COMPUTER PROGRAMS

J. Appl. Cryst. (1999). 32, 1012±1016

XAct: a program for construction, automated setup and bookkeeping of crystallization experiments

D. E. Brodersen, L. B. Jenner, G. R. Andersen and J. Nyborg* at IMSB, Arhus University, Gustav Wieds Vej 10c, DK-8000Aarhus C, Denmark. E-mail: jnb@imsb.au.dk

(Received 22 April 1999; accepted 28 April 1999 )

Abstract

XAct is a stand-alone application for Microsoft Windows2 95/98 that, through a spreadsheet-like interface, allows the user toconstruct crystallization experiments in a highly ¯exible way.The object-oriented data structure helps to maintain anoverview of many experiments and provides reusable stockand tray objects. The program features a hardware interfacethat enables automatic setup of experiments by use of a Gilson222 autosampler (Gilson Inc., Wisconsin, USA). Additionally,experimental information can be imported from externalspreadsheets and World Wide Web applications.

1. Introduction

In recent years, determination of macromolecular crystalstructures has been greatly facilitated by the development ofimproved methods for data collection and computation.Crystallization of macromolecules, however, is still a bottle-neck and the process of crystallization is not well understoodtheoretically. In order to explore the crystallization space in anef®cient manner, sparse-matrix methods have been developed(Carter & Carter, 1979; Jancarik & Kim, 1991; Segelke &Rupp, 1998).

The characteristic feature of these methods is the need toproduce a large number of precipitant solutions of widelyvarying composition and concentration. Mixing these solutionsby hand is a tedious, time-consuming and repetitive task, whichis also prone to errors. Mainly for this reason, the manuallabour involved has frequently been eschewed by constructionof automated crystallization apparatus. Automated crystal-lization equipment (`crystallization robots') has been designedto perform the time-consuming work involved in the repeti-tious mixing of precipitating solutions as well as the setup ofprotein droplets. Some approaches use commercially availablerobotics equipment, customized via its setup software andlocally developed software (Ward et al., 1988; Rubin et al.,1991; Cox & Weber, 1987), while other systems started asexperimental prototypes and later developed into commercialproducts [for example, that of Chayen et al. (1990, 1994), nowavailable through Douglas Instruments, Berkshire, UK (http://www.douglas.co.uk)]. The employed instrumentation rangesfrom small motorized and computer-controlled pipettes(EiseleÂ, 1993) to large automated laboratories that take care oftasks as diverse as liquid handling, dispensing of proteinsolution onto cover slips, and application of vacuum grease toseal experiments (Soriano & Fontecilla-Camps, 1993; Sadaouiet al., 1994).

With an almost exponential increase in the number of newprotein crystal forms reported during the past three to fourdecades, several attempts have been made to aid the

researcher in recording and analysing the results of the vastamount of experiments that are being carried out in most X-ray crystallography laboratories. Hassell et al. (1994) used aworksheet-based system combined with an extensive databasefor recording the outcome of many crystallization trials basedon the quality indicator originally devised by Carter & Carter(1979). An even more advanced approach was taken by Wardet al. (1988), who used computer analysis of digital images toassess the quality of individual crystallization experiments.Furthermore, the use of arti®cial intelligence to extract rele-vant information from a large database containing informationon successful crystallization experiments has been attempted(Roussel et al., 1990).

Our laboratory has for a number of years used a low-costautomated crystallization system based on autosampler anddiluter hardware available from Gilson [Gilson Inc.,Wisconsin, USA (http://www.gilson.com)]. This solutionprovides an ef®cient system that can easily be accommodatedin most academic laboratories, both spatially as well as ®nan-cially. The use of Gilson autosampler hardware in the contextof protein crystallization was ®rst described by Old®eld et al.(1991) and later improved by Zeelen and co-workers (Zeelenet al., 1994). The implementation of the Gilson autosampler asa crystallization robot in our laboratory was originallyperformed via a spreadsheet approach that used MicrosoftExcel2² and a set of prede®ned macros to control the hard-ware (Andersen & Nyborg, 1996). This setup allowed not onlyprecise dispensing of precipitant solutions, but also quick andreproductive mixing of protein droplets. The crystallizationrobot is now being used on a daily basis. In a continued effortto improve its usefulness, we have developed a new program,called XAct, for setting up experiments, controlling the hard-ware and recording the results of the experiments.

2. Program features

2.1. Overview

XAct is a stand-alone application running under theMicrosoft Windows2 95/98 operating system and has beenwritten using the Delphi2 3.0³ implementation of object-oriented Pascal. The program is compiled as 100% native 32-bit code and requires no separate run-time libraries. The mainapplication (Xactpro.exe) is based on a semi-hardware-independent architecture, so that all direct communicationwith the Gilson hardware is performed through a separatedynamic link library (DLL), the gilson222.dll. This

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# 1999 International Union of Crystallography Journal of Applied CrystallographyPrinted in Great Britain ± all rights reserved ISSN 0021-8898 # 1999

² Microsoft Excel and Microsoft Windows 95/98 are trademarks of theMicrosoft Corp., Redmont, WA, USA.³ Delphi is a trademark of Inprise Corp., Scotts Valley, CA, USA.

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approach makes future support for other types of hardwarepossible. The program is designed to accommodate multipleusers, one of which is the system administrator (or siteadministrator) responsible for user control and maintenanceof tray and stock templates (see below).

2.2. Hierarchical and object-oriented design

In addition to accommodating many users, XAct features anextensive object-oriented data structure. Each user is equippedwith a personal login for which data and overall settings arepreserved between work sessions. Since a single user mightundertake several hundred experiments, these are divided intoa number of subcategories based on project, type of proteinand protein batch (see Fig. 1). Thus, a single user can work onmultiple projects, each comprising several different proteinsunder various conditions (batches). The hierarchical structureis re¯ected in a so-called `treeview' component (the Overview)that can be used to browse through previous experiments,create new experiments, control the attached hardware andrecord results (Fig. 2). New experiments (crystallization trays)can be created from scratch by entering information such asthe type of experiment, temperature, number of individualwells, and other information speci®c to the setup. Alter-natively, a new tray can be based on a template that can be anykind of blank or even prede®ned experiment. Thus, commonlyused screens or sparse-matrix experiments, for example, can beadded to the list of available templates for easy access.

2.3. The Tray Editor

The central component of XAct is the Tray Editor, which isused to design the crystallization experiments (Fig. 2). Theinformation contained within the Tray Editor describes howthe various crystallization solutions (well solutions) in theexperiment are mixed from the given stock solutions. Theeditor is similar to a spreadsheet ®le; it contains one column foreach stock solution and one row for each well solution to mix.The user can edit the contents of the tray by entering eithervolumes (in ml) or concentrations (in any unit that applies) in

the spreadsheet cells. The appearance of the tray can bechanged to display volumes, concentrations, or both at thesame time. A special column is dedicated to the water stocksolution and cannot be edited since the values are calculatedautomatically from the known total well volume. The right-most column shows the approximate pH of each well solutionprovided that a buffer with de®ned pH value is present amongthe stock solutions. New stock solutions can be added to ablank or existing tray by using the context-sensitive right-mouse button menu or by conveniently dragging prede®nedstock objects from the Stock Repository (see below).

2.4. The Stock Repository

Stock solutions are treated as separate entities in the object-oriented data model and can as such be transferred betweenexperiments. The Stock Repository provides a means forreusing previously de®ned stock objects. The repository isdivided into two parts: the public and the personal repository.Whereas only the site administrator is able to modify thecontents of the public repository (which all users can access ona read-only basis), each user is allowed to maintain his owncollection of stock objects (in the personal repository) that canbe modi®ed and reused in later sessions. Stock objects can beadded to the personal repository from any previous experi-ment or copied from the public repository. Any stock objectcan be used simply by dragging it onto the Tray Editorcontaining the experiment in question.

2.5. Automatic dispensing of solutions

If the Gilson 222 autosampler is connected to the PC via aserial port and the GSIOC device driver (supplied by Gilson)is installed, XAct is capable of controlling the autosampler tomix solutions for a given experiment as speci®ed in the TrayEditor. Through the setup dialog (see x2.6 below) the userinforms the program about what types of racks are placed inthe ®ve available slots on the tray of the Gilson 222. The threeleft-most slots are used for the mixed well solutions, whereasthe two right-most slots are reserved for the stock solutions(see Fig. 3). Depending on the types of racks used and thevolumes that should be mixed, this extremely ¯exible setup canaccommodate several hundred solutions in a single run. Thehardware driver controlling the Gilson 222 is invoked from thecontext-sensitive menu attached to each tray in the Overviewwindow. Upon invocation, the user is prompted to identifywhich of the well solutions in the tray should be mapped to theavailable tube positions in the mounted racks. Next, XActcalculates the amount used of each stock solution and tells theuser into which slots and positions they should be placed. Afterstarting the actual dispensing of the solutions, the overallprogress can be monitored continuously using the Job Statusdialog, which also shows which stock solution is currently beingprocessed. The job can be interrupted at any time, either byletting the autosampler ®nish the current solution or byabruptly resetting the hardware in case of unexpected beha-viour.

2.6. Gilson 222 setup

As mentioned above, XAct gives the user full control of thesetup of the Gilson 222 autosampler. The autosamplerprovides space for ®ve different racks, each of which cancontain as many as 108 individual tubes (using the Gilsonstandard rack codes 20 or 28). By a simple drag-and-drop

Fig. 1. The XAct object hierarchy. The extensive hierarchy of dataallows many users to maintain hundreds of experiments withoutlosing overview. The object-oriented model shown here is not onlyan imaginary idea but is re¯ected in the directory structure on thedisk and in the data structure within the program.

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functionality, XAct allows the user to indicate which rack typesare to be placed in the ®ve available slots (Fig. 3). A number ofthe more useful standard racks available from Gilson (rackcodes 21 through 29) are prede®ned within the program, butthe possibility of creating custom rack types allows the user toaccommodate virtually any possible rack in the tray of theautosampler. Blank racks (rack code 99) are available fromGilson and can be modi®ed in the local workshop to suitalmost any need. Construction of custom racks requires theuser to enter values for the origin in (X, Y) (i.e. the centre ofthe ®rst tube in the rack) and values for the X and Y steps (i.e.the distance between consecutive tubes in the rack horizon-tally and vertically, respectively). To help the user in thissituation, a so-called wizard is provided (the New Rackwizard), which guides the user through the necessary steps inorder to create a custom rack. Also, this wizard offers real-timecontrol of the (X, Y, Z) arm of the autosampler (using a virtualremote control) allowing a precise measurement of the originand step coordinates for the rack.

2.7. Recording results

Apart from allowing precise mixing of the crystallizationsolutions, XAct also enables the user to record results of earlierexperiments. Once set up, the experimental informationremains within the program and can be accessed at any time.An unlimited number of individual result observations can beattached to each crystallization solution within a tray. For each

recorded result, XAct stores the date of observation, type ofobservation [using the ÿ5 to +7 Q-number descriptor intro-duced by Hassell et al. (1994)] in addition to any commentsthat the user wishes to include.

2.8. Specialized import ®lters

XAct includes advanced import ®lters that allow theextraction of experimental information from external sources.Of these, the Excel2 wizard enables import of MicrosoftExcel2 worksheets written in tab-separated text format. Thisprovides backward compatibility with the previous spread-sheet-based crystallization automation system used in ourlaboratory (Andersen & Nyborg, 1996). In addition, theCRYSTOOL wizard now provides support for importof sparse-matrix experiments constructed using theCRYSTOOL World Wide Web system available at http://www-structure.llnl.gov/crystool/crystool.htm (Segelke &Rupp, 1998). Since XAct signi®cantly expands the amount ofrecorded information for each experiment, these wizards guidethe user through the process of converting external informa-tion into an XAct tray. The manual provides detailed addi-tional information about the required format of these external®les.

Fig. 2. The main window of XAct. To the left, the Overview window that shows the experimental setup for the user `ding'. For the project`Psoriasis-related proteins', the user has created the protein `Psoriasin' with two different batches `HoPsB' and `CaPsA'. Each of these containsthree experiments (trays). On the right is shown an open Tray Editor displaying the tray `HoPsB07'.

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2.9. Printing

XAct implements a full-featured Windows2 printing systemthat allows the user to preview and print any experiment. Theprinted output contains a table showing the amounts (or ®nalconcentrations) of each stock solution in the various wellsolutions. The output can furthermore be customized toinclude additional information about the experiment, such asdate, user and project name.

2.10. Requirements and installation

XAct will run on any PC running Microsoft Windows2 95/98and requires no additional run-time libraries. A 9/25-pin RS-232 serial port and a GSIOC device driver (available fromGilson) are necessary for hardware control of the Gilson222 autosampler. The program automatically detects the driverand hardware and if either is not present, XAct will start in off-line mode. In off-line mode, experiments and stock objects canbe set up as usual, but all features involving hardware controlof the Gilson 222 are disabled.

The instrumentation needed for automatic mixing of solu-tions comprises the Gilson 222 autosampler, which isconnected to a Gilson 401 diluter equipped with a 5 ml syringethrough a manufacturer-supplied 9-pin serial cable. The Gilson

222 autosampler is connected to the PC through a GilsonM506B serial modem. Optionally, the supplied keypad can beattached to the autosampler to gain further control of itsbehaviour. The hardware setup is identical to that describedpreviously (Andersen & Nyborg, 1996).

Installation of the program is carried out by a standardWindows2 installation program (Setup.exe) which installsthe program to hard disk, places a link on the Windows2 StartMenu and registers the program in the list of installedprograms.

3. Distribution and documentation

XAct is distributed free of charge on an `as is' basis, whichmeans that a limited amount of support can be provided by theauthors. The complete program distribution, which can beobtained from http://zombie.imsb.au.dk/xact, contains theprogram installation ®les in addition to the manual. A numberof example ®les (an example user pro®le) have been included.Also, the distribution contains various template ®les for use inthe construction of new experiments and examples of customrack de®nitions. Source code is not included but is availableupon request by contacting the authors.

Fig. 3. Program customization. The upper-left window shows the Stock Repository with the available standard (public) stock objects. The lowerright window is the Gilson 222 setup dialog that allows the user to specify which racks are used in the autosampler. In this case, a standard rackcode 23 (with 44 mini-vials) is used for well solutions and two custom racks are used for a total of 20 large 50 ml stock tubes. The list on the leftshows the available standard and custom rack types.

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