25-31 October 2010 EMBO Course 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 2 − ρ δρ 2 I(s) = A( s ) = A( s ) E( s ) + B( s ) s b Ω Ω ♦ A( s ) : atomic scattering in vacuum ♦ E( s ) : scattering from the excluded volume ♦ B( s ) : scattering from the hydration shell Svergun, D.I., Barberato, C. & Koch, M.H.J. (1995). J. Appl. Crystallogr. 28 , 768-773.
25-31 October 2010 EMBO Course The use of multipole expansion 2 − ρ δρ 2 s s s s I(s) = A( ) = A ( ) E( ) + B( ) a s b Ω Ω If the intensity of each contribution is represented using spherical harmonics ∞ l ∑ ∑ = π 2 2 I ( s ) 2 A ( s ) lm = = − l 0 m l the average is performed analytically: L l ∑ ∑ = π − ρ + δρ 2 2 I ( s ) 2 A ( s ) E ( s ) B ( s ) lm 0 lm lm = = − 0 l m l This approach permits to further use rapid algorithms for rigid body refinement
25-31 October 2010 EMBO Course Scattering from a macromolecule in solution: programs CRYSO L/ CRYSO N L l ∑ ∑ = π − ρ + δρ 2 2 ( ) 2 ( ) ( ) ( ) I s A s E s B s lm 0 lm lm = = − l 0 m l • 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
25-31 October 2010 EMBO Course CRYSOL input parameters 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) :
25-31 October 2010 EMBO Course CRYSOL output files 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)
25-31 October 2010 EMBO Course CRYSOL : Scattering from lysozyme lysozyme CRYSOL : Scattering from 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. Scattering amplitudes from individual subunits are evaluated using CRYSOL/CRYSON Not interconnected arrangements of subunits and those with steric clashes should be penalized.
25-31 October 2010 EMBO Course Scattering from a complex particle Shift: x, y, z A B C Rotation: α , β , γ The partial amplitudes of arbitrarily rotated and displaced subunit are analytically expressed via the initial amplitudes and the six positional parameters: C lm (s) = C lm (B lm , α , β , γ , x, y, z). The scattering from the complex is then rapidly calculated [ ] ∞ l ( ) ∑∑ = + + π 2 * I s I ( s ) I ( s ) 4 Re A ( s ) C ( s ) A B lm lm − 0 l Svergun, D.I. (1991). J. Appl. Cryst. 24 , 485-492
25-31 October 2010 EMBO Course Rigid body refinement: two possible strategies I(s) • Domain organization of the complex can I m (s) be found by fitting the experimental data in two ways: • Method 1: Automatic minimization by an s exhaustive search in 6-dimensional space A taking into account biochemical restrains, B interconnectivity, absence of steric clashes and information on contacts (SASREF) • Method 2: Interactive subunit positioning utilizing visual and biochemical criteria and local search C around the selected positions (MASSHA)
25-31 October 2010 EMBO Course Program MASSHA- - Modelling Modelling of Atomic of Atomic Program MASSHA 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 MASSHA transformation hot keys 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
25-31 October 2010 EMBO Course MASSHA graphic presentation hot keys 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
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 MASSHA: Rigid body refinement of heterodimer Refine Refine
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 MASSHA: Rigid body refinement of oligomers with symmetry Compute Compute
25-31 October 2010 EMBO Course MASSHA: Rigid body refinement of oligomers with symmetry Compute Compute
25-31 October 2010 EMBO Course MASSHA: Rigid body refinement of oligomers with symmetry Refine Refine
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 MASSHA: multiple-domain rigid body refinement Compute Compute
25-31 October 2010 EMBO Course MASSHA: multiple-domain rigid body refinement Refine Refine
25-31 October 2010 EMBO Course Examples for rigid body modelling using MASSHA Go to directory /Examples/Massha/ ../pdb_flm - pdb files and shapes (*.flm) of hsa and lysozyme ../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
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