Microbatch Mixing: âShaken not Stirredâ, a Method ... - ACS Publications

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Microbatch mixing: “Shaken not Stirred,” a method for macromolecular microcrystal production for serial crystallography Brian P. Mahon, Justin J. Kurian, Carrie L. Lomelino, Ian Smith, Lilien Socorro, Antonette Bennett, Alex M. Hendon, Paul R. Chipman, Daniel A Savin, Mavis Agbandje-McKenna, and Robert McKenna Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b00643 • Publication Date (Web): 28 Sep 2016 Downloaded from http://pubs.acs.org on September 29, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Crystal Growth & Design is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Crystal Growth & Design

General schematic outlining MBM outlined in 3 steps. Step 1: Tray is set up similar to a batch crystallization method through dispensing crystallization target directly into mother liquor. Step 2: Ligands, or cocrystallization material is added to the well. Note this step is relative to their experiment. Step 3: This step entails sealing the crystallization well with a coverslip and “shaking” the tray to induce constant mixing. This causes induction of secondary nucleation and thus microcrystal growth. Fgiure 1 232x137mm (150 x 150 DPI)

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Crystals of hCA II and HEWL with or without applying MBM. A) Crystals of hCA II and B) HEWL grown using a standard batch crystallization method. Microcrystalline slurries of C) hCA II and D) HEWL after application of MBM. Negative stain EM images of microcrystals of E) hCA II and F) HEWL. Figure 2 151x156mm (150 x 150 DPI)


Page 2 of 35

Page 3 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


X-ray diffraction image from hCA II microcrystal slurry at room temperature. Marked are the resolution shells indicating a high resolution of ~3 Å. Figure 3 137x89mm (220 x 220 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Comparison of lysozyme crystal growth at different shaking speeds. This includes incubation at A) static, B) 200 rpm shaking, and C) 400 rpm shaking. Note, 50 mg/ml HEWL was used in A and B, and 30 mg/mL in C. This was due to precipitate formation observed in wells containing 50 mg/mL and shaking at 400 rpm. All wells prepared at 1:5 protein to precipitant ratio. 222x73mm (300 x 300 DPI)


Page 4 of 35

Page 5 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Crystals of AAV8 grown using MBM. A). Crystals grown with a 1:3, protein to precipitant ratio. B) Crystals grown with a 1:1 protein to precipitant ratio. 182x63mm (150 x 150 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC - graphic TOC 239x127mm (150 x 150 DPI)


Page 6 of 35

Page 7 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Microbatch mixing: “Shaken not Stirred,” a method for macromolecular microcrystal production for serial crystallography Brian P. Mahona, Justin J. Kuriana, Carrie L. Lomelinoa, Ian R. Smithb, Lilien Socorroa, Antonette Bennetta, Alex M. Hendona, Paul Chipmana, Daniel A. Savinb, Mavis AgbandjeMcKennaa and Robert McKennaa,*

a

Department of Biochemistry and Molecular Biology, University of Florida College of Medicine,

Gainesville, Florida 32610 b

Department of Chemistry, University of Florida, Gainesville, Florida 32611

*

Corresponding Author. R.M. Telephone: (352) 392-5696; e-mail: [email protected].

Key words: microcrystals, serial crystallography, microbatch crystallization, nucleation



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract

The advances of serial-crystallography techniques at synchrotron and X-ray free electron laser (XFEL) facilities have made possible the acquisition of useable data sets to determine 3dimensional structures of macromolecules from micro- to nano-sized crystals. In addition, the same technological hallmarks have contributed significantly to the field of time-resolved crystallography. However, production of usable crystalline slurries for serial-crystallographic experiments has been one of the limiting factors and contributes to an alternative sample “bottleneck” in crystal growth. In this study, we propose a method: labelled microbatch-mixing (MBM), which has the capability to produce large quantities of microcrystals of macromolecules suitable for serial-crystallographic experiments. This is shown to be successful for producing lysozyme, carbonic anhydrase, and adeno-associated virus crystals. MBM takes advantage of secondary nucleation induced by mixing via the application of steady agitation during the crystallization process. This leads to excessive nucleation, resulting in large quantities of welldiffracting microcrystals. MBM therefore presents a method that can potentially be applied to a range of macromolecules and a possible simple protocol to produce microcrystals for serialcrystallographic experiments.

2 ACS Paragon Plus Environment

Page 8 of 35

Page 9 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Utilization of X-ray crystallography to determine the atomic resolution structures of macromolecules has long been a dominant method of structural biology.1 The data quality and often experimental feasibility of X-ray diffraction studies is limited by crystal quality, with inherent links between crystal size, order, and radiation damage; whereby it has been previously determined that crystals with diameter of >1 µm were needed to obtain a usable data set.2–4 Recently, with the advances of serial-crystallography techniques at synchrotron and X-ray free electron laser (XFEL) sources, diffraction data sets obtained from micro- and nanocrystals can be collected, and used to determine 3-D structures.5–7 Furthermore, development of serialfemtosecond X-ray diffraction (SFX) experiments made possible by XFELs have made significant progress in the field of time-resolved experiments, which are now being applied and implemented at synchrotron sources.7–12 The excitement and interest sparked by these technological advancements has once again put pressure on sample preparation. Specifically, the use of SFX requires large quantities of micro- to nano-sized crystals.13,14 However, for production of usable crystals, determining crystallization conditions for many macromolecular targets, using complex setups or alterations in protein expression and purification schemes, have been some of the rate limiting factors, thus contributing to an “alternative bottleneck” for sample preparation.13–15 Typically, this has included varying crystallization conditions to increase supersaturation points by modulating precipitant concentration, pH, temperature, or protein



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 35

concentration.16 Furthermore, using micro-seeding techniques or in the case of membrane proteins, growing crystals in lipid cubic phase (LCP), have shown to be viable options.16 More recently, it has been shown that vigorously stirring a sample of photoactive yellow protein (PYP) has shown also to induce micro-crystallization.17 As SFX becomes more routinely used, this presents a significant need to develop standard, and more easily implemented methods to produce suitable microcrystal samples. Previously, it has been shown that formation of small or micro-sized crystals is a result of the induction of secondary nucleation points within the crystallization drops.18 In the process of chemical crystallization of non-biological molecules, nucleation kinetics have been well defined and described through the empirical power law expression:

B = kBNpMqSr

Eq. 1

Where B is the nucleation rate defined by the product of the nucleation rate coefficient (kB), or mixing speed (N), crystal concentration (M), and supersaturation that accounts for thermodynamics of the solution (S), where p, q, and r represent power law exponents.19,20 This indicates that applying agitation via different mixing speeds during the crystallization process can influence the formation of secondary nucleation in a linear relationship.20 Interestingly, the mixing speed applied during crystallization had a greater influence on secondary nucleation than the supersaturation of the solution, a phenomena also noted in PYP after vigorous stirring was implemented prior to crystallization.17,20 In this study, we utilize previous observations of the influence of mixing speed on the formation of secondary nucleation of protein crystalline samples to develop a simple method that


Page 11 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


has shown the capability to produce micro- to nano-sized crystals suitable for SFX. Our method utilizes the in situ mixing of samples during the crystallization process, labelled microbatchmixing (MBM), which can be applied to a range of macromolecular samples. We apply MBM to hen egg-white lysozyme (HEWL), human carbonic anhydrase II (hCA II), and adeno-associated virus serotype 8 (AAV8).21–23 This work proposes a simple method that shows the potential to reliably produce large amounts of crystals of required size for SFX experiments.

Experimental Sample Preparation Lyophilized HEWL was purchased from Sigma Aldrich (USA) and resuspended in 50 mM sodium acetate pH 4.5 to a final concentration of 30 mg/ml or 50 mg/mL prior to use in crystallization. Recombinant hCA II was expressed and purified as described by Mahon et al.24 Briefly, BL21DE3 E.coli cells that contain the plasmid encoding hCA II were grown in 1 liter of Luria broth, supplemented with 100 µg/mL ampicillin, to an OD600 between 0.6 - 1.0 at which point hCA II expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside for ~ 4 hours at 37oC in the presence of 1 M zinc sulfate. Cells containing hCA II were harvested, resuspended using a glass homogenizer, and enzymatically lysed overnight at 4oC. Expressed hCA II was purified by affinity separation using a gravity-fed column containing agarose resin coupled to the inhibitor p-(aminomethyl)benzenesulfonamide (p-AMBS; Sigma). Sodium dodecyl sulfate (SDS) –polyacrylamide gel electrophoresis (PAGE) of eluents was used to estimate purity. Prior to crystallization, hCA II was buffer exchanged in 50 mM Tris-HCl pH 7.8 using centrifugation, and concentrated to 25 mg/ml.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 35

AAV8 samples were generated in a recombinant baculovirus-sf9 expression system (bacmid was generously donated by Sergei Zolotukhin, University of Florida) according to the manufacturer’s protocol and similar to that performed by Lane et al.25 Briefly, an AAV8 containing pellet was resuspended in lysis buffer (50 mM Tris pH 8, 100 mM NaCl, 2 mM EDTA, and 0.2% Triton), and freeze thawed three times in an ethanol/dry-ice slurry and a 37°C water-bath. The sample was then benzonase (Novagen) treated at 37°C for 1 hr. At the end of the incubation period, the sample was then clarified by centrifugation at 10K rpm in a JA-20 rotor for 20 mins. The clarified supernatant was loaded onto a discontinuous iodixanol gradient followed by an ionexchange chromatography step, according to the previously established protocol.25 The purity of the AAV8 containing fractions was determined by coomassie stain SDS-PAGE, and virus integrity determined by negative stain EM. The purified sample was buffer exchanged into a final crystallization buffer (50 mM Tris pH 7.4, 300 mM NaCl and 6% glycerol) and concentrated to 2.5 mg/ml.

Microbatch-Mixing for production of microcrystals MBM utilizes the effect of applying agitation via mixing during the crystallization process to intentionally increase, by orders of magnitude, the secondary nucleation points for micro- and nano-sized crystal formation (Eq. 1). The general procedure consists of a batch crystallization setup that is followed by agitation by mixing.26 For our study, we utilize 24-well VDX plates with sealant (Hampton Research, USA) for crystallization of all samples. Briefly, 300 - 500 µl of precipitant solution was pipetted into each well. Then the protein or virus sample was subsequently added to the wells in ratios of 1:5, 1:10, and 1:15 (sample:precipitant) to determine effects of sample concentration on microcrystallization. Trays were then sealed with a cover slip


Page 13 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and placed at room temperature (RT) on a tabletop shaker set to either 72, 200, or 400 rpm and left overnight. Shaking speeds were determined by 1) minimal speed required to induce significant mixing of sample and 2) highest speed capable of the table-top shaker (Daigger Scientific Inc. USA). A schematic outlining the MBM procedure is presented in Figure 1. The crystallization condition for HEWL consisted of 50 mM sodium acetate pH 4.5, with either 0.8, 1.0, or 1.5 M NaCl.27 Concentrations of NaCl were varied to determine if crystal size, or quantity, could be manipulated in combination with mixing. For hCA II, the crystallization condition used was 1.6 M sodium citrate 50 mM Tris-HCl pH 7.8.28–30 In order to determine if crystal size and quantity could be manipulated (similar to HEWL) we set up several trays containing crystallization buffer at a pH of either 7.8 or 11.0 and with varied concentrations of sodium citrate from 1.6 – 2.0 M (Table 1). In addition, to determine if hCA II microcrystals could be formed via co-crystallization with an inhibitor (as the enzyme is a prominent drug target)21, we repeated these experiments with the addition of acetazolamide (AZM) at a final well concentration of 2 mM and 1% DMSO (Figure 1). Results of hCA II crystal screening are summarized in Table 1, where a “hit” is defined by visible production of crystals that are 10 µm. It should be noted that crystals with sizes > 0.2 µm were observed even after filtering, as determined by the apparent Bragg peaks in the diffraction image and measurements from TEM images (Figure 2 and 3). This is most likely due to the rectangular shape of observed crystalline samples allowing for passage through filter pores, or, due to the viscosity of crystal slurry solution, damage to the syringe filter allowing for larger crystals to pass through. Nonetheless, from negative stain EM images it is predicted that this method of filtering did not induce any damage to the crystals within slurry solutions (Figure 2E and F). Prior to collecting a diffraction image, microcrystal slurries were pelleted by centrifugation at 15,000 rpm for 30 mins, with minimal buffer left to ensure the crystals would not dry out while reducing potential for background scattering from excess solvent. The hCA II slurry was loaded into a quartz capillary and sealed with wax. Diffraction patterns of the hCA II crystal slurries were collected at the Cornell High Energy Synchrotron Source (CHESS) at beamline F1 using a wavelength of 0.98 Å (Figure 3). The images were collected at RT using a Dectris Pilatus 6M detector at a crystal-todetector distance of 500 mm, where (based on the detector radius) the theoretical maximum resolution is determined to be ~3 Å, with exposure times of 0.5, 1, and 5 sec per image. To reduce noise from background scatter, several images (for each exposure time) were summed and



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 35

smoothed background subtracted. The resolution of diffraction was obtained by measuring the distance from beam center to outer most visible concentric ring of diffraction scattering.

Results and Discussion Utilizing MBM, crystals from all samples were successfully grown. For HEWL and hCA II, the microcrystal slurries formed overnight and, using negative stain EM, crystals within these slurries were of sub-micron size (Figure 2E and F). Based on previous studies, overnight (~24 hour) shaking was chosen as the optimum time to induce secondary nucleation.20 Further, it was shown that ~24 hour shaking followed by “static” incubation of the crystallization tray, still resulted in a high level of secondary nucleation and formation of microcrystalline products.20 This is similar to what we have observed in AAV8, whereby 24 hour shaking was utilized and crystals did not form until 5 days after. Compared to a standard batch crystallization method, the crystal size and growth rate were greatly decreased using MBM (Figure 2A-D). This is directly observed by comparing crystals of hCA II and HEWL that were grown using both methods. More specifically, the average crystal size of hCA II and HEWL grown using MBM were estimated (using negative stain EM images) at 1.5 µm3 and 0.008 µm3, respectively (Figure 2E and F). This is compared to hCA II and HEWL grown in a standard batch crystallization setup which yielded crystals with average sizes estimated at 0.008 mm3 and 0.064 mm3, respectively (Figure 2A and B). In addition, MBM resulted in overnight crystal formation in hCA II and HEWL, where as a standard batch method saw crystals in 3 – 5 days. With the usage of dynamic light scattering (DLS), we attempted to characterize the size range of the hCA II microcrystals generated via MBM. The scattering from filtered hCA II


Page 17 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


crystals (Supplemental Figure 1) correlated to a hydrodynamic radius (Rh) of 4 nm, which most closely approximates the size of hCA II monomers/dimers in solution, rather than micron or submicron sized crystals within the sample. It is possible that the hCA II crystals were too large to pass through the filter despite the necessity of this step for DLS experiments to reduce nonspecific scattering from dust and debris.32 As such we were unable to record an accurate measurement of crystal size from DLS and therefore have relied on images presented by negative stain EM. Salt and sample concentration appeared to have little effect on HEWL crystal size using MBM (Figure 2F). Instead, sample concentration (i.e. increasing protein:precipitant ratio) had a more prevalent influence on crystal density after MBM. This is similar to that observed in hCA II where salt and protein concentration had little effect on crystal size during MBM and only contributed to crystal density (Table 1). Interestingly, both of these parameters had a more significant effect on crystal size when crystals of both hCA II and HEWL were grown under standard batch methods. This may suggest that successful crystallization conditions determined using a standard batch method may be transitioned with limited manipulation to produce microcrystals using the MBM method. The pH of the crystallization conditions appeared to have limited effect on secondary nucleation as microcrystals were grown in both pH 7.8 and 11 without significant difference in crystal size or quantity (Table 1). It was observed that in sodium citrate concentrations of ≥1.8 M, salt crystals readily form when the sample is exposed to air. In fact, hCA II crystals did not form easily in sodium citrate of 2.0 M (in both a standard batch method and MBM), most likely due to the over saturation of the well with salt, inhibiting hCA II crystal growth (Table 1). As previously described, increasing salt concentrations is a useful method to increase microcrystal growth.13,23 However, in SFX, this has the potential to be



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 35

problematic during sample delivery as salt crystals may form prior to data collection and could clog or damage liquid injectors.5,6 In this study, we have shown that even at lower salt concentrations, applying MBM can produce usable microcrystals for SFX and reduce the chances of forming salt crystals during sample handling and delivery (Table 1). We further applied this technique to a PEG 4000 containing crystallization condition that, in the past, has shown to yield crystals of hCA II.31 In conventional hanging-drop vapor diffusion crystallization setups, using this condition (see Experimental), large diffracting crystals of hCA II often formed after ~3 weeks of incubation at room temperature.31 Applying MBM under the same conditions however, exhibited microcrystal formation overnight. This result suggests a possibility of translating protein crystallization conditions determined by hanging drop vapor diffusion, directly to MBM for microcrystal production. As this is only one example, the extent of manipulating a “traditionally” determined crystallization condition to be used in MBM for microcrystal production is currently unknown and will require application to several proteinaceous targets. In the case of hCA II, it appeared that mixing speed had a significant influence on microcrystal formation using MBM (Table 1). Specifically, it was shown that fast mixing speeds often resulted in a reduction in crystallization “hits”. The reason for this is unclear especially since it has previously been shown that increasing the mixing speed is directly proportional to secondary nucleation rates (Eq.1) and thus results in an increase in microcrystal formation.20 Most likely, the reason for this is related to protein stability, where at 200 rpm there is a significant amount of shear force that potentially causes hCA II to lose conformational stability and thus reduces the propensity to crystallize. This trend was slightly different in the case of HEWL, where we observed room-temperature crystal formation when applying shaking speeds of either 200 or 400


Page 19 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


rpm (Figure 4). As seen before, lack of shaking results in formation of large, well-formed crystals (Figure 4A). Shaking at 200 and 400 rpm however, induced formation of numerous microcrystals of similar morphology (Figure 4B and C). There appeared to be no significant difference between crystals formed at either 200 or 400 rpm shake speeds, which is a similar result to that previously observed.20 Quantification of HEWL crystals formed as a result of shaking was estimated by approximating the total number of crystals in each condition. In control wells, 3 large crystals were observed while over 200,000 crystals were estimated in the wells shaken at both 200 and 400 rpm (approximated from counting crystals in a 0.5 µL volume). It should be noted that due to the difficulty in accurately counting crystals that are too small to be resolved by light microscopy, the given estimate is rather conservative, and the actual number of crystals may be two to three times higher than reported. Microcrystals of hCA II were also shown to be grown in the presence of a ligand, which in this case was a clinically used inhibitor, AZM (Figure 1, Step 2). This implies that co-crystallization protocols can be incorporated with MBM. Interestingly, the presence of AZM in crystallization wells resulted in microcrystal growth at high shaking speeds (200 rpm) versus those without (Table 1). Most likely, this is due to a stabilizing effect of AZM binding to hCA II, similar to that observed in other protein:ligand interactions.33,34 This coincides with our prediction that higher mixing speeds exhibit a destabilizing force on hCA II causing inhibition of crystal growth. To determine if hCA II microcrystal slurries were of proteinaceous content and capable of diffracting to high resolution we collected powder diffraction images on hCA II microcrystal slurries (Figure 3). Diffraction of hCA II microcrystal slurries indicate a pattern consistent with those previously observed for protein samples.35 In addition, samples diffracted to a high resolution of ~3 Å,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 35

which presumably, utilizing a more brilliant source or altering the crystal-to-detector distance, would be much improved and usable for high-resolution SFX (Figure 3). After application of MBM, AAV8 crystals began to appear within 5 days, and were fully grown by ~3 weeks (Figure 5). Unlike HEWL and hCA II, AAV8 crystal size and density appeared to be more sensitive to changes in protein:precipiant ratio, with a lower virus content resulting in fewer, yet larger crystals not suitable for SFX (Figure 5). Although the reason for this is unclear, it should be noted that due to sample quantity, only one mixing speed (72 rpm) was utilized. If the mixing speed was increased however, and due to the intrinsic stability of the viral capsid, it is predicted that this would create an increase in secondary nucleation and we would observe a larger quantity of smaller crystals.36 Interestingly, crystals of AAV8 were only observed in trays where MBM was applied. After ~5 weeks, standard batch crystallization trays containing AAV8 yielded no crystals despite keeping both the crystallization condition and protein:precipitant ratios the same as AAV8 crystals grown using MBM. Despite these observations, solutions from both MBM applied and standard batch crystallization of AAV8 samples were observed using negative stain EM to determine if nano-sized crystals were present that were not visible under the light microscope. Initial analysis with negative stain EM revealed that an intact crystal approximately 200 nm in diameter was present in a sample prepared using MBM (Supplemental Figure S2A). Observing the low resolution fast Fourier transform (FFT) revealed distinct Bragg peaks and thus confirmed that these were AAV8 crystals rather than artifacts from salt or stain (Supplemental Figure S2B). This further suggests the potential for MBM to not only produce nano- to microsized crystals of viruses, but also to expedite their crystallization. Conclusions


Page 21 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In this study we have shown that inducing secondary nucleation, using MBM, during macromolecular crystallization by applying agitation via different mixing speeds to standard batch crystallization methods, caused the formation of microcrystals suitable for SFX. Furthermore, we have shown that after application of MBM, salt and protein concentration, along with pH, have limited effect on crystal size, with only protein concentration having an observable effect on crystal density. Instead, mixing speed had the greatest influence on microcrystal formation, whereby an increase in speed resulted in a reduction in hCA II crystallization. This however, could be overcome through stabilization with a ligand. In summary, MBM presents a potentially useful method for microcrystal growth of a number of macromolecular targets that can be applied to a range of SFX experiments.

Supporting Information. Includes DLS data and negative stain EM and FFT of AAV8 MBM prepared samples.

Acknowledgements We would like to acknowledge the staff at the Cornell High Energy Synchrotron Source (CHESS) and specifically Drs. Sol M. Gruner and Marian Szebenyi (Cornell University) for synchrotron data collection on hCA II microcrystal samples.

Funding Sources

I.R.S. and D.A.S. were support from NSF CHE 1539347; J. J. K., A. B., M. A.-M and R. M. were support from NIH/NIGMS R01 GM109524 and C. L. L. was supported by National Center for Advancing Translational Sciences of NIH under University of Florida Clinical and Translational Science Awards TL1TR001428 and UL1TR001427. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 35

Footnotes The authors declare that they have no conflicts of interest with the contents of this article.

Nonstandard abbreviations. hCA II, human carbonic anhydrases isoform II; HEWL, hen eggwhite lysozyme; AAV8, adeno-associated virus serotype 8; MBM, Microbatch Mixing; SFX, serial-femtosecond crystallography; XFEL, X-ray free electron laser.


Page 23 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


References.

(1)

Nature 2014, 505 (7485), 586–586.

(2)

McPherson, A. Methods 2004, 34 (3), 254–265.

(3)

Durbin, S. D.; Feher, G. Annu Rev Phys Chem 1996, 47, 171–204.

(4)

Holton, J. M.; Frankel, K. A. Acta Crystallographica Section D Biological Crystallography 2010, 66 (4), 393–408.

(5)

Boutet, S.; Lomb, L.; Williams, G. J.; Barends, T. R. M.; Aquila, A.; Doak, R. B.; Weierstall, U.; DePonte, D. P.; Steinbrener, J.; Shoeman, R. L.; Messerschmidt, M.; Barty, A.; White, T. A.; Kassemeyer, S.; Kirian, R. A.; Seibert, M. M.; Montanez, P. A.; Kenney, C.; Herbst, R.; Hart, P.; Pines, J.; Haller, G.; Gruner, S. M.; Philipp, H. T.; Tate, M. W.; Hromalik, M.; Koerner, L. J.; van Bakel, N.; Morse, J.; Ghonsalves, W.; Arnlund, D.; Bogan, M. J.; Caleman, C.; Fromme, R.; Hampton, C. Y.; Hunter, M. S.; Johansson, L. C.; Katona, G.; Kupitz, C.; Liang, M.; Martin, A. V.; Nass, K.; Redecke, L.; Stellato, F.; Timneanu, N.; Wang, D.; Zatsepin, N. A.; Schafer, D.; Defever, J.; Neutze, R.; Fromme, P.; Spence, J. C. H.; Chapman, H. N.; Schlichting, I. Science 2012, 337 (6092), 362–364.

(6)

Chapman, H. N.; Fromme, P.; Barty, A.; White, T. A.; Kirian, R. A.; Aquila, A.; Hunter, M. S.; Schulz, J.; DePonte, D. P.; Weierstall, U.; Doak, R. B.; Maia, F. R. N. C.; Martin, A. V.; Schlichting, I.; Lomb, L.; Coppola, N.; Shoeman, R. L.; Epp, S. W.; Hartmann, R.; Rolles, D.; Rudenko, A.; Foucar, L.; Kimmel, N.; Weidenspointner, G.; Holl, P.; Liang, M.; Barthelmess, M.; Caleman, C.; Boutet, S.; Bogan, M. J.; Krzywinski, J.; Bostedt, C.; Bajt, S.; Gumprecht, L.; Rudek, B.; Erk, B.; Schmidt, C.; Hömke, A.; Reich, C.; Pietschner, D.; Strüder, L.; Hauser, G.; Gorke, H.; Ullrich, J.; Herrmann, S.; Schaller, G.; Schopper, F.; Soltau, H.; Kühnel, K.-U.; Messerschmidt, M.; Bozek, J. D.; Hau-Riege, S. P.; Frank, M.; Hampton, C. Y.; Sierra, R. G.; Starodub, D.; Williams, G. J.; Hajdu, J.; Timneanu, N.; Seibert, M. M.; Andreasson, J.; Rocker, A.; Jönsson, O.; Svenda, M.; Stern, S.; Nass, K.; Andritschke, R.; Schröter, C.-D.; Krasniqi, F.; Bott, M.; Schmidt, K. E.; Wang, X.; Grotjohann, I.; Holton, J. M.; Barends, T. R. M.; Neutze, R.; Marchesini, S.; Fromme, R.; Schorb, S.; Rupp, D.; Adolph, M.; Gorkhover, T.; Andersson, I.; Hirsemann, H.; Potdevin, G.; Graafsma, H.; Nilsson, B.; Spence, J. C. H. Nature 2011, 470 (7332), 73– 77.

(7)

Stellato, F.; Oberthür, D.; Liang, M.; Bean, R.; Gati, C.; Yefanov, O.; Barty, A.; Burkhardt, A.; Fischer, P.; Galli, L.; Kirian, R. A.; Meyer, J.; Panneerselvam, S.; Yoon, C. H.; Chervinskii, F.; Speller, E.; White, T. A.; Betzel, C.; Meents, A.; Chapman, H. N. IUCrJ 2014, 1 (4), 204–212. 17 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(8)

Levantino, M.; Yorke, B. A.; Monteiro, D. C.; Cammarata, M.; Pearson, A. R. Curr. Opin. Struct. Biol. 2015, 35, 41–48.

(9)

Spence, J. C. H. Faraday Discuss. 2014, 171, 429–438.

Page 24 of 35

(10) Coppens, P.; Fournier, B. Journal of Synchrotron Radiation 2015, 22 (2), 280–287. (11) Barends, T. R. M.; Foucar, L.; Ardevol, A.; Nass, K.; Aquila, A.; Botha, S.; Doak, R. B.; Falahati, K.; Hartmann, E.; Hilpert, M.; Heinz, M.; Hoffmann, M. C.; Köfinger, J.; Koglin, J. E.; Kovacsova, G.; Liang, M.; Milathianaki, D.; Lemke, H. T.; Reinstein, J.; Roome, C. M.; Shoeman, R. L.; Williams, G. J.; Burghardt, I.; Hummer, G.; Boutet, S.; Schlichting, I. Science 2015, 350 (6259), 445–450. (12) Pande, K.; Hutchison, C. D. M.; Groenhof, G.; Aquila, A.; Robinson, J. S.; Tenboer, J.; Basu, S.; Boutet, S.; DePonte, D. P.; Liang, M.; White, T. A.; Zatsepin, N. A.; Yefanov, O.; Morozov, D.; Oberthuer, D.; Gati, C.; Subramanian, G.; James, D.; Zhao, Y.; Koralek, J.; Brayshaw, J.; Kupitz, C.; Conrad, C.; Roy-Chowdhury, S.; Coe, J. D.; Metz, M.; Xavier, P. L.; Grant, T. D.; Koglin, J. E.; Ketawala, G.; Fromme, R.; Šrajer, V.; Henning, R.; Spence, J. C. H.; Ourmazd, A.; Schwander, P.; Weierstall, U.; Frank, M.; Fromme, P.; Barty, A.; Chapman, H. N.; Moffat, K.; van Thor, J. J.; Schmidt, M. Science 2016, 352 (6286), 725–729. (13) Kupitz, C.; Grotjohann, I.; Conrad, C. E.; Roy-Chowdhury, S.; Fromme, R.; Fromme, P. Philosophical Transactions of the Royal Society B: Biological Sciences 2014, 369 (1647), 20130316–20130316. (14) Liu, W.; Ishchenko, A.; Cherezov, V. Nature Protocols 2014, 9 (9), 2123–2134. (15) Neutze, R. Philosophical Transactions of the Royal Society B: Biological Sciences 2014, 369 (1647), 20130318–20130318. (16) Schlichting, I. IUCrJ 2015, 2 (2), 246–255. (17) Tenboer, J.; Basu, S.; Zatsepin, N.; Pande, K.; Milathianaki, D.; Frank, M.; Hunter, M.; Boutet, S.; Williams, G. J.; Koglin, J. E.; Oberthuer, D.; Heymann, M.; Kupitz, C.; Conrad, C.; Coe, J.; Roy-Chowdhury, S.; Weierstall, U.; James, D.; Wang, D.; Grant, T.; Barty, A.; Yefanov, O.; Scales, J.; Gati, C.; Seuring, C.; Srajer, V.; Henning, R.; Schwander, P.; Fromme, R.; Ourmazd, A.; Moffat, K.; Van Thor, J. J.; Spence, J. C. H.; Fromme, P.; Chapman, H. N.; Schmidt, M. Science 2014, 346 (6214), 1242–1246. 18 ACS Paragon Plus Environment

Page 25 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(18) Gernert, K. M.; Smith, R.; Carter, D. C. Analytical Biochemistry 1988, 168 (1), 141–147. (19) Garside, J. Chemical Engineering Science 1985, 40 (1), 3–26. (20) Tait, S.; White, E. T.; Litster, J. D. Crystal Growth & Design 2009, 9 (5), 2198–2206. (21) Frost, S. C.; McKenna, R. Carbonic anhydrase mechanism, regulation, links to disease, and industrial applications; Springer: Dordrecht, 2014. (22) Jude Samulski; Nicholas Muzyczka. Annual Review of Virology 2014, 1, 427–451. (23) Falkner, J. C.; Al-Somali, A. M.; Jamison, J. A.; Zhang, J.; Adrianse, S. L.; Simpson, R. L.; Calabretta, M. K.; Radding, W.; Phillips, G. N.; Colvin, V. L. Chemistry of Materials 2005, 17 (10), 2679–2686. (24) Mahon, B. P.; Hendon, A. M.; Driscoll, J. M.; Rankin, G. M.; Poulsen, S.-A.; Supuran, C. T.; McKenna, R. Bioorg. Med. Chem. 2014. (25) Lane, M. D.; Nam, H.-J.; Padron, E.; Gurda-Whitaker, B.; Kohlbrenner, E.; Aslanidi, G.; Byrne, B.; McKenna, R.; Muzyczka, N.; Zolotukhin, S.; Agbandje-McKenna, M. Acta Crystallographica Section F Structural Biology and Crystallization Communications 2005, 61 (6), 558–561. (26) Rayment, I. Structure 2002, 10 (2), 147–151. (27) Judge, R. A.; Jacobs, R. S.; Frazier, T.; Snell, E. H.; Pusey, M. L. Biophysical Journal 1999, 77 (3), 1585–1593. (28) Fisher, Z.; Kovalevsky, A. Y.; Mustyakimov, M.; Silverman, D. N.; McKenna, R.; Langan, P. Biochemistry 2011, 50 (44), 9421–9423. (29) Fisher, S. Z.; Tu, C.; Bhatt, D.; Govindasamy, L.; Agbandje-McKenna, M.; McKenna, R.; Silverman, D. N. Biochemistry 2007, 46 (12), 3803–3813. (30) Domsic, J. F.; Williams, W.; Fisher, S. Z.; Tu, C.; Agbandje-McKenna, M.; Silverman, D. N.; McKenna, R. Biochemistry 2010, 49 (30), 6394–6399.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(31) Fisher, Z.; Boone, C. D.; Biswas, S. M.; Venkatakrishnan, B.; Aggarwal, M.; Tu, C.; Agbandje-McKenna, M.; Silverman, D.; McKenna, R. Protein Engineering Design and Selection 2012, 25 (7), 347–355. (32) Kim, J.; Ahn, S.; Lee, H.; Lee, M. Opt Lett 2013, 38 (11), 1757–1759. (33) Bruylants, G.; Wouters, J.; Michaux, C. Curr. Med. Chem. 2005, 12 (17), 2011–2020. (34) Pinard, M. A.; Aggarwal, M.; Mahon, B. P.; Tu, C.; McKenna, R. Acta Crystallogr F Struct Biol Commun 2015, 71 (Pt 10), 1352–1358. (35) Von Dreele, R. B. Meth. Enzymol. 2003, 368, 254–267. (36) Rayaprolu, V.; Kruse, S.; Kant, R.; Venkatakrishnan, B.; Movahed, N.; Brooke, D.; Lins, B.; Bennett, A.; Potter, T.; McKenna, R.; Agbandje-McKenna, M.; Bothner, B. Journal of Virology 2013, 87 (24), 13150–13160.


Page 26 of 35

Page 27 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Microcrystal Na-citrate condition screening results for hCA II

Na-citrate (M) 1.6 1.6 1.6 1.6 1.6 1.6 1.8 1.8 1.8 1.8 1.8 1.8 2.0 2.0 2.0 2.0 2.0 2.0 a b

pH 7.8 7.8 7.8 7.8 11.0 11.0 7.8 7.8 7.8 7.8 11.0 11.0 7.8 7.8 7.8 7.8 11.0 11.0

RPM 200 72 200 72 200 72 200 72 200 72 200 72 200 72 200 72 200 72

AZMa N N Y Y N N N N Y Y N N N N Y Y N N

Hitb N Y Y Y N Y N Y Y Y N Y N N Y Y N N

Wells contained 2 mM AZM and 1 % DMSO Hit is defined as the appearance of a microcrystalline with crystals of

Microbatch Mixing: âShaken not Stirredâ, a Method ... - ACS Publications

Recommend Documents

Microbatch Mixing: âShaken not Stirredâ, a Method ... - ACS Publications