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 !