Sitting drop protein crystallization is not used as commonly as the hanging drop

method for crystal optimization due to the limitations of commercially available sitting

drop bridges, particularly when they are used in conjunction with 24-well

crystallization plates. The commercially available sitting drop bridge, containing

space for only a single drop, restricts their wider use. Proteins that preferentially

crystallize under sitting-drop conditions therefore require more work, time and

resources for their optimization. As a result of these limitations and using 3D-printing,

we designed and developed a new sitting drop bridge where five crystallization drops

can be placed simultaneously in each well of a 24-well crystallization plate. This

significantly simplifies and increases the potential of sitting drops in crystal

optimization, reducing costs and hence overcomes existing limitations of current

approaches

Hilton, ST

Kozielski, F

Penny, M

Talapatra, S

UCL Discovery

electronic reprintISSN: 1600-5767journals.iucr.org/jDesign, 3D printing and validation of a novel low-costhigh-capacity sitting-drop bridge for protein crystallizationSandeep K. Talapatra, Matthew R. Penny, Stephen T. Hilton and FrankKozielskiJ. Appl. Cryst. (2019). 52, 171–174IUCr JournalsCRYSTALLOGRAPHY JOURNALS ONLINECopyright c© International Union of CrystallographyAuthor(s) of this paper may load this reprint on their own web site or institutional repository provided thatthis cover page is retained. Republication of this article or its storage in electronic databases other than asspecified above is not permitted without prior permission in writing from the IUCr.For further information see http://journals.iucr.org/services/authorrights.htmlJ. Appl. Cryst. (2019). 52, 171–174 Sandeep K. Talapatra et al. · A sitting-drop bridge for protein crystallizationshort communicationsJ. Appl. Cryst. (2019). 52, 171–174 https://doi.org/10.1107/S1600576718017545 171Received 30 August 2018Accepted 11 December 2018Edited by F. Meilleur, Oak Ridge NationalLaboratory, USA, and North Carolina StateUniversity, USA‡ Current address: Discovery Biology,Discovery Sciences, IMED Biotech Unit,AstraZeneca, Alderley Park, UK.Keywords: protein crystallization; sitting-dropcrystallization; hanging-drop crystallization;vapour-diffusion crystallization; microbridges;3D printing.Supporting information: this article hassupporting information at journals.iucr.org/jDesign, 3D printing and validation of a novel low-cost high-capacity sitting-drop bridge for proteincrystallizationSandeep K. Talapatra,*‡ Matthew R. Penny, Stephen T. Hilton and Frank Kozielski*Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University College London, 29–39Brunswick Square, London WC1N 1AX, UK. *Correspondence e-mail: s.talapatra@ucl.ac.uk, f.kozielski@ucl.ac.ukSitting-drop protein crystallization is not used as commonly as the hanging-dropmethod for crystal optimization owing to the limitations of commerciallyavailable sitting-drop bridges, particularly when they are used in conjunctionwith 24-well crystallization plates. The commercially available sitting-dropbridge, containing space for only a single drop, restricts their wider use. Proteinsthat preferentially crystallize under sitting-drop conditions therefore requiremore work, time and resources for their optimization. In response to theselimitations, and using 3D printing, a new sitting-drop bridge has been designedand developed, where ﬁve crystallization drops can be placed simultaneously ineach well of a 24-well crystallization plate. This signiﬁcantly simpliﬁes theprocess and increases the potential of sitting drops in crystal optimization,reducing costs and hence overcoming the limitations of current approaches.1. IntroductionProtein crystallization using the vapour-diffusion method isthe most commonly employed crystallization technique(McRee, 1993). Among several bottlenecks in protein crys-tallography, the optimization of protein crystals is one of thekey factors in obtaining high-resolution protein structures(McRee, 1993; McRee & David, 1999). The majority ofproteins crystallize under both sitting- and hanging-dropvapour-diffusion experiments (Dessau & Modis, 2011).However, a few proteins undergo nucleation and crystalformation preferentially or exclusively in sitting-drop condi-tions (Fresco et al., 1968; Rayment, 2002; Soundararajan et al.,2013; Kozielski et al., 1999). Common practice in these cases isthe use of well established and commercially available 24-wellcrystallization plates such as Linbro plates, in conjunction witha one-position sitting-drop bridge. Hanging-drop crystal-lization experiments are set up using a siliconized coverslip,allowing the simultaneous preparation of multiple crystal-lization drops on the same coverslip. Ideally, in this case, up toﬁve different drops, each having a ﬁnal volume of up to 3 mlunder a single crystallization condition (or per crystallizationwell), can readily be prepared. Crystallization drops composedof larger volumes reduce the overall number of drops whichcan be placed on the same coverslip. Initial optimizationtypically involves the variation of drop volumes, proteinconcentration, crystallization buffer, additives etc. Therefore,the need to place more than a single drop per well is highlydesired for quickly obtaining optimized high-quality and welldiffracting crystals. However, current commercially availablesitting-drop bridges based on the published design (Harlos,1992; Huba´lek et al., 2003) do not allow this ﬂexibility andISSN 1600-5767# 2019 International Union of Crystallographyelectronic reprintmultiple numbers of wells or plates have to be set up toachieve the same throughput as obtained with the hanging-drop method.In order to streamline the sitting-drop method and align itsefﬁciency with the hanging-drop method, and on the basis ofour experience in developing low-cost equipment using 3Dprinting (Tyson et al., 2015; Mohmmed et al., 2016), we havedesigned a ﬁve-position sitting-drop bridge to be used inconjunction with widely used and commercially available 24-well Linbro crystallization plates. In addition, we have testedour newly designed bridge with a well characterized protein todemonstrate the effectiveness of the new ﬁve-position bridge.2. Concept and designThe ﬁve-position sitting-drop bridge design we present herewas originally conceptualized keeping in mind the presentlyavailable sitting-drop methods used in 24-well plates forprotein crystallization and optimization. Our ﬁrst designworked on the basis of a four-position sitting-drop bridge witha compact ﬁt within the well [Fig. 1(a)]. Initially, to increasethe capacity of the sitting-drop bridge we looked at twopotential approaches. The ﬁrst focus was on the design of anew crystallization plate rather than using the established 24-well Linbro plate. The second approach was based on modi-fying the design of the sitting-drop bridge to have increasedcapacity, without undermining the subsequent processes toobtain high-quality crystals. Given the wide use of the 24-wellplate and the established design and setup, we decided topursue the second approach. Using 3D printing, we initiallyfabricated a four-position bridge, and we tested it with denguevirus RNA-dependent RNA polymerase serotype 3 (DENV3RdRp). This protein is well characterized to form crystalsirrespective of the type of method used and it consistentlydiffracts to a high resolution (Noble et al., 2016). Although weobtained crystals using the four-position bridge, there weretwo underlying concerns we wanted to address further. Firstly,we wished to optimize the maximum space available in a singlewell without compromising the stability of the bridge.Secondly, we wanted to generate a better shape andcontouring of the drops so that crystals were easier to observeand harvest under a normal light microscope similar to the oneused for crystals grown in hanging drops.Using 3D printing, we then designed a ﬁve-position sitting-drop bridge, which improved the ﬁt within the individual welland further increased the number of crystallization drops thatcan be set up simultaneously. In addition, the shape of eachposition was modiﬁed in such a fashion that the observation ofprotein crystals under an optical microscope was improvedand the process of ‘ﬁshing’ for crystals using loops wassigniﬁcantly simpliﬁed by allowing this to be done at a moreconvenient angle.3. 3D-printing detailsThe sitting-drop bridges were designed using either Tinkercador Autodesk 123D (Autodesk, San Rafael, California, USA)and exported as .STL ﬁles. 3D printing was carried out using aFormlabs (Somerville, Massachusetts, USA) Form 2 SLA 3Dprinter using Formlabs Clear Resin V4 with a layer height of0.1 mm. Care was taken to ensure that support attachmentpoints were not located underneath wells. After printing, thesitting-drop bridges were immersed in propan-2-ol for 15 min,dried and then cured using Formlabs Form Cure for 30 min at333 K.Although the sitting-drop bridges were printed using clearresin, the layers of the 3D print hampered visualization of thecrystals under the microscope owing to refraction of lightunderneath the well. To improve transparency, a thin layer ofClear Resin V4 was added to the inside surface of the wellsshort communications172 Sandeep K. Talapatra et al.  A sitting-drop bridge for protein crystallization J. Appl. Cryst. (2019). 52, 171–174Figure 1Conceptualization, design and structure statistics. (a) CAD image of (i) the initial four-position design and (ii) the optimized ﬁve-position design. (b)Photographs of (i) the initial four-position microbridge and (ii) the optimized ﬁve-position microbridge. (c) The dimensions of the optimized design. (d)Photographs showing the microbridge (left) before and (right) after treatment with additional resin to improve transparency.electronic reprintand the models were cured for a further 30 min at 333 K. Thisprocedure was also carried out for the surface directlyunderneath the wells (after detachment of support structures)and repeated twice until a satisfactory clarity was achieved.Although we adapted our new ﬁve-position sitting-dropbridge to the widely used 24-well Linbro crystallization plate,our design can easily be tailored to ﬁt other less frequentlyused crystallization plates. The cost of materials for fabricationis just USD 0.32 per microbridge, which includes the FormlabsClear Resin and propan-2-ol. We are able to print up to 25microbridges per printer at one time, taking a total of 5 h(including printing time, washing and ﬁnishing), which equatesto less than 15 min per microbridge. Manual procedures forthe whole process take less than 2 min per microbridge. The3D printing ﬁle for our microbridge design is provided withthis article.4. Application and future use4.1. Set up of crystallization trials and verification of volumeand ratio of protein versus reservoir solutionTo verify and validate the new ﬁve-position sitting-dropbridge that we designed, we carried out crystallizationexperiments in 24-well Linbro plates using both hanging-dropand sitting-drop experiments with the conventional one-position bridge and our new ﬁve-position bridge. Crystal-lization trials were set up using ratios of 1:1, 1:2, 2:1, 2:2 and3:1 ml of DENV3 RdRp and the reservoir solution (crystal-lization condition), respectively. For the one-position sitting-drop bridges, the standard 1:1 ml condition was used. DENV3RdRp crystals were obtained under all conditions, with sizesvarying between 10 and 40 mm [Fig. 2(a)].4.2. Comparison of crystal quality from hanging drops andfrom one-position and five-position sitting-drop bridgesTo compare the quality of the crystals obtained in the ﬁve-position sitting-drop bridge with the hanging drop or one-position sitting-drop bridge, ten to 15 crystals from eachexperiment with sizes ranging between 10 and 40 mm wereharvested for diffraction measurements. The crystals wereselected by simple visual examination under a light micro-scope.We collected data sets and processed them with either XDS(Kabsch, 2010) or iMOSFLM (Battye et al., 2011). Usingstringent criteria for data-collecting statistics, we were able tocompare the three different approaches to obtaining crystals.We initially selected data sets with Rmerge values lower than0.12. Next, we selected data sets which had an Imean/Imean ofat least 2.0 in the outer shell and an overall completeness of atleast 99%. The average resolution of these data sets was 2.0 A˚.short communicationsJ. Appl. Cryst. (2019). 52, 171–174 Sandeep K. Talapatra et al.  A sitting-drop bridge for protein crystallization 173Figure 2Crystallization experiments using the new ﬁve-position sitting-drop bridge. (a) RdRp crystals obtained in each individual position of the new ﬁve-position sitting-drop bridge. Red arrows point to DENV3 RdRp crystals used for diffraction measurements. (b) Crystals from individual drops wereselected for diffraction measurements and crystal quality assessments. Shown here are comparisons of various collection statistics [panel (i) overallRmerge, (ii) resolution, (iii) Imean/Imean of the outer shell and (iv) completeness] of ten different crystals of DENV3 RdRp obtained using the hanging-drop method, a one-position sitting-drop bridge and our new ﬁve-position sitting-drop bridge. There is no signiﬁcant difference in crystal quality.electronic reprintWe were able to obtain at least ten good data sets for all threesetups for comparison, as shown in Fig. 2(b), panels (i)–(iv).Therefore, we conclude that our novel ﬁve-position sitting-drop bridges do not affect the overall quality of the crystals orsubsequent data-collection statistics.AcknowledgementsWe would like to thank Dr Michella Mazzon (UCL MRCLaboratory for Molecular Cell Biology) for providing theexpression plasmid for DENV3 RdRp. We also thankDiamond Light Source for access to beamline I24 (MX17201)that contributed to the results presented here.ReferencesBattye, T. G. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie,A. G. W. (2011). Acta Cryst. D67, 271–281.Dessau, M. A. & Modis, Y. (2011). J. Vis. Exp. 16, 47.Fresco, J. R., Blake, R. D. & Langridge, R. (1968). Nature, 220, 1285–1287.Harlos, K. (1992). J. Appl. Cryst. 25, 536–538.Huba´lek, F., Binda, C., Li, M., Mattevi, A. & Edmondson, D. E.(2003). Acta Cryst. D59, 1874–1876.Kabsch, W. (2010). Acta Cryst. D66, 125–132.Kozielski, F., De Bonis, S., Burmeister, W. P., Cohen-Addad, C. &Wade, R. H. (1999). Structure, 7, 1407–1416.McRee, D. E. (1993). Practical Protein Crystallography. San Diego:Academic Press.McRee, D. E. & David, P. R. (1999). Practical Protein Crystal-lography, 2nd ed. San Diego: Academic Press.Mohmmed, S. A., Vianna, M. E., Penny, M. R., Hilton, S. T., Mordan,N. & Knowles, J. C. (2016). Dent. Mater. 32, 1289–1300.Noble, C. G., Lim, S. P., Arora, R., Yokokawa, F., Nilar, S., Seh, C. C.,Wright, S. K., Benson, T. E., Smith, P. W. & Shi, P. Y. (2016). J. Biol.Chem. 291, 8541–8548.Rayment, I. (2002). Structure, 10, 147–151.Soundararajan, M., Roos, A. K., Savitsky, P., Filippakopoulos, P.,Kettenbach, A. N., Olsen, J. V., Gerber, S. A., Eswaran, J., Knapp, S.& Elkins, J. M. (2013). Structure, 21, 986–996.Tyson, A. L., Hilton, S. T. & Andreae, L. C. (2015). Int. J. Pharm. 494,651–656.short communications174 Sandeep K. Talapatra et al.  A sitting-drop bridge for protein crystallization J. Appl. Cryst. (2019). 52, 171–174electronic reprint

Design, 3D printing and validation of a novel low-cost high-capacity sitting-drop bridge for protein crystallization

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Design, 3D printing and validation of a novel low-cost high-capacity sitting-drop bridge for protein crystallization

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