Lise Arleth

BioXTAS RAW, a software program for high- throughput automated small-angle X-ray scattering data reduction and preliminary analysis

A fully open source software program for automated two-dimensional and one-dimensional data reduction and preliminary analysis of isotropic small-angle X-ray scattering (SAXS) data is presented. The program is freely distributed, following the open-source philosophy, and does not rely on any commercial software packages. BioXTAS RAW is a fully automated program that, via an online feature, reads raw two-dimensional SAXS detector output files and processes and plots data as the data files are created during measurement sessions. The software handles all steps in the data reduction. This includes mask creation, radial averaging, error bar calculation, artifact removal, normalization and q calibration. Further data reduction such as background subtraction and absolute intensity scaling is fast and easy via the graphical user interface. BioXTAS RAW also provides preliminary analysis of one-dimensional data in terms of the indirect Fourier transform using the objective Bayesian approach to obtain the pair-distance distribution function, PDDF, and is thereby a free and open-source alternative to existing PDDF estimation software. Apart from the TIFF input format, the program also accepts ASCII-format input files and is currently compatible with one-dimensional data files from SAXS beamlines at a number of synchrotron facilities. BioXTAS RAW is written in Python with C++ extensions.

Reconciliation of opposing views on membrane–sugar interactions

It is well established that small sugars exert different types of stabilization of biomembranes both in vivo and in vitro. However, the essential question of whether sugars are bound to or expelled from membrane surfaces, i.e., the sign and size of the free energy of the interaction, remains unresolved, and this prevents a molecular understanding of the stabilizing mechanism. We have used small-angle neutron scattering and thermodynamic measurements to show that sugars may be either bound or expelled depending on the concentration of sugar. At low concentration, small sugars bind quite strongly to a lipid bilayer, and the accumulation of sugar at the interface makes the membrane thinner and laterally expanded. Above ∼0.2 M the sugars gradually become expelled from the membrane surface, and this repulsive mode of interaction counteracts membrane thinning. The dual nature of sugar–membrane interactions offers a reconciliation of conflicting views in earlier reports on sugar-induced modulations of membrane properties.

Elliptical Structure of Phospholipid Bilayer Nanodiscs Encapsulated by Scaffold Proteins: Casting the Roles of the Lipids and the Protein

Phospholipid bilayers host and support the function of membrane proteins and may be stabilized in disc-like nanostructures, allowing for unprecedented solution studies of the assembly, structure, and function of membrane proteins (Bayburt et al. Nano Lett. 2002, 2, 853-856). Based on small-angle neutron scattering in combination with variable-temperature studies of synchrotron small-angle X-ray scattering on nanodiscs in solution, we show that the fundamental nanodisc unit, consisting of a lipid bilayer surrounded by amphiphilic scaffold proteins, possesses intrinsically an elliptical shape. The temperature dependence of the curvature of the nanodiscs prepared with two different phospholipid types (DLPC and POPC) shows that it is the scaffold protein that determines the overall elliptical shape and that the nanodiscs become more circular with increasing temperature. Our data also show that the hydrophobic bilayer thickness is, to a large extent, dictated by the scaffolding protein and adjusted to minimize the hydrophobic mismatch between protein and phospholipid. Our conclusions result from a new comprehensive and molecular-based model of the nanodisc structure and the use of this to analyze the experimental scattering profile from nanodiscs. The model paves the way for future detailed structural studies of functional membrane proteins encapsulated in nanodiscs.

A new small-angle X-ray scattering set-up on the crystallography beamline I711 at MAX-lab.

A small-angle X-ray scattering (SAXS) set-up has recently been developed at beamline I711 at the MAX II storage ring in Lund (Sweden). An overview of the required modifications is presented here together with a number of application examples. The accessible q range in a SAXS experiment is 0.009-0.3 A(-1) for the standard set-up but depends on the sample-to-detector distance, detector offset, beamstop size and wavelength. The SAXS camera has been designed to have a low background and has three collinear slit sets for collimating the incident beam. The standard beam size is about 0.37 mm x 0.37 mm (full width at half-maximum) at the sample position, with a flux of 4 x 10(10) photons s(-1) and lambda = 1.1 A. The vacuum is of the order of 0.05 mbar in the unbroken beam path from the first slits until the exit window in front of the detector. A large sample chamber with a number of lead-throughs allows different sample environments to be mounted. This station is used for measurements on weakly scattering proteins in solutions and also for colloids, polymers and other nanoscale structures. A special application supported by the beamline is the effort to establish a micro-fluidic sample environment for structural analysis of samples that are only available in limited quantities. Overall, this work demonstrates how a cost-effective SAXS station can be constructed on a multipurpose beamline.

Structure Parameters of Synaptic Vesicles Quantified by Small-Angle X-Ray Scattering

Synaptic vesicles (SVs) are small, membrane-bound organelles that are found in the synaptic terminal of neurons, and which are crucial in neurotransmission. After a rise in internal [Ca(2+)] during neuronal stimulation, SVs fuse with the plasma membrane releasing their neurotransmitter content, which then signals neighboring neurons. SVs are subsequently recycled and refilled with neurotransmitter for further rounds of release. Recently, tremendous progress has been made in elucidating the molecular composition of SVs, as well as putative protein-protein interactions. However, what is lacking is an empirical description of SV structure at the supramolecular level-which is necessary to enable us to fully understand the processes of membrane fusion, retrieval, and recycling. Using small-angle x-ray scattering, we have directly investigated the size and structure of purified SVs. From this information, we deduced detailed size and density parameters for the protein layers responsible for SV function, as well as information about the lipid bilayer. To achieve a convincing model fit, a laterally anisotropic structure for the protein shell is needed, as a rotationally symmetric density profile does not explain the data. Not only does our model confirm many of the preexisting ideas concerning SV structure, but also for the first time, to our knowledge, it indicates structural refinements, such as the presence of protein microdomains.

Small-angle scattering gives direct structural information about a membrane protein inside a lipid environment.

Monomeric bacteriorhodopsin (bR) reconstituted into POPC/POPG-containing nanodiscs was investigated by combined small-angle neutron and X-ray scattering. A novel hybrid approach to small-angle scattering data analysis was developed. In combination, these provided direct structural insight into membrane-protein localization in the nanodisc and into the protein-lipid interactions. It was found that bR is laterally decentred in the plane of the disc and is slightly tilted in the phospholipid bilayer. The thickness of the bilayer is reduced in response to the incorporation of bR. The observed tilt of bR is in good accordance with previously performed theoretical predictions and computer simulations based on the bR crystal structure. The result is a significant and essential step on the way to developing a general small-angle scattering-based method for determining the low-resolution structures of membrane proteins in physiologically relevant environments.

Gaussian random fields with two level-cuts—Model for asymmetric microemulsions with nonzero spontaneous curvature?

The microstructure of a microemulsion is dominated by the thermodynamics of the surfactant interface between the oil and water domains. As the spontaneous curvature of this surfactant interface is strongly temperature dependent the microstructure of microemulsions also becomes temperature dependent. In the present work we have assumed that the thermodynamics of the interface is determined by the Helfrich Hamiltonian and that the interface can be described by two appropriately chosen level-cuts of a Gaussian random field. It is then possible to express the free energy density of the interface as a functional of the spectral distribution of the Gaussian random field so that the microstructure which minimizes the free energy can be determined by performing a functional minimization of the free energy with respect to the spectral distribution of the Gaussian random field. The two level-cuts are an important feature of the model since they allow us to model microemulsions with nonzero spontaneous curvature and...

Automated microfluidic sample-preparation platform for high-throughput structural investigation of proteins by small-angle X-ray scattering

A new microfluidic sample-preparation system is presented for the structural investigation of proteins using small-angle X-ray scattering (SAXS) at synchrotrons. The system includes hardware and software features for precise fluidic control, sample mixing by diffusion, automated X-ray exposure control, UV absorbance measurements and automated data analysis. As little as 15 µl of sample is required to perform a complete analysis cycle, including sample mixing, SAXS measurement, continuous UV absorbance measurements, and cleaning of the channels and X-ray cell with buffer. The complete analysis cycle can be performed in less than 3 min. Bovine serum albumin was used as a model protein to characterize the mixing efficiency and sample consumption of the system. The N2 fragment of an adaptor protein (p120-RasGAP) was used to demonstrate how the device can be used to survey the structural space of a protein by screening a wide set of conditions using high-throughput techniques.

WillItFit: a framework for fitting of constrained models to small-angle scattering data

A software framework for analysis of small-angle scattering data is presented. On the basis of molecular constraints and prior knowledge of the chemical composition of the sample, the software is capable of simultaneously fitting small-angle X-ray and neutron scattering data to analytical or semi-analytical models of biomacromolecules. The software features various fitting routines along with the possibility of incorporating instrumental resolution effects on the fit. Finally, trust region estimation, based on the profile likelihood strategy, is implemented. The algorithms and models are written in C, whereas the user interface is written in Python. Parallelization is implemented using the OpenMP extensions to C. The source code is available for free upon request or via the associated code repository. The software runs on Linux, Windows and OSX and is available as an open-source initiative published under the General Publishing License.

Recent advances in X-ray compatible microfluidics for applications in soft materials and life sciences

The increasingly narrow and brilliant beams at X-ray facilities reduce the requirements for both sample volume and data acquisition time. This creates new possibilities for the types and number of sample conditions that can be examined but simultaneously increases the demands in terms of sample preparation. Microfluidic-based sample preparation techniques have emerged as elegant alternatives that can be integrated directly into the experimental X-ray setup remedying several shortcomings of more traditional methods. We review the use of microfluidic devices in conjunction with X-ray measurements at synchrotron facilities in the context of 1) mapping large parameter spaces, 2) performing time resolved studies of mixing-induced kinetics, and 3) manipulating/processing samples in ways which are more demanding or not accessible on the macroscale. The review covers the past 15 years and focuses on applications where synchrotron data collection is performed in situ, i.e. directly on the microfluidic platform or on a sample jet from the microfluidic device. Considerations such as the choice of materials and microfluidic designs are addressed. The combination of microfluidic devices and measurements at large scale X-ray facilities is still emerging and far from mature, but it definitely offers an exciting array of new possibilities.