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Malvern PANalytical
Characterizing Biological
Macromolecules by SAXSDetlef Beckers, Jörg Bolze, Bram Schierbeek, PANalytical B.V., Almelo, The Netherlands
This document was presented at PPXRD -Pharmaceutical Powder X-ray Diffraction Symposium
Sponsored by The International Centre for Diffraction Data
This presentation is provided by the International Centre for Diffraction Data in cooperation with the authors and presenters of the PPXRD symposia for the express purpose of educating the scientific community.
All copyrights for the presentation are retained by the original authors.
The ICDD has received permission from the authors to post this material on our website and make the material available for viewing. Usage is restricted for the purposes of education and scientific research.
ICDD Website - www.icdd.comPPXRD Website – www.icdd.com/ppxrd
Malvern PANalytical
Empyrean Nano edition
Atomic PDF analysis
Ultra-SAXS
GISAXS
Transmission diffraction
Powder diffraction
v
v
SAXS / WAXS
Bio-SAXS
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
Empyrean Nano edition
160
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
Protein powder screening and purity
Crystal
system:
Tetragonal
a (Å) 79.120(3)
c (Å) 37.935(1)
V (Å3) 237474(6)
Tetragonal P43212
Crystal
system:
Monoclinic
a (Å) 28.20(2)
b (Å) 54.50(1)
c (Å) 71.68(4)
(o) 95.495(3)
V (Å3) 109660(60)
Monoclinic P1211 (*)
Pawley fit within HighScore Plus
(*) Highest probability
acc. ExtSym
Malvern PANalytical
The X-rays interact with the electrons in the sample.
Image taken from Glatter & Kratky, "Small Angle X-ray Scattering", Academic Press, 1982
• Electrons oscillate in the electric field of the X-rays
• They emit secondary, coherent waves that interfere
with each other
• For X-rays, all electrons can be treated as free
electrons (cf. light scattering: only outer electrons
scatter).
λ1
λ2
λ1 = λ2
X-r
ay s
ourc
e
The interference pattern is measured at small angles 2θ,
very close to the direct beam.
Thomson scattering
The scattering process
Image taken from Glatter & Kratky,
"Small Angle X-ray Scattering", Academic Press, 1982
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
SAXS from spheres of different size
10-6
10-4
10-2
100
0 0.2 0.4 0.6 0.8 1.0
10 20 40
Particle Radius [nm]
q [nm-1
]
I(q
) /
I(q
=0
)
R = 40 nm
R = 20 nm
R = 10 nm
P(q
) =
I(q
) / I(
q=
0)
Part
icle
form
facto
r P
(q)
Scattering vector q [nm-1]
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
Trimodal particle size distribution
Data analysis was done without any assumptions
about the shape or modality of the size distribution curve.
Background-corrected data
Particle size distribution
Malvern PANalytical
Pair distance distribution function p(r)
for different particle shapes
0
0.25
0.50
0.75
1.00
0 10 20 30
r [nm]
p(r
)
Model Calculation p(r) functions
0
0.25
0.50
0.75
1.00
0 10 20 30
r [nm]p
(r)
Model Calculation p(r) functions
0
0.25
0.50
0.75
1.00
0 10 20 30
r [nm]
p(r
)
Model Calculation p(r) functions
Intraparticle distance r [nm] Intraparticle distance r [nm]Intraparticle distance r [nm]
p(r) p(r) p(r)
Overall spherical Elongated Hollow
Dmax DmaxDmax
Dmax Dmax Dmax
Model calculations
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
scattering vector q [nm-1]
BioSAXS data analysis
Small-Angle Scattering Biological Data Bank, Valentini et al., Nucl Acids Res (2014) 43, D357
Log
Inte
nsi
ty I experimental
SAXS data
q [nm-1]
Kratky plotIq2
compactnessfolding/unfolding
sin
4q
λ wavelength of X-rays
2θ scattering angle
Guinier plotln(I)
q2 [nm-2]
Rg
I(0)
Mw
Radius of gyration
Pair distance distribution fct.
intraparticle distance r [nm]
p(r)
FT
Dmax
overall shape &
max. dimension
FT-1
scattering vector q [nm-1]
Log
Inte
nsi
ty I experimental
SAXS data
Malvern PANalytical
SAXS setup
X-ray tube
X-ray mirror
Sample Area
detector
Evacuated beam path
WAXS
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
Advantages of SAXS on proteins
• Protein characterization can be done in situ, with the proteins in solution
and under near physiological conditions.
• Effects of e.g. pH, salt concentration, temperature, added ligand
can be systematically studied.
• Easy sample preparation as compared to e.g. cryo-EM or SC-XRD.
• Also applicable in case protein doesn’t crystallize!
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
Information obtainable from Bio-SAXS experiments (I)
Radius of gyration Rg : overall size parameter
Dmax : maximum dimension
Overall shape: e.g. overall spherical vs. elongated
Molecular weight: differentiate between oligomeric forms
3D envelope shape
DAMMIN, DAMMIF
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
Information obtainable from Bio-SAXS experiments (II)
folded unfoldedDegree of compactness / flexibility
- Protein folding / unfolding
Protein stability
- Detect protein aggregation
- Differentiate repulsive and attractive protein-protein interactions
Protein dynamics- Time-resolved measurements
Validation of atomic structures
- Protein in crystal vs. in solution
CRYSOL
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
Protein size shape and structure
102
103
104
105
106
107
0 1 2 3
measurement time: 60 min
sample
buffer
differential
Inte
ns
ity
[co
un
ts]
Scattering vector q [nm-1]
Apoferritin (12 mg/ml)
An iron storage protein.
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
Apoferritin - Guinier plot
Rg = 5.9 nm
Scattering vector q2 [nm-2]
ln I
10.75
11.00
11.25
0.02 0.03 0.04ln
I
103
104
105
0 0.5 1.0 1.5
Scattering vector q [nm-1]
Inte
ns
ity
[co
un
ts]
No aggregates
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
00 5 10
Apoferritin - Pair distance distribution function p(r)
Intraparticle distance r [nm]
p(r)
Dmax
13nm
Rg = 5.3 nm
The characteristic shape of the p(r) function
points to an object with a hollow structure.
102
103
104
105
0 1 2 3
experimental data
back-transform of p(r)
Scattering vector q [nm-1]
Inte
ns
ity
[co
un
ts]
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
Apoferritin - hollow sphere model
Hollow sphere model(*):
Rcore = 37 Å
dshell = 24 Å
(*) PM Harrison, The structure of apoferritin: molecular size, shape and symmetry from x-ray data, J. Molec. Biol. 6 (1963), 404-22
From the X-ray data apoferritin molecules have a molecular weight of 480,000 and a form approximating on the average at a
resolution of 26 Å to a spherical shell having an external radius of 61 ± 3 Å and internal to external radius ratio about 0.6.
102
103
104
105
0 1 2 3
q [nm-1
]
Inte
nsity [
a.u
.]
solid sphere
hollow sphere
Scattering vector q [nm-1]
Inte
ns
ity
[co
un
ts]
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
3D protein shape reconstruction (ab initio)
start end
cross-sectional
structure
front view
DAMMIF / DAMMIN*(*) D. Franke, D.I. Svergun et al., J. Appl. Cryst. 50, (2017)z
Inverse cross-sectional
structure.
Beads indicate the
location of the buffer
Malvern PANalytical
Protein structure in crystal form vs. in solution
102
103
104
105
106
0 1 2 3
PDB 1IER
CRYSOL
Scattering vector q [nm-1]
Inte
ns
ity
[co
un
ts] experimental data
simulated from atomic model
Rg = 5.3 nm (experiment)
Rg = 5.4 nm (atomic model)atomic
structurelow-resolution
structure
hydration
layer added
Calculation of SAXS data
from published atomic coordinates.
The crystal structure of the protein is similar to its structure in solution.
Malvern PANalytical
Glucose isomerase (11 mg/ml)
ScatterX78
102
103
104
105
106
0 1 2 3
protein solution
pure buffer
differential
t = 60 min
Int.
[a.u.]
q [nm-1
]
Inte
nsi
ty [
a.u
.]
100 mM Tris, 1 mM MgCl2, pH = 8
• Enzyme produced by
microorganisms (bacteria)
• Catalyzes the conversion of
glucose to fructose
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Glucose isomerase
no aggregation
Guinier plot Pair distance distribution function
Rg: radius of gyration
Dmax: maximum dimension of protein
Dmax
overall symmetrical
protein shape
Empyrean Nano (10 min)
Empyrean Nano (60 min)
Synchrotron
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
Simulation of SAXS data from SC-XRD data
and comparison with experimental data
CRYSOL software1
Conclusion:
In solution the protein
is forming a tetramer.
The structures in the crystal
and in solution are similar.
1Svergun D.I., Barberato C. and Koch M.H.J. (1995) CRYSOL - a Program to Evaluate X-ray Solution Scattering of Biological
Macromolecules from Atomic Coordinates J. Appl. Cryst. , 28, 768-773.
Glucose isomerase
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Oligomeric mixtures – Bovine Serum Albumin (BSA)
Bovine serum albumin (BSA), 10 mg/ml in 50 mM
Hepes, 50 mM NaCl, pH = 7.5, T = 20 °C
SAXS data for monomer and dimer
were simulated from the published
atomic structures (using CRYSOL).
Protein Data Bank PDB 4F5S
Experimental data
100% monomer
100% dimer
50% monomer, 50% dimer
106
107
0 1 2 3
q
BSA in Hepes 10mg/ml
measurement time: 60 min
Inte
nsit
y[a
.u.]
Scattering vector q [nm-1]
© 2017 PANalytical B.V. All rights reserved.
Malvern PANalytical
Protein folding / unfolding - BSA in Hepes buffer
In pure buffer (no urea):
folded,
globular particle
folded unfolded
In buffer with 9M urea:
unfolded,
dissolved polymer chain
Scattering vector q [nm-1]
Iq2
no urea
9M urea
Kratky plot (Iq2 vs. q)
0
2x105
4x105
0 0.5 1.0 1.5 2.0 2.5
Int. prop. q-4
Int. prop. q-2
SAXS measurements were done without and with added urea (known to be a denaturant).
Confirmed by Rg determination
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Malvern PANalytical
Liposomes
Multi-lamellar
vesicle
Uni-lamellar
vesicle
• Model biomembrane,
can be further functionalized
and structure / properties
can systematically be studied
• Use as drug carrier
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Lipid phase transition temperature
fluid phase
solid phase
("gel phase")
Lipid bilayer structure Alkyl chain orderingtemperature
phase transition
temperature Tm
fully extended,
closely packed,
slightly tilted
alkyl chains
randomly oriented,
fluid alkyl chains
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Malvern PANalytical
SAXS / WAXS on liposomes
Test sample:
Phospholipid DPPC (25 mg/ml in a PBS
buffer), before extrusion.
- Forming multilamellar vesicles.
The SAXS signal contains information
about the lipid bilayer structure and
stacking.
From the WAXS signal information about
the alkyl chain packing can be deduced.
Melting temperature of DPPC = 41°C
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Summary
• Structural studies on proteins were performed to determine:
• Overall size and shape (simulation and ab-initio determination)
• Folding / unfolding (tertiary structure)
• Stability and complex formation (quaternary structure)
• Oligomeric state and oligomeric mixture
• Molecular weight
© 2017 PANalytical B.V. All rights reserved.