relies heavily on in situ observation methods to characterize the (pre- and early) postnucleation stages and the various subsequent intermediate stages of growth. This leads to the second major factor that has driven the field of protein crystallization to its current state: the development of pivotal techniques such as atomic force © 2012 American Chemical Society

Received: February 10, 2012 Revised: May 3, 2012 Published: May 3, 2012 2986

dx.doi.org/10.1021/cg300202f | Cryst. Growth Des. 2012, 12, 2986−2993

Crystal Growth & Design

Article

Figure 1. Polymorphism of xylanase crystals grown from aqueous bicine buffered (NH4)2SO4/NaI solutions: both monoclinic plates (a−c) and orthorhombic rods (e) are obtained for identical mother liquor compositions (respective diffraction patterns (d, f)). Panels a−c illustrate the habit change through various iterations of macroseeding of the monoclinic plates.

■

growth mechanism, coined repeated 2D nucleation, which is characterized by a fixed location of step generation and an ad random interstep distance resulting in the formation of a 2D hillock, much akin to crystals growing under Berg effect conditions.18 As such, repeated 2D nucleation can be considered a crossover growth mechanism between spiral growth and 2D mediated growth. Likewise, yet another new mode of layer generation was first observed on catalase19 and thaumatin crystals20 where massive macrostepped islands were witnessed forming on their surfaces. Later, similar observations were made for lumazine synthase crystals21 which, in combination with dynamic light scattering results, lead to the insight that these macrosteps were the result of the homoepitaxial merging of dense liquid clusters and the crystalline phase, triggering a rapid solidification of the contacting metastable liquid phase. This event leaves behind a massive mound on the crystalline surface that acts as a considerable source of steps. Finally, we highlight another example of protein crystallization research that has led to further innovations concerning the step kinetic coefficient for crystallization from solution (βstep). Indeed, observations made for ferritin22 and quite recently for glucose isomerase23 have shown that the Eyring kinetics inspired model for βstep which was initially developed for vapor crystal growth24 does not apply to protein crystallization and by extension should not apply to any instance of crystalline growth from solution. In such an environment, the momentum of the growth species will be completely dissipated to the solvent and therefore requires an adapted theoretical model to account for this. In this work, we demonstrate the violation of a classic concept in crystal growth, that is, the succession of modes of crystal growth as a function of supersaturation, most wellknown under the form of the Sunagawa diagram.25 Typically, with increasing supersaturation, a crystal will transition from spiral growth to 2D nucleation mediated growth and eventually will lead to a kinetic roughening regime26 after the breakdown of the 2D nucleation barrier. Here we demonstrate that xylanase constitutes an exception to this rule through a peculiar interplay between the dominating layer generation mechanism and the subsequent occurrence and propagation of defects.

MATERIALS AND METHODS

Production and Laser Confocal Imaging of Xylanase Crystals. Five-times recrystallized xylanase (structural details in Anneli Törrönen et al.;27 21 kDa; synonyms: endo-1,4-β-xylanase, xylanase pI 9.0, XYNII) from Trichoderma sp. was purchased from Macrocrystal Oy (Espoo, Finland) and extensively dialyzed against 10 mM bicine buffer pH 9.0. The freshly dialyzed protein solution was concentrated (Amicon Ultra Centrifugal Filters 10K MWCO, Milipore) and subsequently filtered using a 0.2 μm filter (Acrodisc). Final concentrations were determined spectroscopically at 280 nm using ε280 = 2.72 mL·mg−1·cm−1 and were typically in the region of 20−25 mg·mL−1. Crystals were grown at 20 °C by vapor diffusion using the hanging-drop method using 0.6 M ammonium sulfate, 1 M sodium iodide, and 100 mM bicine buffer pH 9.0 as reservoir solution (200 μL). This reservoir solution was then mixed in a 1:1 ratio with a diluted protein solution (1 μL) yielding a final protein concentration of 8 mg.mL−1 in the hanging drop. Within 1−3 weeks, macroscopic (200 μm) rods and highly twinned plates were obtained which were transferred using a microloop to a closed quartz cuvette (Hellma, Müllheim, Germany). The freshly prepared growth solution inside the quartz cuvette was 1−3 mg·mL−1 xylanase, 0.6 M ammonium sulfate, 1 M sodium iodide, and 100 mM bicine buffer pH 9.0. The plate crystals went through several rounds of macroseeding in order to obtain crystals of high optical quality for further imaging. The crystal−liquid interface was imaged using laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) using the setup described in Sleutel et al.28 The analytical gelfiltration was performed using a Superdex75 with 100 mM bicine pH 9.0 as the running buffer. X-ray Diffraction of Xylanase Crystals. We diffracted xylanase crystals with the purpose of unambiguously determining the exact space group. To that end, we selected crystals with a minimum of defects. For data collection at 100 K, crystals were flashfrozen in a cryocooled nitrogen-gas stream after being subsequently immersed in reservoir solutions supplemented with 10% (w/v) and 25% (w/v) glycerol. X-ray diffraction was done using an in-house Rigaku MicroMax-007HF Microfocus rotating anode generator with a Saturn 944+ digital CCD detector. The recorded diffraction patterns were processed (indexed, integrated, scaled, reduced) with the HKL3000R software package.29 Space group and unit-cell dimensions of monoclinic and orthorhombic polymorphs are P21, 40.31 Å, 38.73 Å, 56.76 Å and 90°, 109.96°, 90°; P212121, 48.94 Å, 58.89 Å, 69.89 Å and 90°, 90°, 90°, respectively (see Supporting Information Table 1 for further details). SHAPE 7.3 was used to calculate the growth 2987



Article

morhpology and, in combination with widefield microscopy images of the crystals, tentatively index the faces of both polymorphs (Figure 1).

indentations that are formed during step advancement, which we interpret as impurity induced step pinning (an example of this is indicated by an “X”). Indeed, analyzing the protein sample under both denaturing and native conditions, that is, on SDS gel and using analytical size-exclusion chromatography, we observed both lower and higher molecular weight species in solution with a relative abundance of 5−10% (Figure 4). These are certainly high enough levels to account for the witnessed impurity effects;32 however, a detailed study is required to unambiguously link both observations. We also observed strong lattice discontinuities (Figure 3d,e) protruding the surface from which 2D and/or spiral hillocks originate. The complex nature of such a step source yields nonequidistant step trains and on some occasions even macrosteps. These examples demonstrate that the xylanase crystals grown under these conditions are far from perfect and do not behave like the quasi flawless model systems such as high purity lysozyme and glucose isomerase crystals. Tested on an X-ray home-source, these crystals diffracted to maximum 2.3 Å, significantly lower than the orthorhombic rods, which, grown under identical conditions, diffracted to 1.6 Å. As such, xylanase could be a more relevant and realistic protein crystallization system that more closely resembles the average scenarios experienced by structural biologists. On more rare occasions, we observed a different mechanism of growth on the xylanase crystal surface. The system starts out with a single dislocation center from which one spiral hillock emanates that fully covers the crystal surface (Figure 5a). The off-center concentric elliptical steps that make up this hillock indicate mild step shape anisotropy. This observation combined with the directional dependence of the elementary step velocity (Figure 5) suggests anisotropy in βstep. This is also apparent in the strong orientation dependence of the step density: in the upper right quadrant individual steps cannot be discerned placing an upper limit on the interstep distance of roughly 1 μm, whereas in the lower left quadrant solitary steps are clearly visible with a spacing of ±1.5 μm (see also Figure 2c). But, most notable are the triangular structures scattered across the lower quadrants of the hillock. Such topology, to our knowledge, has never been observed on any protein crystal interface. These structures can be described as deep incisions (high contrast in the confocal images18) into the hillock slope with two steep opposing walls that act as step stoppers and a lower triangular terrace devoid of any steps (also see Figure 6). These areas are effectively zones of zero normal growth that contrast strongly with the adjacent regions of relatively high step density. Time-lapse imaging reveals that these valleys are dynamic and expand both laterally and normally in the sense that the bottom plateau grows by advancement of the last trailing spiral step and that the relative depth increases due to arrival of new steps at the apex (Figure 5b,c). The initiation of these structures is being triggered at the apex; that is, incoming steps cannot pass this point on the surface, split, and continue on either side. There are two possible models that could account for this: (i) impurity-induced pinning of multiple steps or (ii) the occurrence and subsequent propagation of two linked stacking faults. The observed terrace shape contradicts with the scenario of step pinning  in such a case the steps would bend at the pinning point, inevitably merge once a critical curvature is reached, and continue moving. This is not the case here: the walls are relatively straight and the steps are pinned indefinitely. The stacking fault scenario is therefore a much more plausible scenario here: a stacking fault nucleates

■

RESULTS For the combination of precipitants used in this study (ammonium sulfate and sodium iodide), we obtained xylanase crystals with two different morphologies, namely, thin plates and elongated rods (Figure 1a−c,e). X-ray diffraction indicates that the plates and rods have a monoclinic and orthorhombic space group, respectively (Figure 1). The latter space group has already been reported for XYNII;30 however, we found no reports in the literature of the monoclinic space group. Widefield white light microscopy reveals that these monoclinic plates are layered; that is, they are composed of slightly misoriented subunits with well-defined boundaries. In order to find a link between the microscopic growth mechanism and the macroscopic crystal habit, we performed laser confocal microscopy combined with differential interference contrast microscopy on the monoclinic crystals. We begin by demonstrating that these xylanase crystals in many ways do indeed conform to basic crystal growth principles. This is illustrated in the confocal images displayed in Figure 2, which show that both space

Figure 2. Conventional modes of growth on the (011) face of monoclinic (a−c) and the (011̅) face of orthorhombic (d) xylanase crystals growing from solution at 2 mg·mL−1: spiral hillocks (a), (b), (d) and 2D islands (c). Spiral centers are indicated by white arrows.

groups can grow either by spiral dislocations (a), (b), and (d) or 2D nucleation (c). In Figure 2b one can see a microcrystal embedded in a macrocrystal, which, due to the misalignment of both lattices, generates a grain boundary that serves as a step source. These observations confirm early reports by Malkin and co-workers who used AFM to monitor xylanase crystal growth.31 Additionally, using LCM-DIM we determined the concentration at which zero elementary step velocity was observed (no growth, no dissolution) for the monoclinic space group. We conclude that the equilibrium concentration Ce for 0.6 M ammonium sulfate, 1 M sodium iodide, and 100 mM bicine buffer pH 9.0 at 26 °C is 0.4 mg·mL−1. Looking at the dynamics of the system by performing timelapse imaging, we could identify strong impurity effects. This is summarized in Figure 3a−c where three sequential confocal images reveal the jagged nature of the steps and show deep 2988



Article

Figure 3. Clear signs of step pinning on the monoclinic (011) face: (a−c) deep indentations in the step are formed when the steps advance (example indicated by the cross, (b) after 48 min; (c) after 2 h 24 min); (d, e) spiral hillocks are formed at grain boundaries on the surface (time delay of 25 min; protein concentration 1 mg·mL−1, σ = ln(1/0.4) = 0.9).

sporadic stacking faults impeding step advancement. The defect density is already considerably higher than compared to the crystal in Figure 5 and continues to increase as growth proceeds, until finally an end-state is reached where a vast network of stacking faults is formed which completely abolishes any advancement of steps emanating from the dislocation source (Figure 7b). Note that this is a metastable kinetic trap (no thermodynamic equilibrium has been reached) and that it is lateral growth that has pushed the system toward this intermediary state of normal growth inhibition. Surprisingly, a secondary phase of growth is reinitiated under the form of 2D nucleation of circular islands on the newly formed terraces (Figure 7c−f). This is contradictory to the typical succession of modes of growth: spiral dislocations at low supersaturation followed by 2D nucleation mediated growth at intermediate supersaturation.25 Given that one or multiple crystals were present in the experimental cell (no evaporation, temperature constant within 1 °C, fixed precipitant concentration), we can conclude that the supersaturation will be equal or slightly lower at the onset of this secondary phase that ushers in the recovery of growth. One likely explanation is that there is a crossover supersaturation regime at which both growth modes can occur,18 but why do we not observe any 2D nucleation on the flanks of the spiral hillock? The density of advancing steps generated by the spiral hillock is presumably of such an order that the surface adsorbed and bulk protein molecules in the vicinity of the surface are locally depleted rendering the 2D nucleation activation barrier insurmountable.12 The stacking faults and the accompanying terraces then create local “hotspots” where, given enough induction time, (heterogeneous) 2D nucleation can occur thus reinitiating growth. Note that the observations presented here require the mesoscopic field-of-view of a confocal microscope.35 High resolution methods such as AFM lack the size in observation area (≤40 × 40 μm2) necessary to obtain the complete picture, and macroscopic methods such as widefield microscopes (mmrange) would have simply recorded temporal fluctuations of the normal growth rate.36

Figure 4. Assessment of the purity levels of the xylanase solution: (a) intentionally overloaded SDS gel of xylanase solution: both higher and lower molecular weight impurities are present; (b) chromatogram of an analytical gelfiltration-run under native conditions (Superdex75 10/ 30, 100 mM bicine buffer pH 9.0) also revealing the presence of higher and lower molecular weight impurities.

on or protrudes through the surface, splits into two opposing faults that start to propagate and serve as step stoppers. A 2D mesoscopic model is shown in Figure 6 which demonstrates initiation and subsequent growth of these defects. Obviously, stacking faults are not a novel feature on protein crystal surfaces;10,33,34 however, the splitting of a stacking fault into two branches is new to our knowledge and has some serious implications for the further modes of growth of these crystals as we discuss below. We also stress that these particular defects were not observed in our study on the faces of the orthorhombic xylanase crystals (even after extensive confocal screening), which does suggest it to be space group dependent. The global impact of these stacking faults on growth can reach a crystal wide scale as shown in Figure 7a. In Figure 7a we see a crystal in the intermediate state of spiral inhibition with 2989



Article

Figure 5. Unconventional mode of growth of monoclinic xylanase crystals on the (011) face: a central spiral hillock with stacking faults that make deep incisions into the slope creating triangular terraces devoid of any steps. The spiral center is at the intersection of the white arrows that indicate the directions for which step velocities were determined (a); two zoom-ins of (a) at different points in time (Δt = 19 min) illustrating the expanding of the triangular valleys (b, c). Protein concentration was 2 mg·mL−1 (σ = 1.6). Time-lapse movies of these still images are available as Supporting Information.

■

DISCUSSION

the model for internal stress induced elastic energy build-up and its subsequent elimination by the creation of misfit dislocations. Both theoretical considerations37 and experimental observations38 for protein crystallization support the notion of such a critical crystal size (and with it a critical level of stress) for the onset of dislocation network formation. Although patterns and networks of stacking faults are known to occur on, and in, protein crystals,39 in most cases however, stacking faults remain highly localized and generate microscopic misorder. Typically, this will not affect the B-factor (measure of atomic displacement) mainly because the short-range lattice order is preserved throughout most of the crystal. Here, the total length of the stacking fault network can reach macroscopic dimensions, which, as suggested by Malkin and Thorne,10

We reiterate that these xylanase crystals do not conform with previous protein crystal observations on two key points: their defect density and the observation that these stacking faults significantly grow in size. The density of these defects is of a scale that is previously unseen in protein crystal growth. Partially this can be explained due to the relative high impurity content of the solution. Additionally, there does seem to be a correlation between the surface defect density and the crystal’s dimensions. In our study, crystals with no detectable stacking faults (Figure 2) are 110 μm or smaller, whereas the crystals with numerous amounts of stacking faults (Figures 5 and 7) are 150 μm or larger. This suggests a critical size that complies with 2990



Article

Figure 6. Mesoscopic model for the occurrence and propagation of stacking faults: on an unperturbed spiral hillock (a) two joining stacking faults are formed (b) that both propagate as well as inhibit advancement of incoming steps. This leads to the sequential accumulation of steps along these fault lines (c−g) giving rise to two opposing steep ridges that envelop a lower terrace upon which a secondary growth phase is initiated under the form of 2D nucleation (g); multiple stacking faults lead to a group of deep grooves that “carve” into the spiral slope (f).

crystals. To what extent do these lattice discontinuities have an impact on the X-ray diffraction quality? The fact that highresolution structures of xylanase from Trichoderma are present in the protein databank (e.g., PDB-id: 2DFB)30 suggests that these defects are not necessarily an intrinsic part of xylanase’s growth modes. But remarkably, all high resolution structures were obtained from the orthorhombic polymorph; no structures obtained from the mononclinic polymorph are reported. Taking into account the observation done in this work, it seems reasonable to assume that the high density of defects is related to a specific space group, monoclinic in this case, and does significantly affect the diffraction properties. To unequivocally confirm this, additional experimental data are required. For instance, high defect density crystals might display telltale diffraction patterns from which structural information on the stacking faults can be inferred. This will require an initial confocal imaging screening prior to X-ray diffraction/topography to first confirm the presence of and select crystals with the highest defect density and then collect the diffraction patterns in order to achieve maximum results. Other questions include what triggers the occurrence of the stacking faults, why are they linked, how do they propagate, and are they related to the presence of impurities in solution? High resolution AFM imaging on both purified and unpurified samples could yield insights into these questions. These are nontrivial experiments the extent of which is beyond the scope

will significantly increase the mosaic widths of the diffraction spots (we observe a 3-fold increase in mosaicity between the orthorhombic and monoclinic space group; see Supporting Information Table 1). Such an extensive defect length scale is accomplished by the continuous occurrence of new and the propagation of existing defects. Defect growth proceeds until two defects meet or the crystal edge is reached. In this way and given enough time, they can fully cover the total crystal surface. We found no records of propagating stacking faults in the crystal growth literature: they are always reported as stagnant line defects that obstruct steps on the surface. Here we show experimental evidence that they can be dynamic and that they progressively inhibit growth as time proceeds. In many cases, protein crystal stacking faults display defined crystallographic orientations that correspond to the orientation of the crystal edges.33 This is not what we observe for xylanase: based on Figures 5 and 7 we conclude that the orientation and the angle between two interlinked stacking faults (ranging between 40 and 110°) are a function of the angular position around the spiral center and do not coincide with any particular crystallographic directions.

■

CONCLUDING REMARKS AND PERSPECTIVES Several questions still remain open and need to be answered to fully elucidate the events observed on the monoclinic xylanase 2991



Article

Figure 7. Progression of the stacking fault formation on the (011) face at 3 mg·mL−1 (σ = 2.0): (a) intermediate state where step formation remains dominated by the spiral dislocation with sporadic stacking faults impeding step advancement; (b) end-state where a vast network of stacking faults is formed which completely abolishes any advancement of steps emanating from the dislocation source (recorded 30 min after (a)); (c−f) sequential images (∼3 min interval) illustrating the recovery of growth through 2D nucleation resulting in the formation of circular islands on the freshly formed terraces. The spiral center is at the intersection of the white arrows that indicate the directions for which step velocities were determined, and black arrows indicate regions where new islands are formed. Time-lapse movies of these still images are available as Supporting Information.

the improvement of the manuscript’s quality. M.S. and D.M. are grateful for the support by the Belgian PRODEX Programme under contract number ESA AO-2004-070. A.V.D. acknowledges the support by Grant No. AYA2009-10655 of the Ministry of Science and Innovation, Spain, and the ConsoliderIngenio 2010 project “Factoriá Española de cristalización” (A.V.D.). X-ray infrastructure was funded by the Herculesstichting, Grant No. UABR/09/005.

of this article. Ultimately they can, however, lead to a molecular-scale model that explains both the occurrence and propagation of these fault lines.

■

ASSOCIATED CONTENT

S Supporting Information *

The space group details, unit cell dimensions, and statistics of data collection of both Xylanase polymorphs can be found in Supporting Table 1. Time-lapse movies of Figure 5a, Figure 5b,c, and Figure 7a,b are available free of charge via the Internet at http://pubs.acs.org.

■

■

REFERENCES

(1) Lewis, E., K. J. Magn. Reson. 2011, 213, 477−491. (2) Nietlispach, D.; Gautier, A. Curr. Opin. Struct. Biol. 2011, 21, 497−508. (3) Chapman, H. N.; et al. Nature 2011, 470, 73−77. (4) Fratalocchi, A.; Ruocco, G. Phys. Rev. Lett. 2011, 106, 105504. (5) Durbin, S. D.; Carlson, W. E. J. Cryst. Growth 1992, 122, 71−79. (6) Sazaki, G.; Matsui, T.; Tsukamoto, K.; Usami, N.; Ujihara, T.; Fujiwara, K.; Nakajima, K. J. Cryst. Growth 2004, 262, 536−542. (7) Durbin, S.; Feher, G. J. Mol. Biol. 1990, 212, 763−774. (8) McPherson, A.; Malkin, A. J.; Kuznetsov, Y. G.; Plomp, M. Acta Crystallogr., Sect. D 2001, 57, 1053−1060. (9) McPherson, A.; Kuznetsov, Y. G.; Malkin, A.; Plomp, M. J. Struct. Biol. 2003, 142, 32−46 DA - 20030429.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (M.S.); [email protected] (A.V.D). Phone: +32 2 629 1932. Fax: +32 2 629 1963. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors would like to acknowledge the suggestions and critical comments of Reviewers 1 and 2 which contributed to 2992



Article

(10) Malkin, A. J.; Thorne, R. E. Methods 2004, 34, 273−299. (11) Burton, W. K.; Cabrera, N.; Frank, F. C. Philos. Trans. R. Soc. London 1951, 243, 299−358. (12) Chernov, A. A. Modern Crystallography III. Crystal Growth; Springer-Verlag: Berlin, 1984. (13) Saito, Y. Statistical Physics of Crystal Growth; World Scientific: Singapore, 1996. (14) Markov, I. V. Crystal Growth for Beginners: Fundamentals of Nucleation, Crystal Growth and Epitaxy; World Scientific Publishing: Singapore, 2003. (15) Vekilov, P. G.; Alexander, J. I. D. Chem. Rev. 2000, 100, 2061− 2089. (16) Vekilov, P. G.; Chernov, A. A. Solid State Phys. 2002, 57, 2−149. (17) McPherson, A. Crystallization of Biological Macromolecules; Cold Spring Harbor Laboratory Press: New York, 1999. (18) Van Driessche, A. E. S.; Sazaki, G.; Otalora, F.; Gonzalez-Rico, F. M.; Dold, P.; Tsukamoto, K.; Nakajima, K. Cryst.Growth Des. 2007, 7, 1980−1987. (19) Malkin, A. J.; Kuznetsov, Y. G.; McPherson, A. Surf. Sci. 1997, 393, 95−107. (20) Malkin, A. J.; Kuznetsov, Y. G.; Glantz, W.; McPherson, A. J. Phys. Chem. 1996, 100, 11736−11743. (21) Gliko, O.; Pan, W.; Katsonis, P.; Neumaier, N.; Galkin, O.; Weinkauf, S.; Vekilov, P. G. J.Phys.Chem.B 2007, 111, 3106−3114. (22) Petsev, D. N.; Chen, K.; Gliko, O.; Vekilov, P. G. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 792−796. (23) Sleutel, M.; Van Driessche, A. E. S.; Pan, W.; Reichel, E. K.; Maes, D.; Vekilov, P. G. J. Phys. Chem. Lett. 2012, 1258−1263. (24) Chernov, A. A. Soviet Phys. Uspekhi 1971, 4, 116−148. (25) Sunagawa, I. In Morphology of Crystals; Sunagawa, I., Ed.; Terra Scientific Publishing Company: Dordrecht, 1987; pp 509−587. (26) Sleutel, M.; Maes, D.; Wyns, L.; Willaert, R. Cryst. Growth Des. 2008, 8, 4409−4414. (27) Törrönen, A.; Harkki, A.; Rouvinen, J. EMBO J. 1994, 13, 2493−2501. (28) Sleutel, M.; Willaert, R.; Wyns, L.; Maes, D. Cryst. Growth Des. 2009, 9, 335−343. (29) Otwinowski, Z.; Minor, W.; Charles W. Carter, J. Macromolecular Crystallography Part A; Academic Press: New York, 1997; Vol. 276; pp 307−326. (30) Watanabe, N.; Akiba, T.; Kanai, R.; Harata, K. Acta Crystallogr., Sect. D 2006, 62, 784−792. (31) Malkin, A. J.; Kuznetsov, Y. G.; McPherson, A. J. Cryst. Growth 1999, 196, 471−488. (32) Nakada, T.; Sazaki, G.; Miyashita, S.; Durbin, S. D.; Komatsu, H. J. Cryst. Growth 1999, 196, 503−510. (33) Malkin, A. J.; Kuznetsov, Y. G.; McPherson, A. J. Struct. Biol. 1996, 117, 124−137. (34) Land, T. A.; De Yoreo, J. J. J. Cryst. Growth 2000, 208, 623−637. (35) Sleutel, M.; Maes, D.; Van Driessche, A. E. S. Adv. Chem. Phys. 2012, 151, 223−276. (36) Van Driessche, A. E. S.; Otalora, F.; Sazaki, G.; Sleutel, M.; Tsukamoto, K.; Gavira, J. A. Cryst.Growth Des. 2008, 8, 4316−4323. (37) Chernov, A. A. J. Cryst. Growth 1999, 196, 524−534. (38) Yau, S. T.; Thomas, B. R.; Galkin, O.; Gliko, O.; Vekilov, P. G. Proteins 2001, 43, 343−352. (39) Ko, T. P.; Kuznetsov, Y. G.; Malkin, A. J.; Day, J.; McPherson, A. Acta Crystallogr., Sect. D 2001, 57, 829−839.

2993


An Unconventional Mode of Protein Crystal Growth - American

Recommend Documents