ACCESS TO HIGH THROUGHPUT SAXS DATA ...

ACCESS TO HIGH THROUGHPUT SAXS DATA COLLECTION FOR THE STUDY OF BIOLOGICAL MACROMOLECULES

Kevin N. Dyer*, Ashley J. Pratt*, Henry Y. H. Tang, John A. Tainer, Greg L. Hura Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA Henry Y. H. Tang Department of Chemistry, University of California, Berkeley, CA 94720, USA John A. Tainer Department of Integrative Structural and Computational Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA

Abstract SAXS is increasingly utilized to complement crystallographic studies as access to the technique has increased and novel mechanistic insights are reported. Conformational changes underlie most associations in biological metabolic networks and support catalysis or communication of information. Biological macromolecules often undergo multiple conformational changes as part of their mechanism. Crystallography typically provides atomic resolution information on one low energy conformation. As SAXS is a solution-based technique and can be conducted in high throughput, macromolecules can be examined in many context dependent conformational states. Comparisons of SAXS profiles in different conditions are sufficient to identify small conformational changes and, by using available crystallographic results, often sufficient to define how conformation has changed. Thus, specifically pairing SAXS and crystallography provides a unique means to comprehensively couple biologically relevant conformational changes in solution and within stable assemblies. Here we review recent results from three systems: a cellulase (EngD), a human cell cycle regulator (Retinoblastoma protein), and a pathogenic metalloenzyme (Cu,Zn-Superoxide dismutase), with a focus on how SAXS experiments and software utilized crystallographic information for analysis to provide important mechanistic information beyond those available from crystal structures alone. We also describe our most recent efforts to improve access to a growing user community through our web-based application: https://sibyls.als.lbl.gov/htsaxs.

*authors contributed equally

Introduction Small angle X-ray scattering (SAXS) has been increasingly embraced for structural characterization of biological macromolecules in solution over the last ten years because of four principal factors: 1) the need to build upon and extend the information in our mature library of atomic resolution structures; 2) the emergence of improved analysis tools; 3) improved data quality; and 4) improved access to instrumentation. Here we will highlight three recent biological applications, focusing on how SAXS data added value to our understanding of each system. We will also describe a new web-based tool that aid preliminary processing of and enhances access to SAXS data collection at the SIBYLS beamline located at the Advanced Light Source. Through these discussions we hope to promote the growth of this resurgent technique that addresses a central challenge in structural biology. Namely, structural genomics efforts have struggled to keep pace with the throughput of sequence-based methodologies, to the extent that mechanistic insights afforded by structural means are often overlooked. Successes in engineering or modifying biological systems will necessarily arise from a combination of experimental approaches that support a fundamental understanding of mechanism. We anticipate that X-ray scattering techniques will increasingly play an important role in combining mechanistic insights from structure with RNA/DNA sequence based approaches. The Structurally Integrated BiologY for the Life Sciences, SIBYLS, beamline (beamline 12.3.1 https://sibyls.als.lbl.gov), at Lawrence Berkeley National Laboratory, uniquely operates as both a macromolecular crystallography (MX) and SAXS endstation [1]. A 30minute transition time between the modes enables investigators facile access to both techniques on the same day. When SIBYLS was first established (2005), SAXS was a relatively small niche technique applied to a few select systems. In contrast to the current state, the SIBYLS SAXS user base now includes approximately 200 laboratories with applications to widespread pathways and purposes. SIBYLS technology development is problem-based and a primary focus is to integrate MX and SAXS results for insights into biological mechanisms [2]. Reflected in our publications database, most of our investigators will utilize MX and SAXS together to provide a broader understanding of biological mechanism. Attractive aspects of SAXS for structural biologists are that it provides data on most samples without size or crystallization restrictions and it defines mass [3], flexibility [4], and ensemble states. SAXS also provides an experimental basis to model unknown structures from structurally similar ones. A common path forward from SAXS studies is the identification of targets for crystallization. Lastly, following atomic resolution structure determination, SAXS takes crystallographic characterization further by elucidating conformational changes adopted by the protein. This process couples directly to the inputs and outputs of macromolecular crystallography (MX) pipelines like that established by the Protein Structure Initiative (PSI) and their associated centers. Relative to crystallography, SAXS data contain less information. However, crystallographic results and other data provide significant restraints for SAXS analysis so that detailed information (such as the interfaces between elements of a non-crystallizable complex) can be defined and modeled atomistically [5, 6].

An important complementary aspect of SAXS to MX efforts is that it can be carried out with high throughput [7-11]. Biological macromolecules are components of large networks replete with multi-level feedback loops. Characterization of macromolecules in one conformational state has limited impact on our ability to diagnose and engineer the multidimensional circuits that control cellular outcome. Distinguishing possible conformations and the conditions where they occur is required to connect macromolecular structures with the biology occurring at mutlicellular length scales. SIBYLS has operated SAXS in a high throughput mode for three years. In a typical week, we collect 12 x 96 well plates worth of samples. Many samples have been collected via a mail-in/hand-in approach. While work to improve the system continues, several investigations have successfully utilized the high throughput SAXS system in conjunction with MX results to provide novel insights into mechanism, three of which are described below. Mechanistic insights from recent SAXS studies I. Cellulases The study of the structure and dynamics of cellulases is particularly well-suited for combining SAXS and MX [12]. Cellulases are a class of proteins that digest cellulose. Their mechanisms have gained interest as part of efforts to convert plant biomass into renewable energy. Generally, individual domains are very stable and amenable to MX while the extent of the flexibility between domains is capably probed by SAXS. In their study of the cellulase Endoglucanase D (EngD) from Clostridium cellulovarans, Bianchetti et al. [13] were able to solve the crystal structure of the two-domain protein (Fig. 1a). Four EngD molecules were found in the asymmetric unit. A 40 amino-acid linker between the two domains (a catalytic domain and a carbohydrate binding module) was not resolved in the crystallographic density. Thus the connectivity between a particular catalytic domain and carbohydrate module was not apparent. To identify which domain positioning best represented the solution structure, SAXS measurements were conducted. Through analysis of the SAXS data, the following was determined. In solution, EngD is a monomer rather than the tetramer found in the crystal structure. None of the juxtapositions between domains found in the crystal structure fit the SAXS data well. Furthermore, no single structure was able to fit the SAXS data adequately. In order to assess the agreement between crystal structures and SAXS data, the program Fast Open X-ray Scattering (FOXS) was used [14, 15]. An efficient aspect of FOXS is that it calculates SAXS profiles from atomic structures using the Debye formula with amino-acid form factors rather than atomic form factors. Methods for calculating SAXS profiles from atomic coordinates are rapidly developing as new algorithms and optimizations are introduced. Further, SAXS analysis was conducted to better define the solution structure of EngD. Utilizing a molecular dynamics approach, BilboMD [16], an ensemble of conformational states were created. BilboMD is efficient in generating conformations by conducting molecular dynamics at high temperatures without solvation. The SAXS profiles are calculated from over 10,000 generated conformations and compared to the experiment.

An ensemble of the two domains relative to one another provided an excellent fit suggesting that the two domains are flexibly linked. By combining SAXS and MX, a mechanistic understanding of the system was obtained. The catalytic binding module fixes the enzyme to a cellulose surface allowing the catalytic subunit to achieve a high local concentration and probe the nearby surface for its substrate through the flexibility identified by SAXS.

Figure 1. Combining SAXS and crystallography for mechanistic insights from flexibility. (a) SAXS data (black) from a cellulase [13] as compared to the atomic resolution models of a solved crystal structure (dashed grey), the best single model (cyan) from BilboMD [16] and an ensemble (magenta). The ratio of the experimental profiles relative to the models are shown in the inset with the ribbon diagrams shown below. The ensemble is a representative minimal ensemble. (b) The same for Retinoblastoma protein [20]. In this case phosphorylation triggers a change in conformational state.

II. Retinoblastoma protein Flexibility is a generalizable characteristic of many macromolecules, anticipated to be a feature in approximately 40% of human proteins [17]. Flexible regions often play an

important role in the conformational changes required for mechanism. Retinoblastoma protein (Rb) is a hub for many cell cycle control processes [18]. Many types of cancers show aberrations in Rb function [19]. Rb interactions with over 10 proteins are regulated by phosphorylation of Rb in tens of serine or tyrosine sites. While many states and complexes are of interest very few are likely to be crystallized. Through SAXS studies of both the phosphorylated and unphosphorylated states, phosphorylation of tyrosine 373 (Y373) resulted in a relatively compact and rigid Rb structure [20]. The rigidity was assessed by the rate of decay of the SAXS curve which shows a q-4 dependence. In contrast the unphosphorylated state decays with a q-3.3 dependence indicating greater flexibility. Burke et al. [20] were able to solve the structure of an Rb phosphorylated at Y373. The Rb construct is composed of two elongated domains where the phosphorylated Y373 serves as a contact between the two domains (Fig 1b). A crystal structure of the unphosphorylated Y373 has yet to be determined. Using BilboMD with the available crystal structure, the unphosphorylated state was further probed. Both elongated and compact structures were required to fully fit the data. With this information Burke et al. proposed a mechanism by which Rb becomes incapable of binding its diverse set of partners in the phosphorylated state. The compact phosphorylated state closes off binding sites, whereas in the flexible unphosphorylated state large sections of surface area become available for productive binding and therefore further downstream cellular signaling. III. Assaying oligomerization changes of aggregation-prone proteins. Aging diseases, in particular neurodegenerative diseases, have been linked to macromolecular misfolding and aggregation affecting particular regions of the central or peripheral nervous system. A variety of disease-causing proteins (and nucleic acids), under the right circumstances, can form larger molecular weight polymeric species generally referred to as “aggregates”[21]. However, the individual triggers, kinetics and morphologies of the molecular species formed when a native-like fold is disturbed vary considerably – both from protein to protein and depending on the solution conditions, genetic lesions, and interaction partners present. Cu,Zn-Superoxide dismutase (SOD) metalloenzymes are implicated in the fatal motor system disease Amyotrophic Lateral Sclerosis. Most of the >150 genetic lesions in SOD1 documented [22] at present result from point mutations in SOD (Fig 2a), which result in opportunistic defects including framework destabilization (Fig 2b), a precursor to tissuespecific protein aggregation [23]. In our recent studies, we assessed destabilizing effects on SOD stability imparted by the presence, absence or alteration of metal co-factors through mutation and experimental conditions. X-ray structures of disease mutants and engineered monomers are available; yet these mutants often only vary subtly from their respective wild-type counterparts (Fig 2c). Mutations that are solvent exposed are particularly susceptible to crystallographic packing modifications relative to solution states. Basal-state filamentous crystallographic assemblies and fibril-like oligomers bring up the age-old question of biological relevance, as crystalline assemblies are notoriously biased toward low-energy species. With the availability of hundreds of SOD crystal

structures, and the importance of screening many mutations and conditions for inducing or controlling aggregation, we employed high throughput SAXS for comprehensive characterization of aggregation kinetics and nuclei structures. In particular, we recently applied high throughput SAXS to characterize conformational changes manifesting as differences in mutant SOD misfolding trajectories [24]. In our studies, we induced aggregation in a subset of SOD mutant samples by varying levels of metal cofactors and used SAXS as an assay to determine the degree and morphology of aggregation in the sample. We determined optimal and mild conditions to discriminate between mutant and wild-type SOD behavior. One particularly useful tool we developed and employed was the structural comparison heatmap tool [25] (Fig 2d). The tool visually maps pairwise comparisons of SAXS profiles collected with different mutations, conditions and time points. The relative severity and type of aggregation can be visualized at-a-glance.

Figure 2. Using SAXS to study conformational changes of pathogenic Cu,Zn superoxide dismutase proteins in solution. (a) Ribbon diagram of human Cu,Zn Superoxide distmutase (from PDB code 1PU0) is rendered in purple with point mutation sites mapped as red Cα balls. (b) Generalized framework destabilization model for dominant form of SOD-mediated protein fibrillation. (c) Degree of subunit rotation evident in WT (purple backbone trace) and mutant (red) SOD crystallographic structures are similar. (d) A pair-wise comparison map of SAXS data from different SOD mutants and conditions. Each column or row compares one SAXS profile with all others. With wild-type non-aggregated state in the top left corner (sample A). The maximally different and most aggregated state from wild-type is sample Q. The samples have been ordered using a clustering approach.

We recently implemented a webserver (http://sibyls.als.lbl.gov/saxs_similarity) that enables the general SAXS userbase to perform these types of analyses in the absence of any structural model bias. In our experiments, we were able to rapidly test effects of mutations, buffers, solvents and small molecules on SOD misfolding and aggregation. We were able to correlate in vitro trends to the limited available patient outcome data

making our studies relevant to disease etiology of SOD aggregates. The SAXS data helped us revise our framework destabilization hypothesis (Fig 2b) to account for the effects of secondary and auxiliary pathogenic factors. The “high resolution” nature of SAXS-based assays (as compared to other applied light scattering approaches) enabled development of a plausible atomistic model for aggregate nuclei under a given set of conditions. In the studies described above, SAXS analysis was enhanced by available atomic resolution structures. SAXS provided information beyond that which could be attained by crystallography alone. The SAXS data described in the above applications were collected by the high throughput SAXS data collection system offered to users world-wide. Both academic and proprietary interests are welcome to submit samples. Samples are prepared and delivered in a sealed 96-well plate and within a week SAXS results as well as a preliminary analysis of sample quality by SIBYLS staff are made available. Since the service began in August 2010, a total of 160 unique labs have collected mail-in SAXS data at SIBYLS. Over 70% of these users collected more than once. We have increased the accessibility of SAXS data collection through developing our web-based application process described below. Developing a web-based application for access An important improvement in providing access to SAXS at SIBYLS has been the development of the HTSAXS website (https://sibyls.als.lbl.gov/htsaxs). In high throughput applications, a robust system for scheduling and sample tracking is a key necessity. Beyond these basic needs, communication about the quantity and quality of sample required, delivery methods, as well as access to results, have been enabled and facilitated through the development of our website. While description of innovations in hardware is frequently published, an equally important and often overlooked area of development is software enabling control at multiple levels. Designs of organizational processes are underreported while unnecessary reinvention is common. Here we describe the design of a cross-browser compatible Ruby on Rails web application hosted by the SIBYLS web server for high throughput SAXS data collection. Information about SAXS data collection At https://sibyls.als.lbl.gov/htsaxs, general information about SAXS, sample requirements and user statistics are available to interested investigators without restriction. The ‘About’ page introduces SIBYLS staff and describes the capabilities of the SIBYLS high throughput SAXS endstation. Also available is a current total of the number of labs, plates collected and notable publications from our user base. A ‘Contact’ page facilitates direct contact with the SIBYLS staff for prospective users to ask questions, make comments or provide feedback. The ‘Instructions’ page provides a detailed description of sample submission requirements. In brief, SIBYLS currently utilizes a 96-well plate type and sealing mat. The minimum required sample volume is 24 μL. As a rule of thumb at our beamline,

Figure 3. The Calendar page shows upcoming shifts available for mail-in SAXS. The circles under each date icon represent the number of slots in that shift. Occupied slots are red while vacant slots are light gray. (a) Shifts that are completely booked become transparent and can no longer accept reservations.

polypeptide samples provide a high quality signal if their concentration in milligrams per milliliter is greater than 100 divided by the expected molecular weight in kDa. Typically 3 concentrations are submitted for each sample. For robust data collection, duplicate buffers should flank each concentration series, one preceding the series and one following it. Also available on the ‘Instructions’ page are the shipping address and required barcode shipping labels. An explanation of the ALS User office registration process and proposal requirements is also provided and must be adhered to for shipment and subsequent data receipt. Lastly, the steps of data collection are described – namely, what happens after a plate is shipped and is expected back. When plates arrive they are stored at 4° C or -80° C, depending on the temperature maintained during shipment. In preparation for data collection, the plate will be lightly spun at 3700 rpm at 4 °C. When data collection is complete, investigators will get back buffer-subtracted, integrated scattering profiles in ASCII format. Four exposures are taken of each well: 0.5, 1.0, 2.0 and 5.0 seconds in that order, unless otherwise specified. The data are typically collected at 16 °C. The ‘Statistics’ page provides a compilation of sample quality statistics, updated in real time. This page doubles as a tutorial for possible SAXS sample issues. Common annotated comments delivered back with data include evidence of aggregation, radiation sensitivity, detector saturation, low concentration, bad buffer subtraction, bubble present in sample cell, low volume, concentration dependence, repulsion, micro-crystals, and undefined Guinier region. These flags are made through a visual inspection by SIBYLS staff and should be further investigated. Initiating data collection

For investigators to initiate data collection and subsequently obtain access to their data, registration with the ALS user office is required. The registration process collects information about the investigator and sets up a user account. Registration must be accomplished at two levels. The first is in relation to the Advanced Light Source synchrotron whose funding agencies require an understanding of its user population. The second level of registration is required to set up a user account at the beamline to grant users access to data and processing tools. Registration is a one-time process applicable to all subsequent data collections. On the homepage, prospective users can request an account by providing their full name, email, and Principal Investigator. The request is sent to the SIBYLS staff to manually create the account. Accounts are created manually for security reasons. Authentication is handled via an external server. Once created, users will receive an email with instructions to create a password and login to the site. Users with a SIBYLS account have access to the main functionalities of the site and its resources. Each account has the following attributes: the SIBYLS username and password, an email address, affiliation, and a principall investigator. Once registered, users can request a date for data collection.

Figure 4. Representation of the slot model and its attributes. (a) After booking a slot, the ‘Reserved Slot’ page allows users to print their barcode label, upload their plate spreadsheet, track the progress of their data collection and retrieve their data once available. Staff can use this page to download the spreadsheet for data collection, update the slot status, and upload and edit the data package. (b) At the top is an example of the plate QR label. The format of the plate spreadsheet is shown in the middle. The bottom box is a single concentration series from the ‘Quick View’ page of the data package.

Figure 5. Entity relationship diagram of the HTSAXS database. (a) Blue rectangles represent database tables. Red lines indicate users with an admin role. Admins can create and update shifts, which are weeks of mail-in SAXS beamtime. Each shift can hold many slots, which general users can book to mail-in a sealed 96-well plate. Information from a spreadsheet uploaded by the user populates the plate and well tables.(b)The Stats table holds information from the data returned to users. It is used to record sample quality statistics as well as show a quick view page of the data for each plate. (c) The mobile device application interacts with the shift and slot tables.

To select a date for data collection users can go to the ‘Calendar’ page, which shows the available time slots for each week (Fig. 3). If a user critically requires a date that is already full, they can contact the SIBYLS team to request the addition of another slot to the date of interest. Once a user selects a date in the calendar to send their plate, they are directed to a ‘Terms and Agreement’ page, where the most important information from the instructions are reiterated. In addition to the primary email associated with their account, users can enter up to three more email addresses that will receive correspondence regarding the data collection progress for that particular plate. Once the web form is submitted, a data collection slot is created for that user on the selected date. Users must wait at least two weeks before they can book another slot. All slots booked by a given user, both upcoming and past, can be accessed in the ‘My Account’ page, also referred to as the ‘User Show’ page. Sample delivery and data collection progress Once an available date is selected for data collection, a unique identifier is created to associate the user and date. A link to the experiment is added to their ‘My Account’ page wherein information can be uploaded and ultimately SAXS data can be downloaded. By clicking on the specified link the user is taken to the ‘Reserved Slot’ (Fig. 4) page, which

provides more detail about the state of data collection during that time slot. To communicate the order of data collection and streamline post-processing, a critical upload to the ‘Reserved Slot’ page is a formatted spreadsheet to be provided by the user. Spreadsheet rows correlate with each well in a plate and provide a collection order, a buffer or sample label, and a file name. An example spreadsheet template is available on the instructions page. In order to check and validate fields in the spreadsheet, the contents are parsed into a web form that validates the format before saving the information to the server database. First, the ‘order of collection’ column must not repeat or skip any numbers. Second, the well locations must exist in a 96-well plate (A1 - H12). Lastly, the sample IDs must be under 12 characters and can only contain letters, numbers and underscores. As sample IDs will be used to name output files, names with spaces in them are prohibited. Once the spreadsheet passes all validations, the contents are saved to the database. Administrators can then download the validated spreadsheet for data collection. Sample plates are delivered to the beamline via one of several routes. The most facile is overnight shipment. Locally, samples may also be hand-delivered to the Advanced Light Source for data collection by SIBYLS staff. Self-collection is also possible and encouraged. In each of the ‘Reserved Slot’ pages, a unique Quick Response (QR) barcode label is generated (Fig. 4b). This label is printed and affixed with tape to the side of the plate for sample tracking. A mobile device-based application was developed and is currently utilized to scan the QR barcode on a plate to match it with the associated data collection slot (Fig 4b). Once this information is synchronized, the status is updated, and can easily be updated using the web interface menu. This addition to the web application allows staff to quickly update plate status in real time without having to login to the web interface. Once the plate has been shipped, users may track the progress of their experiment from delivery to data collection to data becoming available on the ‘Reserved Slot’ webpage. The mobile device QR barcode system (Fig. 5) enables rapid synchronization and updates immediately upon sample delivery and scanning. Communication of Results Once user samples have been collected, users will find their ‘Reserved Slot’ page annotated with the data collection date and a ‘Quick View’ link that displays an interactive view of their data. This data has already been partially processed: the two-dimensional images read out from our CCD detector are first integrated and then buffer subtracted using our beamline’s algorithms to yield SAXS profile data comprised of Intensity vs. momentum transfer (q) for each exposure time. One of three algorithms are typically used for buffer subtraction and sample either: 1) the buffer provided before the sample; 2) the buffer provided after the sample; or 3) the average. Beamline staff visually inspect each buffer subtracted profile and suggest which of the three options is most ideal. If the “before” and “after” buffer signals are nearly identical, then the average is used. If for obvious or indeterminate reasons, one of the buffers produces negative intensities or shows other non-physical behaviors, the other buffer is selected for subtraction. Users

may reprocess data with an alternate buffer by accessing beamline software via remote account login and staff direction. The processed scattering files are displayed in the ‘Quick View’ report section of the site. The display format splits the data into concentration series. For each series, a thumbnail plot displays an overlay of scattering profiles. The thumbnail plot is zoomable for better visibility when clicked, and shrinks back to a thumbnail for compaction upon a second click. Importantly, an photograph of the sample cell is also taken just prior to X-ray exposure and displayed upon clicking the filename to identify bubbles and low volumes that can lead to erroneous data processing. The entire data package can be downloaded as a zip file from the ‘Reserved Slot’ page. Each buffer subtracted scattering file is listed adjacent to associated sample quality comments. The comments are a global initial assessment by staff of data quality assuming the purpose of analysis is for determining the shape of the molecule. The staff-based data quality comments are also used to populate a separate database table which tallies overall userbase quality statistics. These compiled statistics are then used to populate the percentage values presented in the ‘Statistics’ page. An overview of the web based database is shown in Figure 5. Administrators of the HTSAXS site have access to additional functionalities above those available to users. Much of the additional functionality is for scheduling of data collection and management of raw and processed data. Thus, the tight integration accomplished in our HTSAXS database system enables organized, at-a-glance interpretation for our userbase. Conclusion One of the goals at the SIBLYLS program and beamline is to enable facile and integrative data collection and processing for both MX and SAXS data. MX provides the high resolution required to bridge chemistry to biology and has enabled remarkable insights into biological mechanisms. In many cases, like those described above, tens or even hundreds of conformational states, mutations or constructs are necessary to understand the functionality of a macromolecule. Since SAXS is high throughput and a solution-based technique, all thermodynamic states may be interrogated. Building SAXS analysis around an atomic resolution structure is particularly powerful as shown with the three examples above. Opportunities for analysis have increased with recent algorithms [6, 15, 16, 2528]. Ongoing enhancements in SAXS capabilities enable the detection of small conformational changes in solution. We have improved access to the technique and initial results through our web-based system located at https://sibyls.als.lbl.gov/htsaxs. We expect that other user facilities will also increase access to SAXS data collection and our efforts as described here may be helpful. As capabilities to measure and analyze increase, we expect high throughput SAXS will increase the throughput at which we gain an understanding of macromolecular mechanism.

Acknowledgements K.N.D., A.J.P., H.Y.H.T., J.A.T. and G.L.H. are supported by the Integrated Diffraction Analysis Technologies (IDAT) program, the DOE Office of Biological and Environmental Research plus the National Institutes of Health grant MINOS (Macromolecular Insights on Nucleic Acids Optimized by Scattering) GM105404. A.J.P. is funded by an NIH/NIA training grant in Basic Aging Research and Age-related Disease (T32AG000266) at the Buck Institute for Research on Aging. Work on SOD is supported by NIH grant R01GM039345 (to J.A.T.).

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