BioSAXS special applications Martin A. Schroer EMBL Hamburg - - PowerPoint PPT Presentation
BioSAXS special applications Martin A. Schroer EMBL Hamburg Examples for bioSAXS experiments Disclaimer Might not work for all samples Might not make sense for all samples You will likely need more sample solution however
Examples for bioSAXS experiments
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Disclaimer
however
Contact the beamline scientists!
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Robotic sample changer SEC-SAXS/MALS
Stopped Flow Microfluidics (e.g. for THz) Laser excitation
28/02/2020
detection system
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Air gap
Higher X-ray absorption by air Higher background signal (air + windows + sample cell)
In vacuum:
In air
Try to reduce the air gap as much as possible! Use proper window material!
+ BSA + buffer + buffer + BSA + buffer + buffer
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Sample changer temperature range:
Temperature controlled capillary holder
User setup
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Example:
27/05/2020 8
Need High pressure sample cells
Allows to study protein stability
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High pressure effects
⇒ reduced constrast as water gets compressed
⇒ unfolding Guinier plot
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p [bar] p [bar]
Cosolvents can change the pressure – effect
Urea
TMAO
with sea depth Recently more studies (several protein, tRNA, microtubuli) but still more to be explored: terra incognita
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molecules; is present in joints and blood flow
essential part of the extracelluar matrix (joints) Rheo-SAXS: Hyaluronan chain network gets more ordered
D.C.F. Wieland et al., J. Syn. Rad. 24, 646 (2017).
Hyaluronan: 750 kDa 0 - 1500 1/s
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Light can induce different types of reactions
Example:
Tidow group (Hamburg) @ ID09, ESRF
hutch experiments)
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To fibre Direct
Energy per pulse [mJ]
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THz radiation
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Fröhlich‘s prediction
moment (Fröhlich condensation) Such collective vibrations (normal modes) may lead to long-range conformational changes. Such changes can be probed by SAXS. THz excitation & SAXS probe
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Such a noval type of experiment needs
Setup I Setup II
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Combined THz-SAXS measurements demand dedicated sample environment
enough sample excitation
3D printed Polystyrene cell
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Setup installed at P12
The same stop of the sample is illuminated X-ray THz
have been performed on cells
geometrical model
different antibiotics
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E.F Semeraro et al., IUCrJ 4, 751 (2017). A.R. v. Gundlach et al., BBA - Biomembranes 1858, 918 (2016).
Real space maps of scattering intensity
crystals)
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D.C.F. Wieland et al., Acta Biomaterialia 25, 339 (2015).
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Radiolysis of water (99 % of sample volume) → radical formation → interactions with solvent accessible sites → formation of large aggregates
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C.M. Jeffries et al., J. Syn. Rad. 22, 273 (2015).
Radiation damage limits data collection of biological samples For SAXS: Aggregates spoil signal of undamaged proteins
2 H2O + X-ray → H3O+ + •OH + e-
aq
e-
aq + O2 → HO 2
S.D. Maleknia et al., Anal. Biochem. 289, 103 (2001).
Problem for SAXS: Damage not (easy) predictable Different schemes to reduce the effect of radiation damage
(DTT, ascorbic acid; glycerol)
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Coflow
Cryo-cooling
S.P. Meisburger et al., Biophys.
High flux
M.A.Schroer et al., J. Synchrotron Rad. 25, 1113 (2018)
Cell geometry
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Proteins are scattering weakly
However:
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ATP
Josts et al, Structure 28, 348 (2020).
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Continuous change of Rg
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Continuous change of Rg
Continuous change of Rg But NOT overlapping Radiation Damage
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Continuous change of Rg
Continuous change of Rg But NOT overlapping Radiation Damage
Pump – probe scheme
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Continuous change of Rg
Continuous change of Rg But NOT overlapping Radiation Damage
Pump – probe scheme
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No unneeded exposure of sample Example:
+ Allows to determine the kinetics/reaction
Modern BioSAXS experiments
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32 21/02/2019