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DATA ANALYSIS Fitting RDC Data to Structure. Software you should have: Open Babel to convert structure files formats: ( http://openbabel.org/wiki/Install ) NOTEPAD++, excellent text editor for Windows: http://notepad-plus-plus.org/download. DATA ANALYSIS Fitting RDC Data to Structure.

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DATA ANALYSIS Fitting RDC Data to Structure

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Data analysis fitting rdc data to structure

DATA ANALYSIS

Fitting RDC Data to Structure

Software you should have:

Open Babel to convert structure files formats:

(http://openbabel.org/wiki/Install)

NOTEPAD++, excellent text editor for Windows:

http://notepad-plus-plus.org/download


Data analysis fitting rdc data to structure

DATA ANALYSIS

Fitting RDC Data to Structure

Software for performing SVD fitting of structure to RDCs data:

MSpin by Armando Navarro-Vazquez, Commercialized by Mestrelab Research,

Santiago de Compostela, SPAIN.

http://mestrelab.com/software/mspin/

PALES by Markus Zweckstetter, Max Plank Institute for Biophysical Chemistry, Göttingen, GERMANY.

http://www.mpibpc.mpg.de/groups/zweckstetter/_links/software_pales.htm


Data analysis fitting rdc data to structure

DATA ANALYSIS

Fitting RDC Data to Structure

In order to fit the data to a set of judicious structures (all the configurational space of your molecule) you need to know more than five independent (non-parallel internuclear vector) RDCs.

At least three of them have to be out of the plane.

You need a minimun of five RDCs to calculate the alignment tensor (A), but need more than five to perform the fitting using Singular Value Decomposition analysis either with the programs MSpin or PALES.

Each fitting will give you a quality factor Q (Cornilescu Q factor - J. Am. Chem. Soc. 1998, 120, 6836-6837). The lower the Q factor the better the fitting.

Fitting structures in MSpin is very straightforward. You can use the tutorial from Mestre website. MSpin reads any type of PDB files and also the XYZ format.


Data analysis fitting rdc data to structure

On the other side, PALES follows a unique PDB file format.

PALES file format

The structure file:

For Small Molecules, PALES reads a structure file that is an adaptation of the original

PDB file format used for proteins. Instead of having N number of residues

(e.g. aminoacids), the file represents a single residue (N=1).


Data analysis fitting rdc data to structure

PRACTICAL EXAMPLES


Data analysis fitting rdc data to structure

LUDARTIN

- gastric cytoprotective effect

- inhibits the aromatase enzyme

- first isolated in 1972 from Artemisia carruthii by Geissman and Griffin as a mixture with it 11,13-dihydroderivative

- the stereochemistry displayed in 1 based on the chemical shift and coupling constants of H-6 and on the chemical shift of H-15

  • Giordano, O. S.; Guerreiro, E.; Pestchanker, M. J.; Guzman, J.; Pastor, D.; Guardia, T. J. Nat. Prod. 1990, 53, 803-9.

  • Blanco, J. G.; Gil, R. R.; Alvarez, C. I.; Patrito, L. C.; Genti-Raimondi, S.; Flury, A. FEBS Lett. 1997, 409, 396-400.

  • - Geissman, T. A.; Griffin, T. S. Phytochemistry 1972, 11, 833-5.


Data analysis fitting rdc data to structure

Determination of the Stereochemistry of Ludartin (1) Using Chemical Transformations

Sosa, V. E.; Oberti, J. C.; Gil, R. R.; Ruveda, E. A.; Goedken, V. L.; Gutierrez, A. B.; Herz, W. Phytochemistry1989, 28, 1925-9.


Data analysis fitting rdc data to structure

H-3

H-3 and CH3-15

27o above and below

the plane of the five-membered

ring

CH3-15

RMS fit and overlay of the 3D structures of ludartin (1) and 3,4-b-epoxy-ludartin (2) using only the heavy atoms belonging to the 5-member and the 7-member rings, and the lactone ring.

RMS error: 0.039 Å


Data analysis fitting rdc data to structure

PALES Presentation


Data analysis fitting rdc data to structure

This is the methylacetamide PDB file created by HyperChem

PALES can not read this file. It does not recognize HETATM

HETATM 1 C 1 -1.413 -1.626 0.000

HETATM 2 C 2 -1.342 -0.119 0.000

HETATM 3 N 3 -2.536 0.577 -0.000

HETATM 4 C 4 -2.573 2.004 0.000

HETATM 5 O 5 -0.252 0.487 -0.000

HETATM 6 H 6 -0.373 -2.033 -0.000

HETATM 7 H 7 -1.947 -1.993 0.910

HETATM 8 H 8 -1.947 -1.993 -0.910

HETATM 9 H 9 -3.390 0.079 0.000

HETATM 10 H 10 -3.637 2.354 -0.000

HETATM 11 H 11 -2.048 2.408 0.908

HETATM 12 H 12 -2.048 2.408 -0.908

CONECT 1 2 6 7 8

CONECT 2 1 3 5

CONECT 3 2 4 9

CONECT 4 3 10 11 12

CONECT 5 2

CONECT 6 1

CONECT 7 1

CONECT 8 1

CONECT 9 3

CONECT 10 4

CONECT 11 4

CONECT 12 4

END


Data analysis fitting rdc data to structure

Save the structure generate by HyperChem in hin format

Convert the hin format to a PDB file using Open Babel


Data analysis fitting rdc data to structure

PDB file created by Open Babel from hin file

COMPND C:\Users\rgil\SMASH 2010 RDCs Workshop\Methyl Acetamide\methylacetamide.hin

AUTHOR GENERATED BY OPEN BABEL 2.2.3

HETATM 1 C LIG 1 -1.413 -1.626 0.000 1.00 0.00 C

HETATM 2 C LIG 1 -1.342 -0.119 0.000 1.00 0.00 C

HETATM 3 N LIG 1 -2.536 0.577 -0.000 1.00 0.00 N

HETATM 4 C LIG 1 -2.573 2.004 0.000 1.00 0.00 C

HETATM 5 O LIG 1 -0.252 0.487 -0.000 1.00 0.00 O

HETATM 6 H LIG 1 -0.373 -2.033 -0.000 1.00 0.00 H

HETATM 7 H LIG 1 -1.947 -1.993 0.910 1.00 0.00 H

HETATM 8 H LIG 1 -1.947 -1.993 -0.910 1.00 0.00 H

HETATM 9 H LIG 1 -3.390 0.079 0.000 1.00 0.00 H

HETATM 10 H LIG 1 -3.637 2.354 -0.000 1.00 0.00 H

HETATM 11 H LIG 1 -2.048 2.408 0.908 1.00 0.00 H

HETATM 12 H LIG 1 -2.048 2.408 -0.908 1.00 0.00 H

CONECT 1 2 6 7 8

CONECT 2 1 3 5 5

CONECT 3 2 4 9

CONECT 4 3 10 11 12

CONECT 5 2 2

CONECT 6 1

CONECT 7 1

CONECT 8 1

CONECT 9 3

CONECT 10 4

CONECT 11 4

CONECT 12 4

MASTER 0 0 0 0 0 0 0 0 12 0 12 0

END

The Information inside the blue

boxes is not used by PALES.

ERASE IT


Data analysis fitting rdc data to structure

Edit the file with a good text editor accordingly. I recommend NOTEPAD++

HETATM 1 C LIG 1 -1.413 -1.626 0.000 1.00 0.00

HETATM 2 C LIG 1 -1.342 -0.119 0.000 1.00 0.00

HETATM 3 N LIG 1 -2.536 0.577 -0.000 1.00 0.00

HETATM 4 C LIG 1 -2.573 2.004 0.000 1.00 0.00

HETATM 5 O LIG 1 -0.252 0.487 -0.000 1.00 0.00

HETATM 6 H LIG 1 -0.373 -2.033 -0.000 1.00 0.00

HETATM 7 H LIG 1 -1.947 -1.993 0.910 1.00 0.00

HETATM 8 H LIG 1 -1.947 -1.993 -0.910 1.00 0.00

HETATM 9 H LIG 1 -3.390 0.079 0.000 1.00 0.00

HETATM 10 H LIG 1 -3.637 2.354 -0.000 1.00 0.00

HETATM 11 H LIG 1 -2.048 2.408 0.908 1.00 0.00

HETATM 12 H LIG 1 -2.048 2.408 -0.908 1.00 0.00

END

PALES is written in C and generally it is very forgiving in terms of format, i.e. it

does not care whether you use spaces, tabs, ...

However, editors from windows such as NOTEPAD or WORDPAD may intruduce

characters that can make the file unreadeable by PALES. This not always the case

but it may happen.

The only requirement is that the naming convention in the PDB file and the RDC

table are identical. In addition, PALES only takes into account lines in the PDB file

starting with "ATOM". For the MAC and LINUX versions of the GUI will also

include an automatic reformatting step for the PDB file. Then at least in the GUI

these problems should not show up.


Data analysis fitting rdc data to structure

Edit the file with a good text editor accordingly

HETATM 1 C LIG 1 -1.413 -1.626 0.000 1.00 0.00

HETATM 2 C LIG 1 -1.342 -0.119 0.000 1.00 0.00

HETATM 3 N LIG 1 -2.536 0.577 -0.000 1.00 0.00

HETATM 4 C LIG 1 -2.573 2.004 0.000 1.00 0.00

HETATM 5 O LIG 1 -0.252 0.487 -0.000 1.00 0.00

HETATM 6 H LIG 1 -0.373 -2.033 -0.000 1.00 0.00

HETATM 7 H LIG 1 -1.947 -1.993 0.910 1.00 0.00

HETATM 8 H LIG 1 -1.947 -1.993 -0.910 1.00 0.00

HETATM 9 H LIG 1 -3.390 0.079 0.000 1.00 0.00

HETATM 10 H LIG 1 -3.637 2.354 -0.000 1.00 0.00

HETATM 11 H LIG 1 -2.048 2.408 0.908 1.00 0.00

HETATM 12 H LIG 1 -2.048 2.408 -0.908 1.00 0.00

END

  • Editing:

  • Replace all HETATM with ATOM

  • Insert the word TER before END

  • Add the proper number next to each atom label (C, N, O, H, etc)

  • See edited file in next slide


Data analysis fitting rdc data to structure

Edit the file with a good text editor accordingly

ATOM 1 C1 LIG 1 -1.413 -1.626 0.000 1.00 0.00

ATOM 2 C2 LIG 1 -1.342 -0.119 0.000 1.00 0.00

ATOM 3 N3 LIG 1 -2.536 0.577 -0.000 1.00 0.00

ATOM 4 C4 LIG 1 -2.573 2.004 0.000 1.00 0.00

ATOM 5 O5 LIG 1 -0.252 0.487 -0.000 1.00 0.00

ATOM 6 H6 LIG 1 -0.373 -2.033 -0.000 1.00 0.00

ATOM 7 H7 LIG 1 -1.947 -1.993 0.910 1.00 0.00

ATOM 8 H8 LIG 1 -1.947 -1.993 -0.910 1.00 0.00

ATOM 9 H9 LIG 1 -3.390 0.079 0.000 1.00 0.00

ATOM 10 H10 LIG 1 -3.637 2.354 -0.000 1.00 0.00

ATOM 11 H11 LIG 1 -2.048 2.408 0.908 1.00 0.00

ATOM 12 H12 LIG 1 -2.048 2.408 -0.908 1.00 0.00

TER

END

NOTE: The changes are printed in red

Codes uses by the

PALES RDC input file

See next slide

ATOMNAME

RESNAME

RESID

ATOMNAME is the atom number in the structure file

RESNAME is a fake name to simulate a residue (e.g. Lys, Ala, etc, in a peptide), LIG in this case, but you can use any name as long as you use de same name in

RDCs input table.

RESID is the number of the residue in peptide sequence, for a small molecule is just 1.


Data analysis fitting rdc data to structure

The RDCs table file in PALES

VARS RESID_I RESNAME_I ATOMNAME_I RESID_J RESNAME_J ATOMNAME_J D DD W

FORMAT %5d %6s %6s %5d %6s %6s %9.3f %9.3f %.2f

1 LIG C1 1 LIG H6 -6.800 1.000 1.00

1 LIG C1 1 LIG H5 0.800 1.000 1.00

1 LIG C1 1 LIG H8 -25.230 1.000 1.00

1 LIG N3 1 LIG H9 -15.6 1.000 1.00

1 LIG C4 1 LIG H8 -75.5 1.000 1.00

1 LIG C4 1 LIG H10 7.510 1.000 1.00

1 LIG C4 1 LIG H11 -4.530 1.000 1.00

1 LIG C4 1 LIG H12 123.0 1.000 1.00

Experimental

Error (Hz)

RDCs (Hz)


Data analysis fitting rdc data to structure

Fitting data to structure using command line PALES in Windows:

To perform an SVD fitting in PALES you have to execute the following command:

pales –bestFit –pdb –name.pdb –inD rdc_file.tab –outD output.SVD.file

name.pdb is the name of the PDB file

rdc_file.tab is the name of the RDC data file

The output is very well explained in the PALES documentation or in the Nature

Protocols paper (See below). Right now we are only interested on the Q factor.

The lower the Q factor the better the SVD fitting.

See also:

Nature Protocols 2008 3(4) 679-690


Data analysis fitting rdc data to structure

PERSPECTIVES


Data analysis fitting rdc data to structure

Future Directions on RDCs in Small Molecules

  • Alignment Media: more variety (PEO), more deuteration, chiral gels for all solvents, …

  • Low Temperature Alignment Media to resolve conformational

  • average.

  • Commercialization: gels, stretching apparatus, software, etc…

  • Adapted Measurement Techniques: in conjunction with scaling of RDCs novel pulse sequences

  • Software: Individual error treatment, incorporation of RCSA,automation of structure generation, ab initio andMD methods for treating flexible molecules, prediction of alignment (absolute configuration)

  • Technical Improvements: Gradient shimming


Data analysis fitting rdc data to structure

Revisit Underutilized Experiments


Data analysis fitting rdc data to structure

Selective 1D NOESY

NOE buildup curves of H10 while selectively exciting H4 axial of cortisol (1) in DMSO-d6: (a) the ‘‘raw’’ NOE buildup curve; (b) the NOE buildup curve obtained with PANIC (peak amplitude normalization for improved cross-relaxation)


Data analysis fitting rdc data to structure

Series of 1D NOESY Spectra of Ludartin

H-3 is selectively excited

H-15

H-2a

H-2b

600 ms

100 ms

Steps of 100 ms

NOE  1/r6


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