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Determination of apoferritin structure parameters using small
angle neutron scattering
Joint Institute of Nuclear Research, Dubna Summer Student Practice 2014
Daniela Dogaru (ROM), Zuzana Bednáriková (SVK), Mária Huráková (SVK), Veronika Gdovinová (SVK)
Supervisor: Kuklin Aleksandr Ivanovich ( Senior Researcher at Frank Laboratory of Neutron Physics – Department of Neutron Scattering Investigations of Condensed Matter )
OUTLINE
Small Angle Neutron Scattering - introduction to the SANS technique
ATSAS program package - data treatment - structure modeling
determine structural parameters of Apoferritin - radius of gyration, diameter - low resolution structure model
Small Angle Neutron Scattering (SANS) an experimental technique that uses coherent and elastic neutron
scattering at small scattering angles to investigate the structure of various substances at a scale of about 1 - 100 nm
spectrum : Scattering Intensity I as a function of a wavevector Q
)()()( QSQPQI
I(Q) = scattering intensity
Ø = density of particles in volume
P(Q) =<|F(Q)|2>, F(Q)- form factor
S(Q) = structure factor
Q = wavevector Ɵ = scattering angle
λ = Wavelength of incident beam
0,01 0,1
100000
1000000
1E7
1E8
log
I
q
3VVK.pdb
Information obtained by SANS:
- sizes, spatial correlations and shapes of particles in crystalline,
solution or amorphous states
- phase transitions and degree of polydispersity
- molecular weight
Applications:
biology (viruses, proteins, lipids,....)
chemistry (polymers, colloids, gel,...)
material technology (ceramics, powder,...)
EQUIPMENT
YUMO spectrometer
1 – two reflectors; 2 – zone of reactor with moderator; 3 – chopper; 4 – first collimator; 5 – vacuum tube; 6 – second collimator; 7 – thermostate; 8 – samples table; 9 – goniometer; 10-11 – Vn-standard; 12 – ring-wire detector; 13 – position-sensitve edetector "Volga"; 14 – direct beam detector
ATSAS program package
created by Svergun et al. at EMBL in HAMBURG
programs suite for small-angle scattering data analysis from biological macromolecules
1. PRIMUS (manipulations with experimental 1D SAS data)
2. GNOM (indirect transform program that evaluates the particle distance
distribution function p(r))
3. DAMMIN (ab initio shape determination using a dummy atom model)
4. MASSHA (interactive modelling of atomic structures and shape analysis)
DETERMINING of Rg
0,01 0,1
100000
1000000
1E7
1E8
log
I
q, A-1
Fit for SANS experimental data for apoferritin.
Guinier approximation for a globular particle in PRIMUS:
Spherical shape with Rg = 55.0 0.323 1/A
APOFERRITIN
globular protein complex present in every cell type
the primary intracellular iron-storage protein keeping iron in a
soluble and non-toxic form
known structure parameters (X-ray crystalography, NMR)
Image obtained from Protein Data Bank
- size : 450 kDa with 24 subunits
- diameter : 120 Å
- spherical shell → inner radius: 40Å
→ outer radius: 60Å
DISTANCE DISTRIBUTION FUNCTION
The analysis of this function helps to find structural details about the
molecule.
spherical molecule
with shell structure
Distance distribution function obtained using GNOM
2
2
3
21
3
1
23
2
3
1 )]()([)()( qRRqRRRRqI
,3
cossin3)(
t
tttt qRt
DETERMINING of STRUCTURE FACTOR S(q)
)()()( QSQPQI
0,01
1
Str
uctu
re f
acto
r
q, A-1
0,01 0,11E-7
1E-6
1E-5
1E-4
1E-3
0,01
0,1
1
10
experimental data
theoretical fit
Inte
nsit
y,
cm
-1
q, A-1
Projection of obtained apoferritin structure
with programs DAMMIN.
method to restore ab initio low resolution shape of randomly oriented
particles in solution
a search volume which encloses the particle is filled with dummy
atoms
apoferritin → model of spherical shell
DAMMIN 3D MODELING TOOL
CONCLUSIONS
from the experimental data were obtained the experimental
curves (PRIMUS)
using ATSAS package (PRIMUS, GNOM) we determined:
- the radius of gyration – Rg = 55 0.323
- distance between molecules ~ 85Å
- diameter – d ~ 110Å
- structure factor
- model for apoferritin molecule
using DAMMIN - apoferritin has spherical shell structure
which is in agreement with structures obtained with different
methods (X-ray, NMR)
REFERENCES Feigin L.A. & Svergun D.I. (1987). Structure Analysis by Small-Angle X-
Ray and Neutron Scattering. In NY: Plenum Press.
J. Texeira (1992). Introduction to Small Angle Neutron Scattering Applied
to Colloidal Science. In Kluwer Academic Publishers, Netherlands.
D. I. Svergun (1999). Restoring low resolution structure of biological
macromolecules from solution scattering using simulated annealing. In
Biophys J. 2879-2886.
P.V. Konarev, V.V. Volkov, A.V. Sokolova, M.H.J. Koch and D. I. Svergun
(2003). PRIMUS - a Windows-PC based system for small-angle scattering
data analysis. In J Appl Cryst. 36, 1277-1282.
A .I. Kuklin, T. N. Murugova, O. I. Ivankov, A. V. Rogachev, D. V. Soloviov,
Yu. S. Kovalev, A. V. Ishchenko, A. Zhigunov, T. S. Kurkin, V. I. Gordeliy
(2012). Comparative study on low resolution structures of apoferritin via
SANS and SAXS. In J Phys: Conference Series 351.
The team would like to thank the members of the Frank Laboratory of Nuclear
Physics and the YuMO SANS team for all their support and especially to our
supervisor Dr. A.I.KUKLIN for his guidance and patience.
ACKNOWLEDGEMENT
We would like to extend our regards to the organizers of the Summer Student
Practice 2014 and all members of the JINR involved with this project.
THANK YOU FOR YOUR ATTENTION !