Journal of Crystal Growth 232 (2001) 173–183 Viral capsomere structure, surface processes andgrowth kinetics in the crystallization of macromolecular crystals visualizedby in situ atomic force microscopy A.J. Malkin*, Yu.G. Kuznetsov, A. McPherson Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA Abstract In situ atomic force microscopy (AFM) was usedto investigate surface evolution duringthe growth of single crystals of turnip yellow mosaic virus (TYMV), cucumber mosaic virus (CMV) andglucose isomerase. Growth of these crystals proceeded by two-dimensional (2D) nucleation. For glucose isomerase, from supersaturation dependencies of tangential step rates andcritical step length, the kinetic coefficients of the steps andthe surface free energy of the step edge were calculated for different crystallographic directions. The molecular structure of the step edges, the adsorption of individual virus particles and their aggregates, and the initial stages of formation of 2D nuclei on the surfaces of TYMV and CMV crystals were recorded. The surfaces of individual TYMV virions within crystals were visualized, and hexameric andpentameric capsomers of the T ¼ 3 capsids were clearly resolved. This, so far as we are aware, is the first direct visualization of the capsomere structure of a virus by AFM. In the course of recording the in situ development of the TYMV crystals, a profoundrestructuring of the surface arrangement was observed.This transformation was highly cooperative in nature, but the transitions were unambiguous andreadilyexplicable in terms of an organizedloss of classes of virus particles from specific lattice positions. # 2001 Elsevier Science B.V. All rights reserved. PACS: 81.10.Dn; 68.10.Jy; 68.35.Bs; 61.72. Ày Keywords: A1. Atomic force microscopy; A1. Biocrystallization; A1. Surface processes; A1. Surface structure 1. Introduction electron microscopy is now usedas a means of obtaining low-resolution phase information for Recent years have seen the convergence of a complex assemblies such as large viruses [3], which variety of technologies for the determination of the is then extended to high resolution by X-ray structural andeven dynamic properties of supra- diffraction. As we show here, atomic force micro- molecular assemblies [1,2]. For example, cryo- scopy (AFM) may also be a useful tool for obtaining similar information. Because it can be applied under essentially physiological conditions, *Corresponding author. Tel.: +1-949-824-4397; fax: +1- 949-824-1954. in aqueous media, it may in many cases be E-mail address: [email protected] (A.J. Malkin). superior to other microscopic techniques which 0022-0248/01/$ - see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0022-0248(01)01063-6 174 A.J. Malkin et al. / Journal of Crystal Growth 232 (2001) 173–183 require dehydration, freezing, or staining, or the nanometer level. Sixtyfoldsymmetry provided potentially otherwise perturb the structures of by multiple identical protein subunits results the samples. in identical countenance from every direction. The clarity with which structural detail can be These properties along with large size of virions seen on the surfaces of small viruses, here TYMV make icosahedral viruses virtually a perfect system which has a diameter of only about 28 nm, to study surface phenomena and growth mechan- suggests that AFM may be even more broadly isms at molecular resolution, where even useful as an analytical tool. Many viruses cannot events involving an individual virus particle can be crystallizedat all, or have unit cells beyondthe be recorded. Using in situ AFM we demonstrated range of X-ray crystallography. In those cases, as that the growth of the (1 0 1) face of turnip yellow suggestedhere, AFM may providean insightful mosaic virus (TYMV) andthe (1 1 0) face of approach to study, not only large virus structure, cucumber mosaic virus (CMV) crystals proceeds but also dynamic processes such as assembly and strictly by two-dimensional nucleation. We have decapsidation [4,5]. been able to image growth step edges as well as In the past several years scanning tunneling observe the adsorption of individual virus parti- microscopy (STM) has been successfully appliedto cles, their clusters, andrecordthe initial stages of studies of atomic mobility and surface evolution of formation of 2D nuclei on crystalline surfaces. The crystalline materials grown in ultra high vacuum capsomere structures of virions immobilizedwith- [6,7]. These results have been usedin a number of in crystals of TYMV were visualizedalong with practical applications, including development of cooperative restructuring of the TYMV crystal electronic, optoelectronic andsuperconducting surface. We also describe here the results of studies devices. In contrast, experimental data on mole- of the surface morphology andkinetics of growth cular dynamics on surfaces of macromolecular for glucose isomerase crystals. crystals, a governing factor in the development of a quantitative understanding of the crystal growth process, are virtually absent. Scanning probe microscopies, including AFM, 2. Experimental section have also hada major impact on the fieldof nanoscale materials science, where they have Turnip yellow mosaic virus (TYMV), a T ¼ 3 provided information about molecular interac- icosahedral plant virus of 28 nm diameter, is one of tions andconformation [8]. They have also served the most thoroughly studied of all viruses [13–17]. 6 as mechanisms for molecular repositioning [9,10] The TYMV genome is made up of Mr ¼ 1:9 Â 10 andselective cleavage of molecular bonds[11]. single-stranded RNA of 6218 bases [16], and a 694 They have been usedto investigate reconstruction nucleotide subgenomic RNA that is included in of semiconductor surfaces upon cleavage, epitaxial most virions. The structure of the virus was growth, annealing, deposition of surfactant layers, determined by X-ray diffraction analysis [18,19] even controlled manipulation of individual atoms from bipyramidal crystals (space group P6422 with [12]. These kinds of investigations have not been a ¼ b ¼ 515:0 A( and c ¼ 309:4 AA)( grown from widely applied in structural biology because of the virus purifiedfrom Chinese cabbage by conven- fragile character of the materials andtheir liquid tional procedures [13,18]. The capsid of TYMV is environments. composedof 180 identical protein subunits, each This study represents an initial attempt to study of about 20 kDa, organizedinto 12 pentameric and the structural features of macromolecules andtheir 20 hexameric capsomers which project about 40 A( molecular dynamics on the surfaces of macro- above the surface of the virion [17,19]. TYMV molecular crystals using a large particle amenable crystals were grown by the vapor diffusion method to both manipulation anddetailedvisualization. [20] consisting of mixing 10–16 mg/ml TYMV in Because of icosahedral geometry, virions have H2O with 0.8–1.0 M ammonium phosphate in virtually a spherical, completely uniform shape at 100 mM MES at pH. 3.5 andequilibrating A.J. Malkin et al. / Journal of Crystal Growth 232 (2001) 173–183 175 droplets of this mother liquor against reservoirs of supersaturation s was defined as lnðc=ceÞ and 1.0 M ammonium phosphate. ðc À ceÞ where c and ce are the initial andequili- Crystals of cucumber mosaic virus (CMV), a brium protein concentration. T ¼ 3 icosahedral plant virus of 28 nm diameter 6 (Mr ¼ 5:5 Â 10 ) were grown by mixing 3–5 mg/ml CMV in water with 22–25% saturatedammonium 3. Results and discussion sulfate, 50 mM MES, pH 6.5 andequilibrating droplets of this mother liquor against reservoirs 3.1. Surface morphology of TYMV and of 25% saturatedammonium sulfate. CMV CMV crystals crystals studied here diffract X-rays to only 5–6 A( resolution [21] andbelong to the cubic Growth of the (1 0 1) faces of TYMV crystals space group I23 with the unit cell parameter and(1 1 0) faces of CMV crystals occurred a ¼ 336:0 A( [21]. exclusively through two-dimensional nucleation Glucose Isomerase (Mr ¼ 173 000) from Strep- andlayer-by-layer step advancement (Fig. 1). No tomyces rubiginosus was purchasedfrom Hampton dislocation sources were observed. Typically, high Research (Laguna Niguel, CA). The high-resolu- densities of growth steps were consistently ob- tion structure of the orthorhombic crystals studied servedon surfaces of TYMV andCMV crystals. In here (I222, a ¼ 94:01 AA,( b ¼ 99:37 AA,( c ¼ 103:01 AA)( the case of TYMV crystals, two-dimensional has been determined [22]. Crystals of glucose islands exhibited triangular shapes, as seen in isomerase were grown by the vapor diffusion Fig. 1(a), which indicates kinetic anisotropy in step methodconsisting of mixing 20–25 mg/ml of advancement along different crystallographic di- glucose isomerase in water with an equal amount rections. The heights of growth steps were of reservoir solution which consistedof 20% 29 Æ 2nm and25 Æ 2 nm for TYMV andCMV PEG400, 0.1 M Na Hepes, pH 7.5, 0.2 M magne- crystals, respectively. These correspondwell with sium chloride (Hampton Research Crystal Screen I). the (1 0 1) and (1 1 0) interplanar distances deduced Seedcrystals for AFM experiments were nu- by X-ray diffraction [19,21]. cleatedandgrown on glass substrates in a 10 ml Step advancement on the surfaces of growing droplet by vapor diffusion or a batch method. TYMV andCMV crystals proceedsthrough one- Crystals were then transferredinto the AFM fluid dimensional nucleation [23] at the edges, which cell, which was subsequently filledwith a mixture results in kink formation and, subsequently, their of virus, or protein, andprecipitant solution. lateral advancement (Figs. 2a, 3a–c). A similar Images were collectedin tapping modeusing a mechanism was recently observedfor the step Nanoscope III AFM (Digital Instruments, Santa advancement on growing thaumatin and lysozyme Barbara, CA) with Digital Instruments’ oxide crystals [24–28]. sharpenedsilicon nitridetips. Because of the Because of the large size of virions, not only exceptionally fragile character of the TYMV and were the structures of step edges and their move- CMV crystals application of the tapping mode ments visible, but attachment of individual virus methodis essential.