UV/Visible Light Imaging and BioSAXS Mark Benson Rigaku Europe - - PowerPoint PPT Presentation

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UV/Visible Light Imaging and BioSAXS Mark Benson Rigaku Europe - - PowerPoint PPT Presentation

Oct 26, 2022 227 likes •426 views

UV/Visible Light Imaging and BioSAXS Mark Benson Rigaku Europe Automated Drop Imaging Resolution: What is the smallest size crystal I can detect? Detection: Can I see crystals and any other important events even under difficult

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UV/Visible Light Imaging and BioSAXS Mark Benson Rigaku Europe

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Automated Drop Imaging

  • Resolution: What is the

smallest size crystal I can detect?

  • Detection: Can I see crystals

and any other important events even under difficult conditions (precipitate, membrane screens, etc)?

  • Image analysis: How much

time will I have to spend to score all my images?

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New approach to combined UV/visible imaging

Single optical train – Directly compare images Monochromatic camera – Highest resolution Integrated polarisation Integrated UV source Microscope objectives

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Our Solution: High Resolution and Automated Imaging

  • We implemented 3 types of
  • bjectives in our new imager:
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The Result

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Introducing Fluor-Score: UV based auto- scoring

  • Issue with

conventional image analysis approaches: False negatives (missed crystals)

  • Flour-Score focuses
  • n detecting clear

drops

  • Score = Regional

variations of fluorescence signal

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BioSAXS A Complementary Technique

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High Throughput Structure Success

12.0%4.7% 82.6%

X-ray Crystallography NMR No Structural Information

Northeast Structural Genomics Consortium

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77.3% 22.7%

SAXS No Structural Information

High Throughput SAXS Success

Northeast Structural Genomics Consortium

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Profile conversion

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SAXS pattern

q [Å-1] Log(I)

Guinier plot

q2 [Å-2] ln(I)

Kratky plot

q [Å-1] q2I

Pair distribution function

r [Å] P(r)

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Biological applications of SAXS

  • Predictor for crystallizability
  • Ab initio shape determination of native protein state
  • Monitoring conformational changes
  • Ligand binding studies
  • Characterization of oligomeric states and missing

loops/residues

  • Differentiation of monodisperse and aggregated proteins

in solution

  • Differentiation of folded and unfolded protein in solution
  • Long distance constraint in NMR protein structure

refinement

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Predictor of Protein Crystallizability

Too Soluble “Crystallization Slot” Amorphous Precipitate

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BioSAXS-1000

Source Sample Kratky block Pilatus 100K detector

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BioSAXS-1000 optic design

Licai Jiang, Rigaku Innovative Technology US patent: 7,734,011 B2 worldwide patents pending

X-ray Source 3 pin holes Sample Detector Pin hole SAXS 2D Kratky

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BioSAXS-1000 and Synchrotron Data Quality Comparison

Comparison data: BL4-2 at SSRL(red) and BioSAXS-1000 Blue) SSRL data courtesy of T. Grant, J. Luft and E. Snell (HWI) Before scaling After scaling

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SAXS data collected prior to setting up crystallization experiments can tell you something about the probability of success. Crystallization and X-ray analysis can be an iterative process. SAXS following structure determination can be useful for many situations.

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Thank You www.Rigaku.com