Rigid body refinement tutorial (MASSHA case) Petr V. Konarev - - PowerPoint PPT Presentation

Rigid body refinement tutorial (MASSHA case)

Petr V. Konarev European Molecular Biology Laboratory, Hamburg Outstation BioSAXS group

25-31 October 2010 EMBO Course

Scattering from a macromolecule in solution: programs CRYSO L/ CRYSO N

♦ A(s): atomic scattering in vacuum

♦ E(s): scattering from the excluded volume ♦B(s): scattering from the hydration shell

Ω Ω

−

2 b s 2

) B( + ) E( ) A( = ) A( = I(s) s s s s δρ ρ

Svergun, D.I., Barberato, C. & Koch, M.H.J. (1995).

• J. Appl. Crystallogr. 28, 768-773.

25-31 October 2010 EMBO Course

If the intensity of each contribution is represented using spherical harmonics the average is performed analytically: This approach permits to further use rapid algorithms for rigid body refinement The use of multipole expansion

Ω Ω

−

2 b s a 2

) B( + ) E( ) ( A = ) A( = I(s) s s s s δρ ρ

∑ ∑

= − =

+ − =

L l l l m lm lm lm

s B s E s A s I

2 2

) ( ) ( ) ( 2 ) ( δρ ρ π

2 2

) ( 2 ) ( s A s I

lm l l m l

∑ ∑

− = ∞ =

= π

25-31 October 2010 EMBO Course

• The programs:

fit the experimental data by varying the density of the

hydration layer δρ (affects the third term) and the total excluded volume (affects the second term)

predict the scattering from the atomic structure

(when there are no experimental data available)

provide

scattering amplitudes for rigid body refinement routines (binary * .alm files)

compute particle envelope function F(ω) – (* .flm files)

that can be visualized with MASSHA

∑ ∑

= − =

+ − =

L l l l m lm lm lm

s B s E s A s I

2 2

) ( ) ( ) ( 2 ) ( δρ ρ π

Scattering from a macromolecule in solution: programs CRYSO L/ CRYSO N

25-31 October 2010 EMBO Course

Program options : 0 - evaluate scattering amplitudes and envelope 1 - evaluate only envelope and Flms 2 - read CRYSOL information from a .sav file

• - Brookhaven file name < .pdb > : 6lyz
• - Maximum order of harmonics < 15 > :
• - Order of Fibonacci grid < 17 > :
• -Maximum s value < 0.500 > :

The maximum possible s is 1.0 (1/Å)

• - Number of points < 51 > :

Number of points in the theoretical curve (maximum = 201)

• - Fit the experimental curve <Y(es)>/N(o) :

CRYSOL input parameters

25-31 October 2010 EMBO Course

Following file names will be created: 6lyz00.log -- CRYSOL log-file (ASCII) 6lyz00.sav -- save CRYSOL information (binary) 6lyz00.flm -- multipole coefficients (ASCII) 6lyz00.int -- scattering intensities (ASCII) 6lyz00.fit -- fit to experimental data (ASCII) 6lyz00.alm -- partial scattering amplitudes (binary) CRYSOL output files

25-31 October 2010 EMBO Course

CRYSOL : Scattering from CRYSOL : Scattering from lysozyme lysozyme

1-Difference (FINAL curve) 2- Atomic 3- Shape 4- Border

6lyzNN.int output file from CRYSOL

25-31 October 2010 EMBO Course

Rigid body modelling

The high resolution structures of the components (subunits or domains) are known. The tertiary structure assumed to be unchanged upon complex formation. Arbitrary complex can be constructed by moving and rotating one of the subunits. This operation depends on three Euler rotation angles and three Cartesian shifts. Not interconnected arrangements of subunits and those with steric clashes should be penalized. Scattering amplitudes from individual subunits are evaluated using CRYSOL/CRYSON

25-31 October 2010 EMBO Course

Scattering from a complex particle

The partial amplitudes of arbitrarily rotated and displaced subunit are analytically expressed via the initial amplitudes and the six positional parameters: Clm(s) = Clm(Blm, α, β, γ, x, y, z). The scattering from the complex is then rapidly calculated

Rotation: α, β, γ A Shift: x, y, z C B

( )

[ ]

∑∑

∞ −

+ + =

* 2

) ( ) ( Re 4 ) ( ) (

l l lm lm B A

s C s A s I s I s I π

Svergun, D.I. (1991). J. Appl. Cryst. 24, 485-492

25-31 October 2010 EMBO Course

A B C

Rigid body refinement: two possible strategies

• Domain organization of the complex can

be found by fitting the experimental data in two ways:

• Method 1: Automatic minimization by an

exhaustive search in 6-dimensional space taking into account biochemical restrains, interconnectivity, absence of steric clashes and information on contacts (SASREF)

• Method

2: Interactive subunit positioning utilizing visual and biochemical criteria and local search around the selected positions (MASSHA)

I(s) Im(s) s

25-31 October 2010 EMBO Course

Program MASSHA Program MASSHA-

• Modelling

Modelling of Atomic

• f Atomic

Structures and Shape Analysis Structures and Shape Analysis

♦3D representation of atomic structures, bead models and surfaces ♦Geometrical transformations using hotkeys and/or menus ♦Rotations around the geometrical center or specific residue ♦Rigid body refinement against experimental scattering data * interactive and/or automated * heterodimeric or homodimeric models * symmetric oligomers and multi-domain models ♦Saving refined models in PDB format ♦Runs on Windows XP/2000/9x, WIndows NT

P.V. Konarev, M.V. Petoukhov & D.I. Svergun (2001).

• J. Appl. Cryst. 34, 527-532

25-31 October 2010 EMBO Course

MASSHA: 3D representations

25-31 October 2010 EMBO Course

Arrow Up -- Rotate around X axis (clockwise) Arrow Down -- Rotate around X axis (counterclockwise) Arrow Left -- Rotate around Y axis (clockwise) Arrow Right -- Rotate around Y axis (counterclockwise) z -- Rotate around Z axis (clockwise) x -- Rotate around Z axis (counterclockwise) Ctrl + Arrow Up -- Move up along Y Ctrl + Arrow Down -- Move down along Y Ctrl + Arrow Left -- Move left Ctrl + Arrow Right -- Move right

MASSHA transformation hot keys

25-31 October 2010 EMBO Course

F1 - to display a brief HELP about hotkeys F2 - to toggle SLD TYPE (front -> contour -> front+back) F3 - to toggle PDB TYPE (chain -> circles) F4 - to display INFORMATION window F5,F6 ZOOM In/Out the picture in the graphic window F7 - to display residue NUMBERS for PDB objects F8 - to toggle BACKGROUND (black -> white) F9 - to toggle GRID mode F11,F12 to change the angular and space INCREMENTS P,p to FILL in active PDB bodies displayed as circles S,s to FILL in active SLD bodies +/- to increase/decrease the atom RADIUS by 0.5 A 1-9 to select the ACTIVE body from loaded structures 0 to make ALL bodies active

MASSHA graphic presentation hot keys

25-31 October 2010 EMBO Course

MASSHA: Rigid body refinement of heterodimer

Add body Add body

25-31 October 2010 EMBO Course

MASSHA: Rigid body refinement of heterodimer

25-31 October 2010 EMBO Course

MASSHA: Rigid body refinement of heterodimer

Compute Compute

25-31 October 2010 EMBO Course

Refine Refine

MASSHA: Rigid body refinement of heterodimer

25-31 October 2010 EMBO Course

MASSHA: Rigid body refinement of oligomers with symmetry

25-31 October 2010 EMBO Course

MASSHA: Rigid body refinement of oligomers with symmetry

25-31 October 2010 EMBO Course

Compute Compute

MASSHA: Rigid body refinement of oligomers with symmetry

25-31 October 2010 EMBO Course

Compute Compute

MASSHA: Rigid body refinement of oligomers with symmetry

25-31 October 2010 EMBO Course

Refine Refine

MASSHA: Rigid body refinement of oligomers with symmetry

25-31 October 2010 EMBO Course

MASSHA: multiple-domain rigid body refinement

25-31 October 2010 EMBO Course

MASSHA: multiple-domain rigid body refinement

1;3;5 1;3;5

25-31 October 2010 EMBO Course

MASSHA: multiple-domain rigid body refinement

25-31 October 2010 EMBO Course

MASSHA: multiple-domain rigid body refinement

Compute Compute

25-31 October 2010 EMBO Course

Compute Compute

MASSHA: multiple-domain rigid body refinement

25-31 October 2010 EMBO Course

Refine Refine

MASSHA: multiple-domain rigid body refinement

25-31 October 2010 EMBO Course

Examples for rigid body modelling using MASSHA

25-31 October 2010 EMBO Course Go to directory /Examples/Massha/ ../heterodimer

• heterodimer modelling (see exampl1.txt)

../homodimer

• homodimer modelling (see example2.txt)

../oligsym

• symmetric oligomer (see example3.txt)

../multibody

• multibody modelling (see example4.txt)

../homodimer_hc_titanus_p2 – Hc fragment of titanus toxin dimer (see info.txt) hcd2ry.pdb – symmetry axis along Y hcd2rz.pdb – symmetry axis along Z ../oligsym_pyruvate_oxidase_p222 – tetramer structure with P222 symmetry (see info.txt) poxtetr.pdb – Xtal structure ref_tetr.pdb – refined SAXS model ../pdb_flm

• pdb files and shapes (*.flm) of hsa and lysozyme