Nelly R. Hajizadeh

Consensus Bayesian assessment of protein molecular mass from solution X-ray scattering data

Molecular mass (MM) is one of the key structural parameters obtained by small-angle X-ray scattering (SAXS) of proteins in solution and is used to assess the sample quality, oligomeric composition and to guide subsequent structural modelling. Concentration-dependent assessment of MM relies on a number of extra quantities (partial specific volume, calibrated intensity, accurate solute concentration) and often yields limited accuracy. Concentration-independent methods forgo these requirements being based on the relationship between structural parameters, scattering invariants and particle volume obtained directly from the data. Using a comparative analysis on 165,982xa0unique scattering profiles calculated from high-resolution protein structures, the performance of multiple concentration-independent MM determination methods was assessed. A Bayesian inference approach was developed affording an accuracy above that of the individual methods, and reports MM estimates together with a credibility interval. This Bayesian approach can be used in combination with concentration-dependent MM methods to further validate the MM of proteins in solution, or as a reliable stand-alone tool in instances wherexa0an accurate concentration estimatexa0is not available.

Integrated beamline control and data acquisition for small-angle X-ray scattering at the P12 BioSAXS beamline at PETRAIII storage ring DESY

A versatile C++/Python-based data-acquisition and beamline control system for the P12 small-angle X-ray scattering beamline P12 at PETRAIII (DESY, Hamburg, Germany) is presented.

Probing the Architecture of a Multi-PDZ Domain Protein: Structure of PDZK1 in Solution

The scaffolding protein PDZK1 has been associated with the regulation of membrane transporters. It contains four conserved PDZ domains, which typically recognize a 3-5-residue long motif at the C terminus of the binding partner. The atomic structures of the individual domains are available but their spatial arrangement in the full-length context influencing the binding properties remained elusive. Here we report a systematic study of full-length PDZK1 and deletion constructs using small-angle X-ray scattering, complemented with biochemical and functional studies on PDZK1 binding to known membrane protein partners. A hybrid modeling approach utilizing multiple scattering datasets yielded a well-defined, extended, asymmetric L-shaped domain organization of PDZK1 in contrast to a flexible beads-on-string model predicted by bioinformatics analysis. The linker regions of PDZK1 appear to play a central role in the arrangement of the four domains underlying the importance of studying scaffolding proteins in their full-length context.

Study and mitigation of radiation damage on the P12 BioSAXS beamline

With the progress in X-ray sources and increasing X-ray beam flux, radiation damage is a major challenge for biological SAXS measurements on modern synchrotron beamlines. In macromolecular solutions, radiation damage occurs mostly indirectly. Water molecules are dissociated into hydrogenand hydroxyl-radicals by the X-ray beam. In turn, these free radicals oxidise the macromolecules, which eventually start to aggregate. Since the SAXS intensity scales with the square of the particle size, a few large aggregates may quickly spoil the scattering pattern of the intact molecules. The high brilliance P12 BioSAXS beamline at Petra-3 storage ring (DESY, Hamburg) [1], dedicated and optimised for solution scattering, delivers 5*10^12 photons/s in a 120*200 μm^2 beam (full width half maximum) at the sample position. With this flux, aggregates may be rapidly formed and the data become unusable after exposure of a few tens of ms. Different approaches have been explored to reduce radiation damage [2]: attenuation of the beam, “in-flow” sample measurement, addition of free radicals scavengers or additives that stabilizes proteins and prevent their aggregation. All these methods help to reduce the damage, but all have their limits, and their action can vary depending on the nature of the sample. The “in-flow” measurement, where fresh sample is continuously flowed in the beam path during the exposure, is routinely used at many beamlines and may be the most versatile approach using no additives. On the downside, more sample is required compared to the measurement on a fixed sample. To improve the efficiency of in-flow measurements, radiation sensitive samples were measured in capillaries of different diameter (Schreoer et al., 2017, in preparation). SAXS cells are typically dimensioned such that the signal of aqueous sample is maximised, while the limited lifetime of the sample in the beam and the sample consumption are sometimes ignored. Using smaller capillaries (e.g. 1 mm instead of 2 mm diameter), the scattering signal is still decent and can be detected on low background beamline, but the sample volume required drops significantly (with the square of the diameter). In practice, for a given sample volume, in-flow measurement in small capillaries leads to less noisy data compared to the standard capillaries. This exploration of radiation damage is particularly important in the context of high flux operations on the P12 beamline (Blanchet et al., 2017, in preparation) using the recently commissioned double multilayer monochromator. With the intense flux of 4*10^14 photons/s in 85 x 285 μm^2 at the sample position delivered in this setup, proteins aggregate in a couple of ms and macroscopic effects such as bubble formation and very large aggregates are present in less than a second of exposure. Proper characterization and mitigation of radiation damage are required to make optimal use of this powerful beam.

Status of the EMBL BioSAXS beamline P12 at PETRA III

The high-brilliance synchrotron beamline P12 of the EMBL located at the PETRA III storage ring (DESY, Hamburg) is dedicated to biological small-angle X-ray scattering (SAXS) and has been designed and optimized for scattering experiments on macromolecular solutions [1]. P12 offers both automated sample delivery and data processing capabilities plus tailored sample environments to cater for a diverse user community, spanning novices to experts. During the 2016 beam year, P12 had 240 user visits from across the world. The undulator and double crystal monochromator deliver a beam of energy tunable between 4 and 20 keV with up 1*10^13 photons per seconds focused by bimorph mirrors down to the size of 200μmx100 μm. Recently, a multilayer monochromator has been commissioned increasing the total flux by a factor of 40. High throughput solution SAXS measurements are performed in an in vacuum flow through capillary. The samples are automatically loaded by a robotic sample changer, which also cleans and dries the capillary between measurements. The typical exposure time is one second and the full loading/cleaning cycle finished within 1 minute. The flexible sample-detector-distance offers the option for wide angle scattering. Alternatively, an on-line size exclusion chromatography mode is available with additional spectrometers (UV/Vis, refractive index and MALS) attached for online purification and characterization. For these experiments, particular care was taken to automate the measurements such that they can be performed with a minimal input from the user. Fully automated data collection by the sample changer robot is followed by the computation of the overall parameters of the solute (Rg, p(r), MW and 3D low resolution shape) by the data analysis pipeline SASFLOW within minutes after data collection. This high level of automation allows one to conduct and analyze over 1000 measurements per day and also allows for permit remote and mail-in operation. The sample environment can be rapidly exchanged to conduct “non-standard” SAXS experiments such as scanning SAXS, microfluidic chips, etc. The beamline is further being developed to allow for fast time resolved measurements. With the multilayer monochromator and using the newly installed EIGER 4M detector, data can be collected at 750 Hz frame rate. A stopped flow device, already available at the beamline, allows time resolved data collection with a dead time of a few ms. Continuous flow chip and laser triggering devices are developed to further reduce the dead time and allow sub-ms time resolved SAXS experiments.

Validation of biological small-angle scattering data and models in SASBDB

Small angle scattering (SAS) of X-rays and neutrons is actively used to study the global shapes of proteins, nucleic acids, macromolecular complexes and assemblies in solution. Due to recent advances in instrumentation and computational methods, the quantity of experimental scattering data and subsequent publications is increasing dramatically. Small Angle Scattering Biological Data Bank (SASBDB, www.sasbdb.org, [1]) is a curated repository that makes the experimental SAS data and derived models underlying scientific publications discoverable, accessible and citable. SASBDB allows investigators to locate and freely access: • experimental scattering data presented in various ways (logarithmic plot, Guinier plot, dimensionless Kratky plot, derived pair distance distribution function); • overall parameters derived from the data (radius of gyration, maximum intra-particle distance, molecular weight, excluded volume); • associated models of various types (high resolution models from PDB, hybrid models, ab initio models, mixtures) along with scattering patterns computed from them and fitted to SAS data; • technical details about the experiment; • a priori information about the sample (sequence, expected oligomeric state etc.). The information contained in SASBDB is available via a Web interface and the REST API in a number of formats including XML, JSON and sasCIF, an extension of core Crystallographic Information File for SAS [2]. When the number of entries in SASBDB reached a few hundreds it became obvious that the quality of the data and of the biomacromolecular structures deposited in SASBDB needs to be assessed critically using community-accepted validation methods. We propose a number of formal criteria for evaluating the quality of the experimental data, accuracy of data processing, reliability of the models and the extent to which a given model fits SAS data. Our goal is to collect feedback from the SAS community and ultimately obtain agreement for the approaches to SAS data validation and model validation with the perspective of incorporating into the wwPDB validation reports and setting up publication standard for journals. The development of SASBDB is correlated with the activities and recommendations of the SAS and validation Task Forces of the wwPDB.

Automation and inclusion of additional information for biological solution SAXS

Small angle X-ray scattering (SAXS) of biological macromolecules in solution is widely used to assess the size, volume, shape, and flexibility of proteins in near physiological conditions. SAXS is extremely useful as a standalone technique but also in hybrid investigations where information from other methods such as X-ray crystallography, NMR spectroscopy and electron microscopy is combined. Solution SAXS at high-brilliance facilities often allows a large variety of experiments, from high-throughput data collections with robotic sample changers, continuous-flow in-line sample purification and characterization to time resolved applications and even customized sample environments [1]. A dedicated BioSAXS beamline must be able to robustly collect and real-time analyze the output of high-throughput experiments while accommodating the different sample environments and their unique demands on the beamline configuration. The BECQUEREL user interface at the BioSAXS beamline P12 at PETRA III, DESY, Germany, combines a lean layout with the capabilities of experiment-specific profiles and the power of customizable data collection scripts to allow a user to make the most out of the available beam time. Access levels to hardware devices and their functionality help novice users to avoid accidents while a recommender system guides them through the steps to the next data collection. Power users and beamline scientists are given more complex functionality, including automated alignment operations or manual scans across motor ranges, e.g. to align customized sample environments in the beam. The data collection scripts are designed to minimize dead time during the measurement sequence, e.g. an empty capillary shot is taken while the sample is loading, which allows monitoring of the stability of the instrument, and therefore indirectly the quality of the data. During automated data collection, the user is free to prepare more samples or queue additional measurements. Following data collection, the acquired data is processed by the SASFLOW data analysis pipeline [2,3]. The pipeline is freely configurable and provides real-time feedback about the sample characteristics, enabling the user to modify the sample conditions if necessary. SASFLOW executes the primary processing steps, including absolute calibration using water, after which it automatically evaluates the overall geometrical parameters of the macromolecules and reconstructs an ab initio model. Latest additions include shape classification, ambiguity assessment as well as determination of the useful data range [3]. Furthermore, the pipeline is able to exchange information with the laboratory-information management system ISPyB. This integration allows the user to conveniently pass over the metadata (sample and buffer names and compositions, concentrations) as well as information from other methods, e.g. available high resolution models. The additional information stored in ISPyB can also be used to evaluate model fits conduct analysis of mixture, and also process the data from membrane proteins (presently in preparation). The modularity of SASFLOW facilitates adaptability and portability as recently demonstrated by its adaption at the Shanghai Synchrotron Radiation Facility.

High-flux time-resolved experiments and anomalous scattering at EMBL P12 beamline

Andrey Gruzinov1, Clement E. Blanchet1, Martin A. Schroer1, D.C. Florian Wieland2, Alessandro Spilotros1, Daniel Franke1, Nelly Hajizadeh1, Cy M. Jeffries1, Stefan Fiedler1, Sergey Filippov3, Manfred Roessle4, Gergely Katona5, Dmitri I. Svergun1 1EMBL Hamburg, Hamburg, Germany, 2Helmholtz-Zentrum Geesthacht (Außenstelle DESY), Hamburg, Germany, 3Institute of Macromolecular Chemistry, Prague, Czech Republic, 4Luebeck University of Applied Science, Luebeck, Germany, 5University of Goteborg, Goteborg, Sweden E-mail: agruzinov@embl-hamburg.de

ATSAS 2.8 software for small-angle scattering from macromolecular solutions

Alejandro Panjkovich1, Daniel Franke1, Maxim V. Petoukhov2, Petr V Konarev2, Anne Tuukkanen1, Haydyn DT Mertens1, Al G Kikhney1, Nelly Hajizadeh1, Cy M Jeffries1, Dmitri I Svergun1 1European Molecular Biology Laboratory, Hamburg Outstation, Hamburg, Germany, 2Federal Scientific Research Centre Crystallography and Photonics of Russian Academy of Sciences, Moscow, Russian Federation E-mail: apanjkovich@embl-hamburg.de

High-throughput and time-resolved BioSAXS at the P12 beamline of EMBL Hamburg

Last decades saw a growing interest for SAXS from the structural biology community, underlining the need for dedicated instruments able to rapidly collect accurate SAXS data on weakly scattering, sensitive, and scarce samples. The EMBL BioSAXS beamline P12 (PETRA-III ring, Hamburg) is tailored for biological solution SAXS and offers services to about 100 user groups from the entire world every year. The undulator and double crystal monochromator deliver a beam of energy tunable between 4 and 20keV with up two 1013 photons per seconds focused by bimorph mirrors down to the size of 200x100 μm2. High throughput solution SAXS measurements are performed in an in vacuum flow through capillary. The samples are automatically loaded by a robotic sample changer, which also cleans and dries the capillary between measurements. The typical exposure time is one second and the full loading/cleaning cycle finished within 1 minute. Alternatively, an on-line size exclusion chromatography mode is available with additional spectrometers (UV/Vis, refractive index and RALS) attached for online purification and characterization. For these experiments, particular care was taken to automate the measurements such that they can be performed with a minimal input from the user. Fully automated data collection by the sample changer robot is followed by the computation of the overall parameters of the solute (Rg, p(r), MW and 3D low resolution shape) by the data analysis pipeline SASFLOW within minutes after data collection. This high level of automation allows one to conduct and analyze over 1000 measurements per day and also allows for permit remote and mail-in operation. The sample environment can be rapidly exchanged to conduct “non-standard” SAXS experiments such as scanning SAXS, microfluidic chips, etc. The beamline is further being developed to allow for fast time resolved measurements. A multilayer monochromator, presently in commissioning, delivers the flux 5x1014 photons per seconds allowing for data collection on biological samples within a few ms, and using the newly installed EIGER 4M detector, data can be collected at 750 Hz frame rate. A stopped flow device, already available at the beamline, allows time resolved data collection with a dead time of a few ms. Continuous flow chip and laser triggering devices are developed to further reduce the dead time and allow sub-ms time resolved SAXS experiments. Pilot time-resolved experiments conducted at P12 will be presented.