scholarly journals Alpha-synuclein oligomers and fibrils originate in two distinct conformer pools: a small angle X-ray scattering and ensemble optimisation modelling study

2015 ◽  
Vol 11 (1) ◽  
pp. 190-196 ◽  
Author(s):  
Cyril C. Curtain ◽  
Nigel M. Kirby ◽  
Haydyn D. T. Mertens ◽  
Kevin J. Barnham ◽  
Robert B. Knott ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Small Angle ◽  
Alpha Synuclein ◽  
Size Exclusion ◽  
X Ray ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Modelling Study ◽  
Ray Scattering

Size exclusion chromatography with small angle X-ray scattering and ensemble optimisation modelling reveals conformers in random pool of α-synuclein.

scholarly journals Size-exclusion chromatography small-angle X-ray scattering of water soluble proteins on a laboratory instrument

2018 ◽  
Vol 51 (6) ◽  
pp. 1623-1632 ◽  
Author(s):  
Saskia Bucciarelli ◽  
Søren Roi Midtgaard ◽  
Martin Nors Pedersen ◽  
Søren Skou ◽  
Lise Arleth ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Small Angle ◽  
Size Exclusion ◽  
Scattering Data ◽  
X Ray ◽  
Laboratory Instrument ◽  
X Ray Scattering ◽  
Home Institution ◽  
Exclusion Chromatography ◽  
Ray Scattering

Coupling of size-exclusion chromatography with biological solution small-angle X-ray scattering (SEC-SAXS) on dedicated synchrotron beamlines enables structural analysis of challenging samples such as labile proteins and low-affinity complexes. For this reason, the approach has gained increased popularity during the past decade. Transportation of perishable samples to synchrotrons might, however, compromise the experiments, and the limited availability of synchrotron beamtime renders iterative sample optimization tedious and lengthy. Here, the successful setup of laboratory-based SEC-SAXS is described in a proof-of-concept study. It is demonstrated that sufficient quality data can be obtained on a laboratory instrument with small sample consumption, comparable to typical synchrotron SEC-SAXS demands. UV/vis measurements directly on the SAXS exposure cell ensure accurate concentration determination, crucial for direct molecular weight determination from the scattering data. The absence of radiation damage implies that the sample can be fractionated and subjected to complementary analysis available at the home institution after SEC-SAXS. Laboratory-based SEC-SAXS opens the field for analysis of biological samples at the home institution, thus increasing productivity of biostructural research. It may further ensure that synchrotron beamtime is used primarily for the most suitable and optimized samples.

scholarly journals Newly developed Laboratory-based Size exclusion chromatography Small-angle x-ray scattering System (La-SSS)

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Rintaro Inoue ◽  
Tatsuo Nakagawa ◽  
Ken Morishima ◽  
Nobuhiro Sato ◽  
Aya Okuda ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Small Angle ◽  
Size Exclusion ◽  
Scattering System ◽  
X Ray ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Ray Scattering

scholarly journals Methods for analysis of size-exclusion chromatography–small-angle X-ray scattering and reconstruction of protein scattering

2015 ◽  
Vol 48 (4) ◽  
pp. 1102-1113 ◽  
Author(s):  
Andrew W. Malaby ◽  
Srinivas Chakravarthy ◽  
Thomas C. Irving ◽  
Sagar V. Kathuria ◽  
Osman Bilsel ◽  
...  
Keyword(s):  
Linear Combination ◽  
Size Exclusion Chromatography ◽  
Small Angle ◽  
Size Exclusion ◽  
Data Sets ◽  
Saxs Data ◽  
X Ray ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Ray Scattering

Size-exclusion chromatography in line with small-angle X-ray scattering (SEC–SAXS) has emerged as an important method for investigation of heterogeneous and self-associating systems, but presents specific challenges for data processing including buffer subtraction and analysis of overlapping peaks. This paper presents novel methods based on singular value decomposition (SVD) and Guinier-optimized linear combination (LC) to facilitate analysis of SEC–SAXS data sets and high-quality reconstruction of protein scattering directly from peak regions. It is shown that Guinier-optimized buffer subtraction can reduce common subtraction artifacts and that Guinier-optimized linear combination of significant SVD basis components improves signal-to-noise and allows reconstruction of protein scattering, even in the absence of matching buffer regions. In test cases with conventional SAXS data sets for cytochrome c and SEC–SAXS data sets for the small GTPase Arf6 and the Arf GTPase exchange factors Grp1 and cytohesin-1, SVD–LC consistently provided higher quality reconstruction of protein scattering than either direct or Guinier-optimized buffer subtraction. These methods have been implemented in the context of a Python-extensible Mac OS X application known asData Evaluation and Likelihood Analysis(DELA), which provides convenient tools for data-set selection, beam intensity normalization, SVD, and other relevant processing and analytical procedures, as well as automated Python scripts for common SAXS analyses and Guinier-optimized reconstruction of protein scattering.

scholarly journals Adding Size Exclusion Chromatography (SEC) and Light Scattering (LS) Devices to Obtain High-Quality Small Angle X-Ray Scattering (SAXS) Data

Crystals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 975
Author(s):  
Melissa A. Graewert ◽  
Stefano Da Vela ◽  
Tobias W. Gräwert ◽  
Dmitry S. Molodenskiy ◽  
Clément E. Blanchet ◽  
...  
Keyword(s):  
Light Scattering ◽  
Size Exclusion Chromatography ◽  
Small Angle ◽  
Size Exclusion ◽  
Saxs Data ◽  
X Ray ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Set Up ◽  
Ray Scattering

We describe the updated size-exclusion chromatography small angle X-ray scattering (SEC-SAXS) set-up used at the P12 bioSAXS beam line of the European Molecular Biology Laboratory (EMBL) at the PETRAIII synchrotron, DESY Hamburg (Germany). The addition of size exclusion chromatography (SEC) directly on-line to the SAXS capillary has become a well-established approach to reduce the effects of the sample heterogeneity on the SAXS measurements. The additional use of multi-angle laser light scattering (MALLS), UV absorption spectroscopy, refractive index (RI), and quasi-elastic light scattering (QELS) in parallel to the SAXS measurements enables independent molecular weight validation and hydrodynamic radius estimates. This allows one to address sample monodispersity as well as conformational heterogeneity. The benefits of the current SEC-SAXS set-up are demonstrated on a set of selected standard proteins. The processed SEC-SAXS data and models are provided in the Small Angle Scattering Biological Data Bank (SASBDB) and are labeled as “bench-marked” datasets that include the unsubtracted data frames spanning the respective SEC elution profiles and corresponding MALLS-UV-RI-QELS data. These entries provide method developers with datasets suitable for testing purposes, in addition to an educational resource for SAS data analysis and modeling.

scholarly journals Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering

Antibodies ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 48
Author(s):  
Frank Lüdel ◽  
Sandra Bufe ◽  
Willem M. Bleymüller ◽  
Hugo de de Jonge ◽  
Luisa Iamele ◽  
...  
Keyword(s):  
Small Angle ◽  
Antibody Fragment ◽  
Correlation Spectroscopy ◽  
Size Exclusion ◽  
Single Chain ◽  
X Ray ◽  
X Ray Scattering ◽  
Single Chain Fv ◽  
Exclusion Chromatography ◽  
Ray Scattering

Depending on the linker length between the V H and the V L domain, single-chain Fv (scFv) antibody fragments form monomers, dimers (diabodies) or higher oligomers. We aimed at generating a diabody of the anti-MET antibody 3H3 to use it as crystallization chaperone to promote crystallization of the MET ectodomain through the introduction of a pre-formed twofold axis of symmetry. Size exclusion chromatography, however, suggested the protein to be monomeric. Hence, we used scattering techniques applied to solutions to further investigate its oligomerization state. The small angle X-ray scattering (SAXS) curve measured for our protein nicely fits to the scattering curve calculated from the known crystal structure of a diabody. In addition, concentration-dependent photon correlation spectroscopy (PCS) measurements revealed a hydrodynamic radius of 3.4 nm at infinite dilution and a negative interaction parameter k D , indicating attractive interactions that are beneficial for crystallization. Both SAXS and PCS measurements clearly suggest our antibody fragment to be a diabody in solution. Chemical cross-linking with glutaraldehyde and cell motility assays confirmed this conclusion.

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