Online ion-exchange chromatography for small-angle X-ray scattering

Hutin, Stephanie; Brennich, Martha; Maillot, Benoit; Round, Adam

doi:10.1107/s2059798316012833

Cited by 22 publications

(14 citation statements)

References 43 publications

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“…SAXS data were collected at the ESRF beamline BM29 on a Pilatus 2M detector (Dectris) using the standard setup for size-exclusion chromatography coupled SAXS mode 62 . A Superdex 200 column (GE Healthcare) connected to an HPLC system (Shimadzu) was used for size-exclusion chromatography to separate aggregates from monodisperse samples.…”

Section: Methodsmentioning

confidence: 99%

Regulator-dependent mechanisms of C3b processing by factor I allow differentiation of immune responses

Xue

Ricklin

et al. 2017

Nat Struct Mol Biol

102

120

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The complement system labels microbes and host debris for clearance. Degradation of surface-bound C3b is pivotal to direct immune responses and protect host cells. How the serine protease factor I (FI), assisted by regulators, cleaves either two or three distant peptide bonds in the CUB domain of C3b remains unclear. We present a crystal structure of C3b in complex with FI and regulator factor H (FH; domains1–4 with 19–20). FI binds C3b–FH between FH domains 2 and 3 and a reoriented C3b C-terminal domain and docks onto the first scissile bond, while stabilizing its catalytic domain for proteolytic activity. One cleavage in C3b does not affect its overall structure, whereas two cleavages unfold CUB and dislodge the thioester-containing domain (TED), affecting binding of regulators and thereby determining the number of cleavages. These data explain how FI generates late-stage opsonins iC3b or C3dg in a context-dependent manner, to react to foreign, danger or healthy self signals.

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Section: Methodsmentioning

confidence: 99%

Regulator-dependent mechanisms of C3b processing by factor I allow differentiation of immune responses

Xue

Ricklin

et al. 2017

Nat Struct Mol Biol

102

120

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“…In conclusion, online ion-exchange and online size-exclusion chromatography are important biochemical purification methods that can be coupled directly to SAXS 6,7,[9][10][11][12]25,26 . The background subtraction of IEC-SAXS data is slightly more difficult and ambiguous than for SEC-SAXS, but it is nevertheless possible.…”

Section: Discussionmentioning

confidence: 99%

“…Follow step 4.3 to 4.5 to align the models and to identify the most representative one. 12 on the representative models of the SEC (step 4.5) and IEC-SAXS data (step 5.9) with P6 symmetry: supcomb model_sec.pdb model_iec.pdb 2. Import the model_sec.pdb and the model_iec-r.pdb into a molecular visualization software.…”

Section: Iec-saxs Data Reduction Analysis and Modelingmentioning

confidence: 99%

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Brennich¹,

Round²,

Hutin³

2017

JoVE

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Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC-and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

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“…We observed that the use of SEC alone can provide information on the protein aggregation state as molecules approached known precipitation points in their phase diagrams, which is confirmed by the associated in-line SLS measurements. Although chromatographic analyses are nowadays implemented into different characterization methodologies [22][23][24][25][26] and at different beamline facilities [27][28][29][30][31], facilitating the determination of precise information for protein samples at the preparatory stage would certainly improve the efficiency of the results obtained through subsequent characterization methodologies.…”

Section: Introductionmentioning

confidence: 99%

The Use of Size Exclusion Chromatography to Monitor Protein Self-Assembly

Adawy

Groves

2017

Crystals

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High resolution size exclusion chromatography (SEC) coupled with static light scattering (SLS) analyses were conducted to study the effect of the mobile phase ionic strength and protein concentration on the output of SEC experiments. The results highlight the effect of small changes in the mobile phase composition on the estimation of molar masses estimated from retention time-based calibration curve compared with those obtained from SLS analysis. By comparing the SLS data with the SEC chromatograms, we show that SEC can provide helpful information on the protein aggregation state as macromolecules approach known precipitation points in their phase diagrams. This suggests the potential use of SEC as an easily accessible lab-based scanning methodology to monitor protein self-assembly prior to nucleation and crystallization. Implications for the use of SEC to study protein phase diagrams are discussed.

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Online ion-exchange chromatography for small-angle X-ray scattering

Abstract: SAXS coupled with online ion-exchange chromatography allows the collection of high-quality BioSAXS data.

Cited by 22 publications

References 43 publications

Regulator-dependent mechanisms of C3b processing by factor I allow differentiation of immune responses

Regulator-dependent mechanisms of C3b processing by factor I allow differentiation of immune responses

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

The Use of Size Exclusion Chromatography to Monitor Protein Self-Assembly

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