Small Angle X-Ray Solution Scattering of Biological Macromolecules

Transcription

1 Small Angle X-Ray Solution Scattering of Biological Macromolecules Emre Brookes UltraScan Workshop 15 June 2014

2 Overview Experimental method Sample preparation Experimental data analysis

3 Experimental method X-Ray Source Sample Collimation Detector Synchrotrons typically use mirrors

4 APS Synchrotron The Electron Storage Ring The 7-GeV electrons are injected into the 1104-m-circumference storage ring, a circle of more than 1,000 electromagnets and associated equipment, located in a radiation-proof concrete enclosure inside the experiment hall. A powerful electromagnetic field focuses the electrons into a narrow beam that is bent on a circular path...

5 Synchrotron

6 Experimental method Monochromatic Beam Lab X-ray (λ= nm) Why can we do this? Storage Ring (λ= nm) Neutron reactor (λ=0.1 1 nm) From the 1d curve we can (under ideal conditions) obtain Radius of gyration Molecular weight Unfolded vs folded P(r) [Pairwise distribution function] gives shape information Low resolution envelope Validation of putative atomic structures

7 Molecules in solution

8 Experimental method Can study Protein under physiologic conditions Time resolved studies Large protein complexes Unfolded or partially folded proteins Complex systems (protein+dna, protein+lipid etc)

9 Experimental method Low information content (IC) in SAS curves Svergun, D.I. & Koch, M.H.J. (2003) Small-angle scattering studies of biological macromolecules in solution. Rep. Prog. Phys Shannon channels = Dmax * q-range / π the number of [obtainable parameters] typically does not exceed Polydisperse 1d data will have multiple species Monodisperse Maximize IC / species

10 Sample Preparation highly monodisperse at least 5kD concentration mg/ml volume ul (higher for small proteins) much more for time-resolved matching buffer (low salt better) most buffer ok (glycerol < 30%, salt < 0.5M) S-reducing agent can help protein stay intact (e.g. DTT) check for oligomerization with concentration or dissociation under dilution simulate shipping conditions (freeze/thaw) and check sample afterwards review with target beamline scientist prior to preparing samples!

11 Sample Preparation check monodispersity single species on native gells SDS-PAGE shows no contamination single symmetric peak on SEC column DLS AUC Mass spec SEC-MALLS Online HPLC-SAXS may help if you can not get a stable monodisperse sample

12 Experimental data analysis

13 Experimental data analysis

14 Experimental data analysis This plot shows s vs log I(s) Some researchers use s instead of q The q-range and associated structural information is shown in the plot

15 Experimental data analysis I(q) P(r) is an inverse Fourier transformation of I(q)

16 Experimental data analysis

17 Parsimonious modeling Lysozyme

18 Conformational variability / Fibrinogen Mattia Rocco et al. Fibrinogen is an important component of the coagulation cascade, as well as a major determinant of blood viscosity and blood flow A centrosymmetric dimer made by 3 pairs of chains US-SOMO/DMD simulations of the conformational variability for comparison to experimental data Images credit: Mattia Rocco

19 Questions How would you view your experimental data to determine if a protein is folded? Approximately what would expect for Dmax of lysozyme? Name 3 reasons a protein might have trouble with SAXS experiments?

20 Hands on SAXS/SANS module. Methods of I(q) vs. q & P(r) vs. r computation from atomic structures and bead models SAXS/SANS module. Comparing computed and experimental data NNLS and best fit operations Advanced topics: HPLC-SAXS peak separation Advanced topics: Parsimonious spacial models Optional analysis of attendees-supplied structures