Small Angle X-Ray Solution Scattering of Biological...
Transcript of Small Angle X-Ray Solution Scattering of Biological...
Brookes, E. US Workshop 15 June 2014
Emre Brookes
UltraScan Workshop
15 June 2014
Small Angle X-Ray Solution Scattering of Biological Macromolecules
Brookes, E. US Workshop 15 June 2014
Overview
Experimental method
Sample preparation
Experimental data analysis
Brookes, E. US Workshop 15 June 2014
Synchrotrons typically use mirrors
Experimental method
X-RaySource
Collimation
Sample
Detector
Collimation
Brookes, E. US Workshop 15 June 2014
APS
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 ...
Synchrotron
http://www.aps.anl.gov/About/APS_Overview/
Brookes, E. US Workshop 15 June 2014
Synchrotron
http://www.aps.anl.gov/About/APS_Overview/
Brookes, E. US Workshop 15 June 2014
Monochromatic Beam
Lab X-ray (λ=0.1 – 0.2 nm)
Storage Ring (λ=0.03 – 0.4 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
Experimental method
Why can wedo this?
Brookes, E. US Workshop 15 June 2014
N S=DMAX (qMAX −q MIN) /πMolecules in solution
Brookes, E. US Workshop 15 June 2014
Can study
Protein under physiologic conditions
Time resolved studies
Large protein complexes
Unfolded or partially folded proteins
Complex systems (protein+DNA, protein+lipid etc)N S=DMAX (qMAX −q MIN) /πExperimental method
Brookes, E. US Workshop 15 June 2014
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. 66 1735-82
Shannon channels = Dmax * q-range / π
“the number of [obtainable parameters] typically does not exceed 10–15”
Polydisperse → 1d data will have multiple species
Monodisperse
Maximize IC / species
N S=DMAX (qMAX −q MIN) /πExperimental method
Brookes, E. US Workshop 15 June 2014
Sample Preparation
highly monodisperse
at least 5kD
concentration .25 – 10 mg/ml
volume 20-50 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!
Brookes, E. US Workshop 15 June 2014
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
Brookes, E. US Workshop 15 June 2014
Experimental data analysis
Brookes, E. US Workshop 15 June 2014
Experimental data analysis
Brookes, E. US Workshop 15 June 2014
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
Experimental data analysis
Brookes, E. US Workshop 15 June 2014
Experimental data analysis
I(q)
P(r) is an inverse Fourier transformation of I(q)
Brookes, E. US Workshop 15 June 2014
Experimental data analysis
Brookes, E. US Workshop 15 June 2014
Parsimonious modeling – Lysozyme
Brookes, E. US Workshop 15 June 2014
Conformational variability / FibrinogenMattia 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
Brookes, E. US Workshop 15 June 2014
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?
Brookes, E. US Workshop 15 June 2014
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