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SLS Crystallization Platform at Beamline X06DA—A Fully Automated Pipeline Enabling in Situ X-ray Diffraction Screening Published as part of the Crystal Growth & Design virtual special issue on the 13th International Conference on the Crystallization of Biological Macromolecules (ICCBM13). R. Bingel-Erlenmeyer,†,§ V. Olieric,*,† J. P. A. Grimshaw,‡ J. Gabadinho,† X. Wang,† S. G. Ebner,† A. Isenegger,† R. Schneider,† J. Schneider,† W. Glettig,† C. Pradervand,† E. H. Panepucci,† T. Tomizaki,† M. Wang,† and C. Schulze-Briese†,|| †

Swiss Light Source, Paul Scherrer Institut, CH-5232 Villigen Switzerland ESBATech, an Alcon Biomedical Research Unit LLC, Wagistrasse 21, CH-8952 Schlieren, Switzerland

‡

ABSTRACT: The third beamline for macromolecular crystallography at the superbend magnet X06DA of the Swiss Light Source (SLS) was designed for maximum efficiency and a high degree of automation to serve the needs of both academic and industrial users. An integral part of the automation concept is the crystallization facility built adjacent to the X06DA experimental minihutch. This unique configuration allows users to request and evaluate crystallization experiments using nanodispensing robots and automated imaging systems. Moreover, the beamline integration of the platform enables users to test initial crystallization hits for their diffraction behavior in situ, i.e. in the crystallization container, in an automated manner. This new process layout designed at the SLS gives scientists a rapid feedback on important crystal parameters such as diffraction limit, anisotropy, cell parameters, or mosaicity, and thus, it aids prioritization of subsequent optimization steps, which can also be accomplished at the facility. Here we present the design of the SLS crystallization platform with its integrated in situ X-ray diffraction screening (isXds) at beamline X06DA. A user case covering crystallization rescreening and in situ X-ray diffraction experiments to obtain new crystal forms with improved resolution is described.

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A tremendous amount of work has led to automation of most steps in the crystal to structure determination process at many synchrotron sites.4,5 However, so far, the preceding steps in a structural biology project leading to macromolecular crystals were not considered at synchrotron beamline setups. This includes the major bottleneck in macromolecular crystallography, generating macromolecular crystals suitable for final structure determination, which still remains a time-consuming iterative trial-and-error process. Automation of the crystallization process of biological macromolecules has led to higher success rates with reduced sample amounts6,7 and also to the establishment of various crystallization facilities.812 Nevertheless, initial crystallization hits, often yielding low resolution data, have to be evaluated in the X-ray beam before the respective crystallization conditions can be further optimized in a rational way. However, postcrystallization treatment and crystal mounting quite frequently cause damage to the obtained crystals, thus deteriorating their diffraction capabilities.13 In order to overcome this obstacle,

he structural genomics era has provided the scientific community with automation and high-throughput methods streamlining the crystal structure determination process. In this context, the Swiss Light Source (SLS) at the Paul Scherrer Institut (PSI) operating two very successful high performance undulator beamlines (X06SA and X10SA) designed and built a third beamline for macromolecular crystallography, X06DA (www.psi.ch/ sls/pxiii/). The concept of this new beamline is geared toward a high degree of automation and robustness from the optics to the experimental hutch to ease user operation. The optical design of beamline X06DA allows for rapid change of energy with a true fixed exit and minimal aberrations, resulting in an 80 μm  45 μm (h  v) focused beam at the sample position with a total photon flux not significantly lower than that of undulator beamlines (5  1011 photons/s/400 mA at 12.4 keV). The experimental station offers both efficient manual mounting, owing to a minihutch design,1 as well as a sample changer robot (CATS2), additionally enabling in situ X-ray diffraction.3 An online diffraction data processing pipeline, GO.COM (Wang, M., in preparation), combining available structure solution programs, completes the automation concept. r 2011 American Chemical Society

Received: October 15, 2010 Revised: February 18, 2011 Published: March 17, 2011 916

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Figure 1. Layout of the SLS crystallization platform enabling isXds: The floorplan of beamline X06DA (middle) depicts the spatial vicinity of the various modules of the facility. Liquid handling is accomplished on a single Hamilton ML Star robotic platform which covers the whole work-flow for macromolecular crystallization (1). Crystallization plates are stored and imaged in a RI 1000 (2) with its customized shuttle allowing remote plate transport to be requested (3). A seamless transfer from the imaging system into the experimental minihutch where isXds experiments are carried out is accomplished by a robotic transfer system: The crystallization plate is picked up by a SCARA robot from the shuttle or the user plate hotel and delivered into the hutch via a delivery stage (3). isXds is carried out in the minihutch (4).

macromolecular crystals can be tested for X-ray diffraction in situ13,14 beforehand—i.e. in the crystallization container— deploying robotic systems.3 Thus, users can receive a rapid and unambiguous feedback on the crystal quality, including important information such as resolution limit, anisotropy, cell parameters, and mosaicity, without any additional overhead for crystal mounting. In recent years developments in the field of macromolecular crystallography helped to promote in situ X-ray diffraction screening (isXds). Crystallization containers were devised to minimize the background signal in the diffraction pattern originating from its material. In addition, assemblies were developed giving scientists the opportunity to test their initial crystals in situ either in house (PX Scanner, Agilent technologies) or at the synchrotron.3 The implementation of an automated crystallization facility enabling isXds was an integral part of the concept for the new beamline X06DA at the SLS. It allows covering the work-flow from protein crystallization to isXds and subsequent crystal optimization, thereby providing the possibility to accelerate the structure determination process. This novel crystallization platform also complements the state of the art technologies used for protein production and biochemical and biophysical analysis established at the Biomolecular Research Laboratory (BMR) at PSI. The collaborative effort between the BMR and Macromolecular Crystallography group provides a complete gene to structure pipeline.15 Here the protein crystallization user facility enabling in situ X-ray diffraction screening is described. Test and first user cases

are presented underlining the improved work-flow for the structure determination process at the SLS. The realization of a crystallization facility at beamline X06DA imposed severe spatial constraints on the layout of conventional beamline components. Hence, as a prerequisite, the experimental hutch of beamline X06DA was designed as a minihutch,1 allowing installation of the crystallization facility equipment directly adjacent to it (Figure 1). A central part of the SLS crystallization facility is the liquid handling robot to prepare vapor diffusion experiments. The objective was to cover the whole work-flow of the macromolecular crystallization process: this includes initial screening at the 100 nanoliter scale with commercial crystallization screens, optimization experiments generated from stock solutions, and additive screening. Accordingly, a single platform, a Hamilton Star Plus, capable of accomplishing all these working steps, was custom-designed and installed (Figure 1). It is equipped with eight high volume channels (working volumes from 1 to 1000 μL) and a nanopipettor with an 8 þ 1 channel configuration (working volumes from 100 nL to 1000 nL; from Seyonic, licensed and integrated by Hamilton Bonaduz AG). The reproducibility and accuracy of the liquid handling systems are comparable to those of others9 (also cf. ’Protein dispense test’, 2005, NKI Amsterdam, http://www.bioxhit.org/). The deck configuration was chosen such that microbatch under oil and hanging drop vapor diffusion experiments could be implemented at a later stage. However, so far, sitting-drop crystallization trials 917

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Figure 2. Scheme of the software controls system for the plate transfer from the RI 1000 into the experimental hutch. On the software level, the crystallization plate transfer is managed by integration of the RI 1000 Web Services into the beamline controls system EPICS.

proved to be the most compatible method, in light of the isXds experiments. Preparation of a sitting drop crystallization plate with 2  96 drops requires about 30 min on the Hamilton platform; this includes the transfer of the crystallization solutions from the deepwell block into the reservoir cavity and also the automated sealing of the containers. Since the dispensing step of the nanoliter droplets takes 11 min for all 192 drops, a shutter covering the droplets during preparation and a humidifier were implemented to minimize evaporation problems imposed by such small volumes. Customized crystallization screens can also be prepared from stock solutions. Dispensing a PEG screen, with varying pH and salt concentrations, lasts approximately one hour. The single platform Hamilton ML Star is not designed for the highest throughput but has the advantage of providing a completely automated work-flow. Capable of preparing 10 crystallization plates or 10 optimization screens without manual interference, it is clearly sufficient to prove the concept of a crystallization facility integrated with a synchrotron beamline for isXds. A Formulatrix Rock Imager 1000 (RI 1000) operating at 20 °C and capable of accommodating up to 1000 SBS format crystallization containers was installed as a storage and imaging system adjacent to the minihutch of beamline X06DA (Figure 1). Rock Maker 2.0 software with its connected Microsoft SQL database, on one hand, is used to design and track crystallization experiments and, on the other hand, offers a web-based interface for users to observe and evaluate their crystallization droplets. Each experiment designed in Rock Maker 2.0 is converted into an XML file containing all experimental parameters and bar codes. The XML file is then imported by the respective Hamilton Software ’Crystal’, and a work list is generated for the liquid handler. On the Hamilton platform, a complete bar code tracking system is installed to verify all labware and solutions entering the deck. Thus, a seamless transfer of information between the authoritative database with all the experimental information and the actual pipetting parameters is guaranteed. Once users identify initial crystallization hits in the web-based Rock Maker GUI, it is crucial to obtain information on the diffraction behavior of these crystals as quickly as possible in order to optimize them in a rational way. To enable isXds in an automated manner, the plate storage and imaging system RI 1000 was installed directly adjacent to an opening of the experimental hutch of beamline X06DA (Figure 1). In addition,

an automated transfer system was realized for the transport of the respective crystallization containers from the plate storage hotel into the hutch. Therefore, the RI 1000 was customized with a small shuttle, remotely controlled via web services (Figure 2), which allows for any plate, identified by its bar code, to be transported outside of the cabinet (Figure 1). Once delivered outside, the plate of interest is picked up by a St€aubli 4-axis SCARA TS80 robot (http://www.staubli.com/en/robotics/products/6-axis-scara-industrial-robot/low-payload-6-axis-scara-robot/ts80/), equipped with a pneumatic gripper capable of seizing all SBS format containers. The robot moves the plate to a specified position on a delivery stage, which allows its transportation through an access window directly into the mini-hutch. Inside the hutch, the plate is received by a CATS robot (St€aubli 6-axis robot equipped with a gripper3) which transports and positions it in a vertical position in front of the X-ray beam (Figure 1). The whole process of plate selection, transfer in the X-ray beam position, and crystal alignment takes no longer than 2 min (see Supporting Information). Spillover of reservoir solution (which can contain detergents) into crystallization drops due to the vertical positioning of the plate was never observed for any SBS format sitting drop crystallization container as long as the respective volumes were within manufacturers’ recommendations (usually between 50 and 100 μL). In addition to the aforementioned storage system RI 1000 with its shuttle, a supplemental 12 positions plate hotel is also available and accessible for automated transfer (Figure 1). It enables users to bring their own plates and perform isXds experiments during their beam-time at beamline X06DA. The CATS robot is well suited for aligning and rotating the crystal in the X-ray beam for data collection. Linear movements below 5 μm can be achieved, and the reproducibility (moving to a different drop position or plate location and returning to the starting point) is within 25 μm. Currently the accessible rotation range for in situ X-ray data collection reaches from 5 degree to þ35 degrees for the two top rows of a crystallization plate and from 10 degrees to þ50 degrees for all other positions (an ongoing diffractometer redesign will abrogate these limitations). This oscillation range allows a full data set to be recorded for a high symmetry space group. As an example, a data set of 60 degrees oscillation range was collected on tetragonal hen egg white lysozyme (7% NaCl (w/v), 0.1 M NaOAc pH 4.7, 20% 918

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(v/v) ethylene glycol) contained in a ChipX microfluidic device.16 Good statistics up to 1.6 Å resolution were obtained after processing with XDS:17 an overall completeness of 95.4% (87.5%), an R-sym of 5.7% (42.8%), and an overall I/Sigma of 14.64 (2.15)—values in parentheses are for the highest resolution shell. Therefore, isXds is a valuable tool when crystals cannot be handled for cryogenic data collection, even though radiation damage is a limiting factor (the dose limit of protein crystals collected at room temperature is approximately 2 orders of magnitude lower than that at 100 K). In addition to the customized plate hotel with its shuttle and the hardware developments for the transfer system, the existing SLS user software at the macromolecular crystallography beamlines had to be adapted to support isXds. The SLS data collection program RemDaq was modified in such a way that it allows users to choose between classical data collection mode and isXds. Changing the beamline settings to isXds, which requires more space at the sample position to accommodate the SBS crystallization container, is fully automated and takes no more than 2 min. RemDaq also enables users to select plates for isXds based on their bar code or plate ID by establishing a connection to the RI 1000 web services and prompts the transport of the crystallization container into the hutch as previously described. In the background an in-house developed control software implemented on the beamline controls system EPICS (http://www.aps. anl.gov/epics/) guarantees an unobstructed process flow (Figure 2). It manages the individual components such as motors and sensors. A middleware system for the plate-transfer was developed, tiered by the device locations: inside-experimentalhutch, outside-experimental-hutch, and the access-border between them. Only the first two provide an interface (web services) to the user GUI at the beamline which enables request of crystallization plates for isXds. After positioning the plate at the X-ray beam position, the SLS sample alignment GUI is used to select the desired chamber and to align the crystal of interest in the X-ray beam. This last procedure clearly depends on how well the crystal is seen in the drop and on user experience (in most cases, it takes less than 15 s). Data collection is then configured and executed in RemDaq. The recorded diffraction images can be processed in a rapid and semiautomated way, using the data processing pipeline GO.COM. This allows extracting crystallographic information such as resolution limit, mosaicity, and cell parameters by utilizing LABELIT,18 MOSFLM,19 and XDS.17 The functionality of the isXds setup was evaluated by utilizing several in-house research projects. Salt crystals can be detected very easily by the typical diffraction pattern (Figure 3). However, in such a case, the intensity and quality of a synchrotron beam are not necessarily required, since a much weaker X-ray source, such as the PX scanner or other method, such as UV imaging, is usually sufficient. Nevertheless, the characteristics of a synchrotron X-ray beam deployed for isXds are absolutely crucial for crystals which are not single and of particularly small size. Sea-urchin-like crystals, which individually were only 23 μm in thickness, still yielded a diffraction image, clearly indicating that the obtained crystals were biological macromolecules (Figure 3). For the crystals that are not dramatically affected by postcrystallization treatment, we found that isXds at X06DA gives a very good estimate of the diffraction limit when comparing crystals from the same drop under cryogenic conditions (on either the same beamline X06DA or on a dedicated setup such as the microdiffractometer at undulator beamline X06SA).

Figure 3. isXds experiments conducted at beamline X06DA at the SLS. isXds unambiguously aids to identify salt crystals (A, B). Well diffracting crystals such as lysozyme, yield high resolution diffraction images. Microfluidic chips with reduced polymer thickness reduce the background scattering (C, D). isXds also enables obtaining diffraction information from small and weakly diffracting crystals (E, F). Even nanocrystals in high concentrations produce clearly visible powder diffraction patterns (G, H).

As an ultimate test for the isXds capabilities of SLS beamline X06DA, diffraction images of nanocrystals were recorded in the crystallization container. Hence, cross-linked crystals of lysozyme20 which were about 800  400  400 nm3 in size were distributed in varying concentrations in the respective wells of a prototype of the Greiner CrystalQuick X crystallization plate (the nanocrystals were diluted 1:5 from well to well). The resulting diffraction images clearly showed powder diffraction patterns (Figure 3) for crystallization drops containing high concentrations of nanocrystals (the drops appeared turbid). We were, however, not able to detect diffraction in the droplets which were diluted 1:25 compared to the original droplet. In order to improve the signal-to-noise ratio in isXds experiments, the thickness of the crystallization container material has to be reduced. In light of this necessity, several crystallization containers with much thinner or new materials, such as microfluidic chips (e.g., TOPAZ 1.96 Diffraction Capable Chip, Fluidigm), 919

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the crystals were then subjected to isXds at beamline X06DA (Figure 5). In each case, two consecutive images with an oscillation range of 1 degree respectively and exposure times typically between 1 and 5 s (without attenuation of the beam intensity) were recorded. Utilizing MOSFLM,19 the corresponding point groups could be assigned to P222, P2, or P3. In the best case, these crystals diffracted to 2.4 Å resolution in situ, with full beam intensity. Crystals of several other conditions yielded similar resolution. For direct comparison of diffraction properties, crystals from the nanoliter scale drops were subsequently mounted and frozen in the cryogenic nitrogen stream of beamline X06DA. A total of six complete data sets were recorded, processed using both MOSFLM19 and the automated GO.COM pipeline, solved by PHASER,22 and refined using REFMAC5.23 The best crystals under cryogenic conditions diffracted to 1.7 Å, and the space group was finally determined to be P212121 with three molecules per asymmetric unit (Figure 5). The structure was refined to R = 18.6% (Rfree = 22.9%), allowing the unequivocal positioning of a small organic molecule in the omit map which constitutes an essential formulation component. The point group of these crystals had initially been assigned to P222 from isXds experiments (see above), with similar edges as in the cryogenic data set, demonstrating an excellent correlation between data extracted from in situ diffraction experiments at room-temperature and the final cryogenic data set in P212121. isXds thus proved particularly efficient for space group determination and for crystal quality assessment. The crystallization platform at the Swiss Light Source has been in operation since summer 2010. In order to use the services of

counter-diffusion devices (e.g., CrystalHarp, Molecular Dimensions), or crystallization plates for vapor diffusion experiments (e.g., CrystalQuick X plate, Greiner Bioone), were developed over the past few years. The background observed when using these crystallization devices is dramatically reduced compared to that of standard crystallization containers (Figure 4). Hence, weak diffraction patterns arising from very small crystals can be recorded. With such improved crystallization containers, isXds could be a valuable method to detect nonvisible nanocrystals in view of nanocrystallography investigations at free electron X-ray laser facilities.21 The complete work-flow of the SLS crystallization platform with its isXds layout was evaluated with a test case. ESBATech, a Zurich-based biopharmaceutical company, is engaged in the development of single chain antibody fragments (scFv) for ophthalmic indications. Limited crystallization attempts for one of the scFv had resulted in crystals of space group C2, which diffracted to 2.5 Å at the undulator beamline X06SA equipped with a PILATUS 6 M pixel detector (http://dectris.com/sites/ pilatus6m.html). Solving the structure from these crystals proved to be a nontrivial task, as they contained 12 molecules per asymmetric unit. In addition, the space group could not be unambiguously assigned, and twinning was suspected. Hence, it was decided to screen for crystals in a different space group, ideally with less molecules per asymmetric unit and with improved resolution. After setting up four crystallization plates with 200 þ 200 and 200 þ 100 nL droplets in MRC2 plates (Molecular Dimensions) using the Hamilton crystallization robot, several dozens of crystallization hits were obtained; and

Figure 4. Comparison of various crystallization containers used for isXds. The background scattering originating from the material of the containers was recorded at 12.4 keV (exposure time 1 s, detector distance 180 mm, flux 5  1011 photons/s/400 mA). The intensity measured on the detector was plotted versus resolution. It clearly shows that the Greiner CrystalQuick X plate has an improved background scattering behavior compared to that of standard sitting drop crystallization plates (e.g., MRC2, Greiner Low Profile). The CrystalHarp (Molecular Dimensions) used for counterdiffusion experiments and the diffraction capable chip for free interface diffusion (Fluidigm) exhibit an even more reduced background. 920

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isXds experiments can be accomplished directly at the beamline. However, in order to more efficiently streamline the work-flow from crystallization experiments to isXds and crystal optimization, all steps have to be available remotely. PSI is currently working toward solutions enabling remote access to the macromolecular crystallography beamlines at the SLS including isXds. In order to further automate the complete work-flow, the capabilities of the web-viewing GUI xtalPIMS2426 (http:// www.pims-lims.org) used to observe and score crystallization experiments are being extended. It will then enable users not only to evaluate their drops but also to mark the position of crystals in the crystallization container and schedule them for isXds experiments. In addition, data collection settings such as beam intensity, detector distance, exposure time, and rotation angle used for the diffraction experiments can be prompted using the webbased GUI. Upon the next free shift, diffraction experiments for all crystals queued in the work-list will be executed, using the automated plate transfer system. The current focus on improvements of the crystallization platform at the SLS is on automated drop alignment in the X-ray beam after the automated transport of the crystallization container from the imaging system into the experimental hutch of beamline X06DA. The coordinates of the crystals in the RI 1000 will be used as a reference for crystal alignment in the X-ray beam. Once the crystallization drop is positioned in front of the X-ray beam, it can be rasterized with the synchrotron beam using fast read-out detectors such as the PILATUS.27 In order to ensure sensible scheduling for isXds by users, the RI 1000 will be upgraded with Second Order Nonlinear Imaging of Chiral Crystals (SONICC, Formulatrix, http://www.formulatrix.com/products/ crystallization-imaging/sonicc/index.html), which significantly enhances the sensitivity for crystal detection compared to conventional light microscopy and facilitates discrimination of salt from protein crystals at an earlier stage. Recently, there has been significant progress in the expression and crystallization of membrane proteins, including G-coupled receptor (GPCR) proteins. One of the major technical advances for successful structure determination of GPCRs is crystallization in lipidic cubic phases (LCP). In collaboration between the MRC Cambridge and TPP Labtech, the Mosquito nanodispenser has been modified and further developed such that it is now suitable for dispensing LCP (http://www.ttplabtech.com/products/ mosquito/crystallography.html#LCP). Underlying mechanisms in GPCR signaling are one of the main fields of research of the Biomolecular Research group at PSI. Therefore, it was decided to implement a LCP Mosquito nanodispenser into the SLS crystallization facility to enable crystallization of membrane proteins by means of LCP as part of the services offered by the SLS crystallization platform. Since such crystallization in LCP is also carried out in SBS format, initial crystals can be tested by isXds. This is a big advantage, because mounting crystals obtained in LCP is inherently difficult due to its opaqueness and the typically small dimensions of the crystals. isXds will avoid any damage to the crystals and also has the potential to reveal crystals not visible under the light microscope. In addition, the Mosquito nanodispenser will also greatly increase the throughput for preparing sitting drop vapor diffusion experiments. With these additional planned and currently ongoing measures which further expand the capabilities of the SLS protein crystallization platform enabling in situ X-ray diffraction screening, users have a unique opportunity to obtain crystals suitable for structure determination in a much more efficient way. Taken

Figure 5. Screening for crystals with a novel space group: scFv initially crystallized in C2 with 12 molecules per asymmetric unit (a.u.) and diffracted to 2.5 Å. Utilizing the SLS crystallization platform, dozens of crystallization hits were obtained. Several of these conditions were subjected to isXds. The diffraction images were analyzed, and the point groups determined to be P222, P2, and P3, respectively. Finally, full data sets were recorded under cryogenic conditions for six of these crystals. The final space groups were unambiguously assigned to P212121 and P21 with significantly improved resolution and reduced number of molecules per a.u.

the crystallization facility with its integrated in situ X-ray diffraction screening, users have to register with the Digital User Office (DUO; https://duo.psi.ch) and submit a short proposal. A proposal can be written for specific projects or for a long-term group research project. Once it is accepted, crystallization experiments, as well as time for isXds can be requested via DUO. The implementation of a quality control is foreseen (e.g., using SDS PAGE, light scattering methods, and mass spectrometry) for the crystallization samples, as the initial step of any experiment carried out at the SLS crystallization facility. This will ensure compliance with standards for purity and homogeneity of the macromolecular material. With the currently implemented setup, users can request, track, and evaluate crystallization experiments remotely. Also, 921

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together with the beamline automation at X06DA, this is a clear step toward a much more streamlined and successful work-flow in macromolecular crystallography, eliminating significant bottlenecks in the process.

McCarthy, A.; McCarthy, J.; McSweeney, S.; Meyer, J.; Mitchell, E.; Monaco, S.; Nurizzo, D.; Ravelli, R.; Rey, V.; Shepard, W.; Spruce, D.; Svensson, O.; Theveneau, P. Automation of macromolecular crystallography beamlines. Prog. Biophys. Mol. Biol. 2005, 89, 124–152. (5) Gonzalez, A.; Moorhead, P.; McPhillips, S. E.; Song, J.; Sharp, K.; Taylor, J. R.; Adams, P. D.; Sauter, N. K.; Soltis, S. M. Web-Ice: Integrated Data Collection and Analysis for Macromolecular Crystallography. J. Appl. Crystallogr. 2008, 41, 176–184. (6) Chayen, N. E.; Shaw Stewart, P. D.; Baldock, P. New developments of the IMPAX small-volume automated crystallization system. Acta Crystallogr. 1994, D50, 456–458. (7) DeLucas, L. J.; Bray, T. L.; Nagy, L.; McCombs, D.; Chernov, N.; Hamrick, D.; Cosenza, L.; Belgovskiy, A.; Stoops, B.; Chait, A. Efficient protein crystallization. J. Struct. Biol. 2003, 142, 188–206. (8) Luft, J. R.; Collins, R. J.; Fehrman, N. A.; Lauricella, A. M.; Veatch, C. K.; DeTitta, G. T. A deliberate approach to screening for initial crystallization conditions of biological macromolecules. J. Struct. Biol. 2003, 142, 170–179. (9) Mueller-Dieckmann, J. The open-access high-throughput crystallization facility at EMBL Hamburg. Acta Crystallogr. 2006, D62, 1446– 1452. (10) Dimasi, N.; Flot, D.; Dupeux, F.; Marquez, J. A. Expression, crystallization and X-ray data collection from microcrystals of the extracellular domain of the human inhibitory receptor expressed on myeloid cells IREM-1. Acta Crystallogr. 2007, F63, 204–208. (11) Stock, D.; Perisic, O.; L€ owe, J. Robotic nanolitre protein crystallisation at the LMB. Prog. Biophys. Mol. Biol. 2005, 88, 311–327. (12) Joachimiak, A. High-throughput crystallography for structural genomics. Curr. Opin. Struct. Biol. 2009, 19, 573–84. (13) McPherson, A. In situ X-ray crystallography. J. Appl. Crystallogr. 2000, 33, 397–400. (14) Watanabe, N.; Murai, H.; Tanaka, I. Semi-automatic protein crystallization system that allows in situ observation of X-ray diffraction from crystals in the drop. Acta Crystallogr. 2002, D58, 1527–1530. (15) http://www.systemsx.ch/news/systemsxch-publications/xletter/ (16) Dhouib, K.; Malek, C. K.; Pfleging, W.; Gauthier-Manuel, B.; Duffait, R.; Thuillier, G.; Ferrigno, R.; Jacquamet, L.; Ohana, J.; Ferrer, J. L.; Theobald-Dietrich, A.; Giege, R.; Lorber, B.; Sauter, C. Microfluidic chips for the crystallization of biomacromolecules by counter-diffusion and on-chip crystal X-ray analysis. Lab Chip 2009, 9, 1412–1421. (17) Kabsch, W. Integration, scaling, space-group assignment and post refinement. Acta Crystallogr. 2010, D66, 133–144. (18) Sauter, N. K.; Grosse-Kunstkleve, R. W.; Adams, P. D. Robust indexing for automatic data collection. J. Appl. Crystallogr. 2004, 37, 399–409. (19) Leslie, A. G. W. Joint CCP4 þ ESF-EAMCB Newsletter on Protein Crystallography (1992), No. 26. (20) Falkner, J. C.; Al-Somali, A. M.; Jamison, J. A.; Zhang, I.; Adrianse, S. L.; Simpson, R. L.; Calabretta, M. K.; Radding, W.; Philipps, G. N.; Vicki, L. Generation of Size-Controlled, Submicrometer Protein Crystals. Chem. Mater. 2005, 17, 2679–2686. (21) Shapiro, D. A.; Chapman, H. N.; DePonte, D.; Doak, R. B.; Fromme, P.; Hembree, G.; Hunter, S.; Marchesini, S.; Schmidt, K.; Spence, J.; Starodub, D.; Weierstall, U. Powder diffraction from a continuous microjet of submicrometer protein Crystals. J. Synchrotron Radiat. 2008, 15, 593–599. (22) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl. Crystallogr. 2007, 40, 658–674. (23) Vagin, A. A.; Steiner, R. S.; Lebedev, A. A.; Potterton, L.; McNicholas, S.; Long, F.; Murshudov, G. N. REFMAC5 dictionary: organisation of prior chemical knowledge and guidelines for its use. Acta Crystallogr. 2004, D60, 2284–2295. (24) Mayo, C. J.; Diprose, J. M.; Walter, T. S.; Berry, I. M.; Wilson, J.; Owens, R. J.; Jones, E. Y.; Harlos, K.; Stuart, D. I.; Esnouf, R. M. Benefits of automated crystallization plate tracking, imaging and analysis. Structure 2005, 13, 175–182.

’ ASSOCIATED CONTENT

bS

Supporting Information. Movie showing the workflow and isXds at the SLS crystallization platform at beamline X06DA. This material is available free of charge via the Internet at http:// pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Telephone: þ41 56 310 5233. Present Addresses §

)

ESBATech, an Alcon Biomedical Research Unit LLC, Wagistrasse 21, CH-8952 Schlieren, Switzerland. Dectris Ltd., Neuenhoferstrasse 107, CH-5400 Baden, Switzerland.

’ ACKNOWLEDGMENT We thank our industry partners (Actelion, Boehringer-Ingelheim, Mitsubishi Chemicals, Novartis, and Proteros) and especially J. Benz and A. Pautsch for their valuable feedback. We thank M. Knopp and J. Eich for their work in setting up and debugging the Hamilton liquid handling robot; Z. Umanoff, M. Ordonez, and F. Hoedemaker from Formulatrix for their dedication in establishing the customized Rock Imager with the RI 1000 shuttle. The transfer system was constructed at PSI by P. Nagel under the supervision of P. Heimgartner. We are grateful to R. Luescher, C. Harm, and B. Blarer, who made the plate transfer into the experimental hutch possible by modifying the local access control under compliance with the radiation safety regulations. We are grateful to I. Schlichting, who provided us with nanocrystals and to C. Sauter for the in situ lysozyme data set. We also would like to thank P. Richle for his help on the scFv project. This project was initiated and accompanied by helpful discussions by F. K. Winkler, E. Pohl, A. Prota, and S. Russo. We also would like to thank D. Frey and N. Olieric for their support at the SLS crystallization platform. The funding for the SLS crystallization facility was provided by the PSI Forschungskommission. ’ REFERENCES (1) MacDowell, A. A.; Celestre, R. S.; Howells, M.; McKinney, W.; Krupnick, J.; Cambie, D.; Domning, E. E.; Duarte, R. M.; Kelez, N.; Plate, D. W.; Cork, C. W.; Earnest, T. N.; Dickert, J.; Meigs, G.; Ralston, C.; Holton, J. M.; Albers, T.; Berger, J. M.; Agard, D. A.; Padmore, H. A. Suite of three protein crystallography beamlines with single superconducting bend magnet as the source. J. Synchrotron Radiat. 2004, 11, 447–55. (2) Ohana, J.; Jacquamet, L.; Joly, J.; Bertoni, A.; Taunier, P.; Michel, L.; Charrault, P.; Pirocchi, M.; Carpentier, P.; Borel, F.; Kahn, R.; Ferrer, J. L. A new highly integrated sample environment for protein crystallography. Acta Crystallogr. 2004, D60, 888–94. (3) Jacquamet, L.; Ohana, J.; Joly, J.; Borel, F.; Pirocchi, M.; Charrault, P.; Bertoni, A.; Israel-Gouy, P.; Carpentier, P.; Kozielski, F.; Blot, D.; Ferrer, J. L. Automated analysis of vapor diffusion crystallization drops with an x-ray beam. Structure 2004, 12, 1219–1225. (4) Arzt, S.; Beteva, A.; Cipriani, F.; Delageniere, S.; Felisaz, F.; F€orstner, G.; Gordon, E.; Launer, L.; Lavault, B.; Leonard, G.; Mairs, T.; 922

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