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Twilight: A tool for ligand density validation 

Visualizing Ligand Molecules in Twilight Electron Density

Christian X.  Weichenbergera*,  Edwin  Pozharskib* and  Bernhard  Ruppc

aCenter for Biomedicine, European Academy of Bozen/Bolzano (EURAC), Viale Druso 1, Bozen/Bolzano, I-39100, Italy,  bUniversity of Maryland, Baltimore, MD, USA, and  ck.k. Hofkristallamt, 991 Audrey Place, Vista, CA, 92084, USA

Correspondence email:  Christian.Weichenberger@eurac.edu; epozh001@umaryland.edu


We present a software script for facilitating analysis and visual inspection of ligand molecules in context of the electron density maps calculated from experimental data associated with protein structures determined by X-ray crystallography.


Three-dimensional models of protein structures determined by X-ray crystallography are based on the interpretation of experimentally derived electron density maps. The real-space correlation coefficient (RSCC) provides an easily comprehensible, objective measure for residue-based fit of atom coordinates with electron density. Among protein structure models, protein-ligand complexes are of special interest, given their contribution to understanding the molecular underpinnings of biological activity and to drug design. For consumers of such models it is not trivial to determine the degree to which ligand structure modelling is biased by subjective electron density interpretation. We present a standalone script, Twilight, for analysis, visualization, and annotation of a pre-filtered set of 2,815 protein/ligand complexes deposited with the PDB as of January 15, 2012, with ligand RSCC values that are below a threshold of 0.6. The script runs on various platforms and is available for download at http://www.ruppweb.info/twilight/.

Download this Acta Crystallographica F paper and its companion in Acta Crystallographica D describing the actual findings from the IUCr web site (preferred) or request a link by email. Please read also the editorial Expectation bias and information content Z. Dauter, M. S. Weiss, H. Einspahr and E. N. Baker, Acta Crystallogr. D69, 141 (2013).


Techniques, tools and best practices for ligand electron-density analysis and results from their application to deposited crystal structures. E. Pozharski, C. X. Weichenberger and B. Rupp, Acta Crystallogr D69, 150-167 (2013)

Visualizing Ligand Molecules in Twilight Electron Density. C. X. Weichenberger, E. Pozharski and B. Rupp, Acta Crystallogr. F69(2), 195-200(2013)

Download program

The program distribution comes in a zip archive file which can be downloaded here.

Here is the readme file for installing Twilight

Update and current data files

The distribution comes packaged with the original published datafile current to the January 15 2012 PDB release (ligands-2012-01-15.tsv). In the meantime, analysis of the PDB release January 16 2013, has delivered about 500 more entries that match the twilight selection criteria. You can update either

(a) the complete data file current up to January 16, 2013 (ligands-2013-01-16.tsv)

(b) or if you have annotated and wish to keep the old file, concatenate your old file with the file containing only new entries newligands-2012.tsv, which holds the entries that have changed during the last update (be sure to remove the header line of the appended file, though). The new entries are to a great extent those released in 2012, but the file also contains a good number of older PDB entries that became accessible through EDS during last year. Therefore, this 'update' for the year 2012 also presents entries from before 2012.

When you add the update file to the ligand table 'ligands-2012-01-15.tsv' which we have included  in our TWILIGHT distribution package, you will end up with a complete updated file equivalent to 'ligands-2013-01-16.tsv':


ligands-2013-01-16.tsv = ligands-2012-01-15.tsv + newligands-2012.tsv

Here is the readme file for installing Twilight

My texbook

A useful resource if you want to learn more about protein crystallography, ligand structures, and structure validation

For now

You can still use the web tutorial for free!

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