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


Overview

Experimental method

Sample preparation

Experimental data analysis


Synchrotrons typically use mirrors

Experimental method

X-RaySource

Collimation

Sample

Detector

Collimation


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/


Synchrotron

http://www.aps.anl.gov/About/APS_Overview/


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?


N S=DMAX (qMAX −q MIN) /πMolecules in solution


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


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


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!


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


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




I(q)

P(r) is an inverse Fourier transformation of I(q)


Parsimonious modeling – Lysozyme


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


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?


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

Small Angle X-Ray Solution Scattering of Biological...

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