Dear Philippe,

Most often the problem is that the geometry of the input model is so distorted that the procedure fails to find base pairs and therefore restrain them.

Couple of ways to check for this are:

1. Look in the .log into

Finding SS restraints...
Secondary structure from input PDB file:
0 helices and 0 sheets defined
0.0% alpha, 0.0% beta
12 base pairs and 21 stacking pairs defined.
Time for finding SS restraints: 0.04
Creating SS restraints...

No hydrogen bonds defined for protein.
Restraints generated for nucleic acids:
32 hydrogen bonds
64 hydrogen bond angles
0 basepair planarities
12 basepair parallelities
21 stacking parallelities

This part would give a general idea if anything worked at all.

2. Inspect .geo file after refinement and look for "Basepair parallelity restraints" and "Bond-like restraints" (secondary structure H-bonds) sections. Here you can see exactly what restraints were applied and if the basepairs in question were in fact restrained.

If you supply the restraints in the .parameter file, check the nucleic_acid.hbond_distance_cutoff parameter. The default is 3.4, you may need to increase it so your annotations are not filtered out.

I'm happy to look at your files (model and parameter, if any) to figure out what is happening. Please send them off-list indicating problematic residues. Files will be treated confidentially.

Best regards,

Oleg Sobolev.

On Tue, Jan 30, 2024 at 7:20 AM CUNIASSE Philippe <Philippe.CUNIASSE@cea.fr> wrote:

Dear all,

I am currently refining a protein-nucleic acid structure obtained by MDFF (NAMD) with a 2.9 Å cryo-EM map.

Dues to the poor density of the map at the end of the DNA duplex (likely due to breathing at the ends of the duplex), the structure of the DNA tends to be distorted.

I tried to set up a set of nucleic acid restraints (Base pairing and base stacking for the full sequence), however, despite the fact that the syntax was checked and no error message is present in the log file, it seems that the restraints are not taken into account as if the constant restraints were two weak. And indeed when examining the refined structure, the pairing and stacking restraints for the base pairs at the extremities of the DNA duplex are not fulfilled.

Have you a suggestion to solve this problem ?

Thanks in advance for your help.

Best regards.

Philippe.

-----------------------------------------------

Philippe Cuniasse, PhD/HDR.

Institute for Integrative Biology of the Cell (I2BC)

UMR 9198 CNRS-CEA-Univ Paris Sud

Bat 144 CE-Saclay

91191 Gif-sur-Yvette Cedex, France

Tel: (33) 1 69 08 56 35

Fax: (33) 1 69 08 47 12

Email: philippe.cuniasse@cea.fr

Web: http://biodev.cea.fr/rasmot3d/

Web: https://www.i2bc.paris-saclay.fr

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