Naoto  Yagi Hiroshi  Sekiguchi Cheiron School 2014/09/30-10/01 1. Learning what is SAXS ...and WAXS Non-crystalline diffraction …. what is diffraction? what is scattering? Hierarchy in non-crystalline materials Pitfalls in SAXS 2. Visiting three beamlines: BL40XU, BL40B2, BL45XU These three SAXS beamlines in SPring-8 have different x-ray sources and optics.  To have an actual look at these beamlines is a valuable experience. BL40XU: http://www.spring8.or.jp/wkg/BL40XU/instrument/lang-en/INS-0000000353/instrument summary_view BL40B2: http://www.spring8.or.jp/wkg/BL40B2/instrument/lang-en/INS-0000001280/instrument summary  view BL45XU: http://www.spring8.or.jp/wkg/BL45XU/instrument/lang-en/INS-0000000334/instrument summary  view BL03XU
3. Understanding optics for SAXS
Using the above three beamlines as examples, designs of SAXS beamlines are explained. BL40XU: helical undulator --- double focusing mirrors Pink beam! BL40B2: bending magnet --- double crystal monochromator --- bent cylindrical mirror BL45XU: tandem vertical undulators --- double crystal diamond monochromator --- double focusing mirrors BL03XU: undulator --- double crystal Si monochromator --- double focusing mirrors Other beamlines: BL20XU and beamlines in other facilities.
4. Understanding detectors for SAXS
Several different types of detectors are used at t the above three beamlines. Apart from basic detectors such as ion chambers, they are all area detectors. RAXIS: image plate detector X-ray image intensifier + CCD camera: high sensitivity and fast readout CMOS flatpanel: solid-state area imager PILATUS: photon-counting pixel detector 5. Protein solution scattering measurements at BL40B2 A protein solution scattering experiment will be conducted at BL40B2. The target protein is calmodulin, and its conformational change upon binding Ca²+ ion and a peptide will be analyzed by SAXS. messenger protein expressed in most eukaryotic cells. Calmodulin  acts  as an intermediary   protein   that senses the calcium level and relays the signal to various calcium-sensitive  enzymes, ion  channels  and  other proteins. Calmodulin is a dumbbell-shaped protein composed of two globular domains connected together by a linker (Fig. A). Each domain binds two calcium ions as shown in Fig. B. The linker between the domains is a long alpha helix. Calmodulin typically wraps around its target peptide, with the two globular domains gripping either side of it (Fig. C). Brief introduction of calmodulin is in the following website (Protein Data Bank, Molecule of the Month, http://www.rcsb.org/pdb/101/motm.do?momID=44 ). Mastoparan is used as a target peptide for calmodulin in this beamline practical. It is a peptide toxin from wasp venom, and its amino acid sequence is following, Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu. SAXS measurement will be obtained under the following condition, Wavelength of incident X-ray: 1 Å, Sample-to-detector distance 1150mm, Exposure time: 30 sec. Since scattering angle from the sample depends on the size of the molecule and the wavelength of the incident X-ray, the sample-to-detector distance has to be set accordingly. The exposure time has to be determined by considering the quality of data and the X-ray radiation damage on the sample (X-ray radiation damage is common problem in SAXS measurement of proteins.). The actual sample-to-detector distance is calibrated by irradiating a reference sample of known structure. We usually used silever behenate as a reference sample (d oo = 58.38 Å). In the first step, we will obtain the scattering from calmodulin solution in the absence of Ca 2+ ion and mastoparan under different calmodulin concentrations, such as 5.0 mg/ml, 2.5 mg/ml, 1.25 mg/ml, in Buffer A. Scattering from Buffer A will be measured on every calmodulin measurement to obtain the scattering of calmodulin (When we have obtained two experimental scattering profiles, one from the protein solution and one from the buffer, we subtract one from the other to get the scattering of protein only.) Buffer A 50mM Tris-HCl pH=7.2, 100mM NaCl, 1mM EDTA Next, we will obtain the scattering from calmodulin in the presence of Ca 2+ ion and mastoparan under different calmodulin concentrations, such as 5.0 mg/ml, 2.5 mg/ml, 1.25 mg/ml, in Buffer B. Buffer B 50mM Tris-HCl pH=7.2, 100mM NaCl, 1mM EDTA, 5mM CaCl2, 675µM Mastoparan Calmodulin, molecular weight=16706 10mg/mL=599μM Mastoparan, molecular weight =1479 lmg/mL=676μM 6. Practicing data analysis Introduction to widely used SAXS data processing software (fit2D, PRIMUS, etc.) Important formulae: 20 is the scattering angle.
Guinier Plot ... Rg is radius of gyration q ´ R ²
Pair distribution function … Fourier transform of autocorrelation function