Alessandro Spilotros

Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY)

An integrated environment for biological small-angle X-ray scattering (BioSAXS) at the high-brilliance P12 synchrotron beamline of the EMBL (DESY, Hamburg) allows for a broad range of solution scattering experiments. Automated hardware and software systems have been designed to ensure that data collection and processing are efficient, streamlined and user friendly.

Progress in small-angle scattering from biological solutions at high-brilliance synchrotrons

Small-angle X-ray scattering (SAXS) is an established technique that provides low-resolution structural information on macromolecular solutions. This review illustrates the latest progress in the field, highlighting analytical methods for flexible and evolving systems, progress in time-resolved SAXS and developments in archiving and validation.

Multicore iron oxide mesocrystals stabilized by a poly(phenylenepyridyl) dendron and dendrimer: role of the dendron/dendrimer self-assembly.

We report the formation of multicore iron oxide mesocrystals using the thermal decomposition of iron acetyl acetonate in the presence of the multifunctional and rigid poly(phenylenepyridyl) dendron and dendrimer. We thoroughly analyze the influence of capping molecules of two different architectures and demonstrate for the first time that dendron/dendrimer self-assembly leads to multicore morphologies. Single-crystalline ordering in multicore NPs leads to cooperative magnetic behavior: mesocrystals exhibit ambient blocking temperatures, allowing subtle control over magnetic properties using a minor temperature change.

Virus Matryoshka: A Bacteriophage Particle—Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus

Immature human immunodeficiency virus type 1 (HIV-1) is approximately spherical, but is constructed from a hexagonal lattice of the Gag protein. As a hexagonal lattice is necessarily flat, the local symmetry cannot be maintained throughout the structure. This geometrical frustration presumably results in bending stress. In natural particles, the stress is relieved by incorporation of packing defects, but the magnitude of this stress and its significance for the particles is not known. In order to control this stress, we have now assembled the Gag protein on a quasi-spherical template derived from bacteriophage P22. This template is monodisperse in size and electron-transparent, enabling the use of cryo-electron microscopy in structural studies. These templated assemblies are far less polydisperse than any previously described virus-like particles (and, while constructed according to the same lattice as natural particles, contain almost no packing defects). This system gives us the ability to study the relationship between packing defects, curvature and elastic energy, and thermodynamic stability. As Gag is bound to the P22 template by single-stranded DNA, treatment of the particles with DNase enabled us to determine the intrinsic radius of curvature of a Gag lattice, unconstrained by DNA or a template. We found that this intrinsic radius is far larger than that of a virion or P22-templated particle. We conclude that Gag is under elastic strain in a particle; this has important implications for the kinetics of shell growth, the stability of the shell, and the type of defects it will assume as it grows.

Formation of a Secretion-Competent Protein Complex by a Dynamic Wrap-around Binding Mechanism

Bacterial virulence is typically initiated by translocation of effector or toxic proteins across host cell membranes. A class of gram-negative pathogenic bacteria including Yersinia pseudotuberculosis and Yersinia pestis accomplishes this objective with a protein assembly called the type III secretion system. Yersinia effector proteins (Yop) are presented to the translocation apparatus through formation of specific complexes with their cognate chaperones (Syc). In the complexes where the structure is available, the Yops are extended and wrap around their cognate chaperone. This structural architecture enables secretion of the Yop from the bacterium in early stages of translocation. It has been shown previously that the chaperone-binding domain of YopE is disordered in its isolation but becomes substantially more ordered in its wrap-around complex with its chaperone SycE. Here, by means of NMR spectroscopy, small-angle X-ray scattering and molecular modeling, we demonstrate that while the free chaperone-binding domain of YopH (YopHCBD) adopts a fully ordered and globular fold, it populates an elongated, wrap-around conformation when it engages in a specific complex with its chaperone SycH2. Hence, in contrast to YopE that is unstructured in its free state, YopH transits from a globular free state to an elongated chaperone-bound state. We demonstrate that a sparsely populated YopHCBD state has an elevated affinity for SycH2 and represents an intermediate in the formation of the protein complex. Our results suggest that Yersinia has evolved a binding mechanism where SycH2 passively stimulates an elongated YopH conformation that is presented to the type III secretion system in a secretion-competent conformation.

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.

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

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.