Small Angle Scattering (SAXS/SANS) Small Angle Scattering - PowerPoint PPT Presentation

European Molecular Biology Laboratory Hamburg Outstation European Molecular Biology Laboratory Hamburg Outstation European Molecular Biology Laboratory Hamburg Outstation European Molecular Biology Laboratory Hamburg Outstation BioSAXS group BioSAXS group BioSAXS group BioSAXS group Small Angle Scattering (SAXS/SANS) Small Angle Scattering (SAXS/SANS) Small Angle Scattering (SAXS/SANS) Small Angle Scattering (SAXS/SANS) data reduction and overall parameters data reduction and overall parameters data reduction and overall parameters data reduction and overall parameters Dr. Weifeng Shang 尚伟峰 Dr. Weifeng Shang Dr. Weifeng Shang Dr. Weifeng Shang

Outline Outline Outline Outline 1 Sample preparation 1 Sample preparation 1 Sample preparation 1 Sample preparation 2 SAXS in theory 2 SAXS in theory 2 SAXS in theory 2 SAXS in theory 3 SAXS in practice 3 SAXS in practice 3 SAXS in practice 3 SAXS in practice 4 Data collection 4 Data collection 4 Data collection 4 Data collection 5 Data reduction 5 Data reduction 5 Data reduction 5 Data reduction 6 Overall parameters 6 Overall parameters 6 Overall parameters 6 Overall parameters

Sample preparation Sample preparation Sample preparation Sample preparation Exact sequence sequence sequence sequence ---> predicted molecular mass molecular mass molecular mass molecular mass True concentration True concentration True concentration True concentration ( extinction coefficient, absorbance at 280 nm ) Mono-disperse samples Mono-disperse samples Mono-disperse samples Mono-disperse samples ( precipitate, SDS-PAGE, GF/SE chromatography, DLS, AUC, mass spectrometer )

Buffer preparation Buffer preparation Buffer preparation Buffer preparation Matched buffer Matched buffer Matched buffer Matched buffer ( the exact buffer at dialysis equilibrium with the sample ) ( buffer exchange, dialysis ) Good buffer Good buffer Good buffer Good buffer 1. Tris, Hepes, PBS, HBS, etc. 2. salts, additives, small molecules, DTT 3. detergents, < CMC

SAXS in theory SAXS in theory SAXS in theory SAXS in theory

SAXS in practice SAXS in practice SAXS in practice SAXS in practice

Data collection Data collection Data collection Data collection Commonly used detectors for SAXS (0.15 nm, 8.3 keV) Commonly used detectors for SAXS (0.15 nm, 8.3 keV) Commonly used detectors for SAXS (0.15 nm, 8.3 keV) Commonly used detectors for SAXS (0.15 nm, 8.3 keV) 1. Position sensitive gas-filled proportional counter 1. Position sensitive gas-filled proportional counter 1. Position sensitive gas-filled proportional counter 1. Position sensitive gas-filled proportional counter 2. PILATUS detector 2. PILATUS detector 2. PILATUS detector 2. PILATUS detector 3. Image plate 3. Image plate 3. Image plate 3. Image plate 4. CCD detector 4. CCD detector 4. CCD detector 4. CCD detector

Data reduction Data reduction Data reduction Data reduction

Data reduction Data reduction Data reduction Data reduction

Data reduction Data reduction Data reduction Data reduction

Data reduction Data reduction Data reduction Data reduction

Data reduction Data reduction Data reduction Data reduction 1 (buffer, sample, buffer) sequence 1 1 (buffer, sample, buffer) sequence 1 (buffer, sample, buffer) sequence (buffer, sample, buffer) sequence 2 2 2 Radial integration (2D 1D) 2 Radial integration (2D 1D) Radial integration (2D 1D) Radial integration (2D 1D) 3 3 Check for radiation damage Check for radiation damage 3 3 Check for radiation damage Check for radiation damage 4 4 Average frames Average frames 4 4 Average frames Average frames 5 Buffer averaging 5 Buffer averaging 5 Buffer averaging 5 Buffer averaging 6 Buffer subtraction 6 Buffer subtraction 6 Buffer subtraction 6 Buffer subtraction

Data reduction Data reduction Data reduction Data reduction

Data reduction Data reduction Data reduction Data reduction: buffer averaging : buffer averaging : buffer averaging : buffer averaging

Data reduction Data reduction Data reduction Data reduction: buffer averaging : buffer averaging : buffer averaging : buffer averaging

Data reduction Data reduction Data reduction Data reduction: buffer subtraction : buffer subtraction : buffer subtraction : buffer subtraction

Data reduction Data reduction Data reduction Data reduction: buffer subtraction : buffer subtraction : buffer subtraction : buffer subtraction

Overall parameters: Rg Overall parameters: Rg Overall parameters: Rg Overall parameters: Rg

Overall parameters: AutoRg Overall parameters: AutoRg Overall parameters: AutoRg Overall parameters: AutoRg

Overall parameters: Porod Volume Overall parameters: Porod Volume Overall parameters: Porod Volume Overall parameters: Porod Volume

Overall parameters: GNOM Overall parameters: GNOM Overall parameters: GNOM Overall parameters: GNOM

Overall parameters: p(r) Overall parameters: p(r) Overall parameters: p(r) Overall parameters: p(r)

Overall parameters: p(r) Overall parameters: p(r) Overall parameters: p(r) Overall parameters: p(r)

Overall parameters: p(r) Overall parameters: p(r) Overall parameters: p(r) Overall parameters: p(r)

Overall parameters: Dmax Overall parameters: Dmax Overall parameters: Dmax Overall parameters: Dmax

Overall parameters: Dmax Overall parameters: Dmax Overall parameters: Dmax Overall parameters: Dmax

Overall parameters: Dmax Overall parameters: Dmax Overall parameters: Dmax Overall parameters: Dmax

SAXS pipeline SAXS pipeline SAXS pipeline SAXS pipeline