Characterizing Biological Macromolecules by SAXSPharmaceutical Powder X -ray Diffraction Symposium Sponsored by The International Centre for Diffraction Data. This presentation is

$Page 1: Characterizing Biological Macromolecules by SAXSPharmaceutical Powder X -ray Diffraction Symposium Sponsored by The International Centre for Diffraction Data. This presentation is$
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.

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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.

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Empyrean Nano edition

160


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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

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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


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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]


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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

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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)


0

0.25

0.50

0.75

1.00

0 10 20 30

r [nm]

p(r

)


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


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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

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SAXS setup

X-ray tube

X-ray mirror

Sample Area

detector

Evacuated beam path

WAXS


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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!


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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


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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


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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]


Apoferritin (12 mg/ml)

An iron storage protein.


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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


Inte

ns

ity

[co

un

ts]

No aggregates


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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)


Inte

ns

ity

[co

un

ts]


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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


Inte

ns

ity

[co

un

ts]


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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

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Protein structure in crystal form vs. in solution

102

103

104

105

106

0 1 2 3

PDB 1IER

CRYSOL


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.

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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


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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.]



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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


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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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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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



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Characterizing Biological Macromolecules by SAXSPharmaceutical Powder X -ray Diffraction Symposium Sponsored by The International Centre for Diffraction Data. This presentation is