By following DSSR citations, I recently came across the paper by Saon et al. (2025), titled 'Identification and characterization of shifted G•U wobble pairs resulting from alternative protonation of RNA.' This paper provides a detailed analysis of shifted G-U wobble pairs in RNA, characterized by the opposite positioning of G vs. U in the standard G-U wobble pair (see figure below). Conventionally, a G-U wobble has the U located in the major groove, whereas a shifted G-U wobble has the G located in the major groove.

Specifically, the shifted G-U wobble pair involves an H-bond between the N2(G) and N3(U) atoms, which would be donor-donor if U were in its neutral form. There are three ways to rationalize the formation of this H-bond: (1) anionic U as originally proposed by Westhof et al. (2023), (2) U-enolate, and (3) G-imino tautomeric forms as illustrated by Saon et al. (2025). Since the position of the H-atoms cannot be determined from X-ray diffraction and cryo-EM structures, it is not possible (in my understanding) to determine which of these three mechanisms is correct—perhaps it involves a combination of them. What is clear is that the shifted G-U wobble pair is supported by strong experimental evidence from diverse sources. The authors identified 373 high-confidence shifted G-U wobble pairs across four separate structural clusters, spanning all three domains of life.

Structure of standard and shifted G-U wobble pairs. The examples are taken from PDB entry 8B0X (Fromm et al., 2023) and generated using DSSR and PyMOL. Atom names in the Watson-Crick edges are shown in red and blue for oxygen and nitrogen, respectively. Hydrogen bonds are depicted as dashed lines in magenta. The unusual N2(G)...N3(U) hydrogen bond is marked with a star; it would be donor-donor if U were in its neutral form. The shaded illustration at the bottom is taken from Saon et al., showing shifted G-U wobble pairs in anionic, U-enolate, and G-imino tautomeric forms.

I'm glad to see that DSSR has been used in the analysis, as shown in the following excerpts from the paper.

The selected structures were then characterized by Dissecting the Spatial Structure of RNA (DSSR) software [34]. This step output base pair, hydrogen bond, stacking, glycosidic angle, and sugar pucker information for each structure file.

From the DSSR base pair information, all G•U base pairs were identified and filtered as wobble or non-wobble base pairs. All base pairs called by DSSR as G•U wobbles were considered for the next steps of the analysis as standard wobbles. Any base pairs containing hydrogen bonds between G(N1) and U(O4), as well as G(N2) and U(N3) (see Fig. 1) were binned to shifted wobble base pairs.

From the base pair information extracted from the DSSR characterization output, the non-redundant G•U wobbles were binned based on their location in one of the five secondary structure motifs: (1) inside stem, with one WCF base pair above and one below, (2) terminal, with at least one WCF base pair above, (3) terminal, with at least one WCF base pair below, (4) unstructured, where no WCF base pair is right above or below and the wobble does not occur at the closing base pair of a hairpin loop with a maximum of 10 nucleotides, and (5) inside a loop.

Next, for each of the five members, we retrieved the 3D structure of the 20 residues from the respective pdb files and obtained the underlying secondary structures for each of the five files in dot bracket notation using DSSR [34].

DSSR implements a geometric approach to identify hydrogen bonds, including unconventional donor/acceptor combinations (e.g., the N3-to-N3 hydrogen bond in the hemiprotonated cytosine–cytosine base pair in the i-motif). It is capable of identifying all pairs that actually exist in a given structure, whether they are canonical (Watson-Crick or G-U wobble) or non-canonical. The latter pairs may include normal or modified nucleotides, regardless of their tautomeric or protonation state.

Thus, DSSR detects standard G-U wobble pairs and names them as such ('Wobble'). Moreover, it also detects shifted G-U wobble pairs and previously named them as '~Wobble,' meaning similar to a standard wobble pair. Note that the '~Wobble' designation is based on the geometric approach of DSSR, which involves the cW-W relative orientation of the two bases and a large shear value. It is not limited to wobble pairs between G and U.

After reading the Saon et al. paper, I have revised DSSR to specifically characterize shifted G-U wobble pairs and named them as 'sWobble.' The term 'shifted-Wobble' would be too long for the DSSR text output, and using 's' also reflects the shear parameter, which is key in characterizing wobble pairs. As a concrete example, the following DSSR command

x3dna-dssr -i=8B0X.cif --pair-only --more -o=8B0X-pairs.out

would generate the below output in the file 8B0X-pairs.out. Note the name sWobble, the hydrogen bond N3(imino)*N2(amino)[3.26] with a * to indicate an unusual donor/acceptor combination, and the -2.33 shear value.

607 A.U1086        A.G1099        U-G sWobble     --        cWW  cW-W
     [-171.2(anti) ~C3'-endo lambda=33.9] [-170.0(anti) ~C3'-endo lambda=59.2]
     d(C1'-C1')=11.57 d(N1-N9)=9.60 d(C6-C8)=10.07 tor(C1'-N1-N9-C1')=8.8
     H-bonds[2]: "N3(imino)*N2(amino)[3.26],O4(carbonyl)-N1(imino)[2.64]"
     interBase-angle=26  Simple-bpParams: Shear=-2.23 Stretch=0.69 Buckle=22.1 Propeller=-13.8
     bp-pars: [-2.33   0.13    -0.80   24.83   -7.98   -20.38]

The new DSSR version can automatically detect all 373 high-confidence shifted G-U wobble pairs listed in Table S3 of the Saon et al. paper. It will be released soon. This is yet another example of how DSSR is being actively improved to better serve the research community.

References

• Saon,M.S. et al. (2025) Identification and characterization of shifted G•U wobble pairs resulting from alternative protonation of RNA. Nucleic Acids Research, 53, gkaf575.
• Westhof,E. et al. (2023) Anionic G•U pairs in bacterial ribosomal rRNAs. RNA, 29, 1069–1076.
• Fromm,S.A. et al. (2023) The translating bacterial ribosome at 1.55 Å resolution generated by cryo-EM imaging services. Nat Commun, 14, 1095.

As of v2.5.4-2025jun06, DSSR automatically checks for steric clashes or exact duplicates of residues in an input coordinate file. It reports such issues instead of crashing, and will terminate only if an excessive number of overlaps are detected. An simplified example is shown below, which contains two nucleotides (G#1) on chains 0 and 1, respectively

ATOM      1  OP3   G 0   1      -4.270  51.892  37.186  1.00 27.93           O  
ATOM      2  P     G 0   1      -3.834  50.887  37.436  1.00 28.61           P  
ATOM      3  OP1   G 0   1      -4.601  49.700  37.549  1.00 27.02           O  
ATOM      4  OP2   G 0   1      -4.061  52.011  36.684  1.00 25.80           O  
ATOM      5  O5'   G 0   1      -2.906  51.105  38.691  1.00 28.01           O  
ATOM      6  C5'   G 0   1      -1.941  52.126  38.781  1.00 26.76           C  
ATOM      7  C4'   G 0   1      -1.037  51.914  39.967  1.00 26.12           C  
ATOM      8  O4'   G 0   1      -1.822  51.894  41.184  1.00 24.21           O  
ATOM      9  C3'   G 0   1      -0.285  50.591  39.988  1.00 25.12           C  
ATOM     10  O3'   G 0   1       0.884  50.614  39.172  1.00 26.09           O  
ATOM     11  C2'   G 0   1       0.008  50.411  41.462  1.00 26.05           C  
ATOM     12  O2'   G 0   1       1.102  51.209  41.880  1.00 27.46           O  
ATOM     13  C1'   G 0   1      -1.271  50.952  42.083  1.00 28.40           C  
ATOM     14  N9    G 0   1      -2.272  49.904  42.329  1.00 27.27           N  
ATOM     15  C8    G 0   1      -3.470  49.733  41.686  1.00 26.55           C  
ATOM     16  N7    G 0   1      -4.137  48.712  42.125  1.00 25.36           N  
ATOM     17  C5    G 0   1      -3.332  48.176  43.118  1.00 25.64           C  
ATOM     18  C6    G 0   1      -3.529  47.056  43.955  1.00 24.98           C  
ATOM     19  O6    G 0   1      -4.492  46.284  43.991  1.00 24.56           O  
ATOM     20  N1    G 0   1      -2.460  46.862  44.821  1.00 24.78           N  
ATOM     21  C2    G 0   1      -1.346  47.639  44.878  1.00 24.96           C  
ATOM     22  N2    G 0   1      -0.417  47.298  45.782  1.00 23.72           N  
ATOM     23  N3    G 0   1      -1.145  48.689  44.109  1.00 25.74           N  
ATOM     24  C4    G 0   1      -2.171  48.901  43.257  1.00 26.32           C  
ATOM      1  OP3   G 1   1      -6.437  51.060  40.254  1.00 27.81           O  
ATOM      2  P     G 1   1      -5.327  50.209  39.884  1.00 28.55           P  
ATOM      3  OP1   G 1   1      -5.668  48.792  39.652  1.00 26.90           O  
ATOM      4  OP2   G 1   1      -4.838  51.036  38.808  1.00 25.57           O  
ATOM      5  O5'   G 1   1      -4.301  50.297  41.090  1.00 27.94           O  
ATOM      6  C5'   G 1   1      -3.427  51.393  41.257  1.00 26.67           C  
ATOM      7  C4'   G 1   1      -2.528  51.168  42.443  1.00 26.12           C  
ATOM      8  O4'   G 1   1      -3.335  50.964  43.624  1.00 24.16           O  
ATOM      9  C3'   G 1   1      -1.648  49.928  42.372  1.00 25.13           C  
ATOM     10  O3'   G 1   1      -0.467  50.136  41.599  1.00 26.15           O  
ATOM     11  C2'   G 1   1      -1.372  49.649  43.835  1.00 25.96           C  
ATOM     12  O2'   G 1   1      -0.375  50.515  44.354  1.00 27.37           O  
ATOM     13  C1'   G 1   1      -2.714  50.006  44.458  1.00 28.21           C  
ATOM     14  N9    G 1   1      -3.608  48.845  44.581  1.00 27.06           N  
ATOM     15  C8    G 1   1      -4.771  48.614  43.895  1.00 26.37           C  
ATOM     16  N7    G 1   1      -5.340  47.496  44.226  1.00 25.18           N  
ATOM     17  C5    G 1   1      -4.502  46.957  45.190  1.00 25.44           C  
ATOM     18  C6    G 1   1      -4.599  45.755  45.923  1.00 24.77           C  
ATOM     19  O6    G 1   1      -5.480  44.892  45.864  1.00 24.39           O  
ATOM     20  N1    G 1   1      -3.532  45.594  46.796  1.00 24.63           N  
ATOM     21  C2    G 1   1      -2.504  46.469  46.949  1.00 24.81           C  
ATOM     22  N2    G 1   1      -1.560  46.145  47.845  1.00 23.58           N  
ATOM     23  N3    G 1   1      -2.396  47.594  46.280  1.00 25.56           N  
ATOM     24  C4    G 1   1      -3.422  47.779  45.423  1.00 26.12           C  

Running DSSR on the above coordinates will show the following output: [i] 0.G1 and 1.G1 in clashes: min_dist=0.57 where min_dist refers to the minimum distance between heavy atoms of the two nucleotides.

The clash-detection feature in DSSR was added in response to the bioRxiv preprint by Kretsch et al. (2025), titled "Assessment of nucleic acid structure prediction in CASP16" (https://doi.org/10.1101/2025.05.06.652459), which noted that in some predicted RNA models submitted to CASP16, multiple models were not properly delineated with MODEL/ENDMDL in PDB format or _atom_site.pdbx_PDB_model_num in mmCIF format. I communicated with the authors, who kindly provided the PDB files to help debug the issue. For more details, see the blog post Improving DSSR through extreme cases from early June 2025 at https://x3dna.org/highlights/improving-dssr-through-extreme-cases.

The bioRxiv paper by Kretsch et al. was recently published in Proteins: Structure, Function, and Bioinformatics. The relevant citation to DSSR is in Section 2.8 | Secondary Structure Analysis, as follows:

Secondary structures were extracted from CASP16 models with DSSR (v1.9.9-2020feb06) [47]. Some models, in particular due to large clashes, could not be processed by DSSR (Table S1). The base-pair list was extracted from the table in the output file directly because the dot-bracket structure produced by DSSR, in particular for multimers, contained errors. The canonical base pairs were defined as those labeled as Watson-Crick-Franklin (WC) and wobble base pairs (hereafter referred to as ‘base pairs’ or ‘pairs’). All other base pairs are defined as non-canonical base pairs and analyzed separately. Crossed base pairs (pseudoknots) were defined as non-nested canonical base pairs, that is, any canonical base pair (i,j) for which another canonical base pair (k,l) existed with i < k < j < l or k < i < l < j. Singlet base pairs were defined as any canonical base pair that was not part of a stem, that is, (i,j) such that there was no neighboring canonical base pair between i + 1 and j − 1 or between i − 1 and j + 1. Intermolecular base pairs were identified as any canonical base pair between nucleotides in different chains.

It is worth noting that DSSR is actively supported, and I always strive to respond to users’ questions via email or (preferably) on the 3DNA Forum quickly and concretely. If you have any questions about DSSR or need clarifications, please feel free to contact me. Additionally, I monitor 3DNA/DSSR citations in the literature and proactively address issues that come to my attention when necessary.

I recently came across the paper by Zurkowski et al. (2025), titled "Detecting polynucleotide motifs: Pentads, hexads, and beyond.". The authors introduce LinkTetrado, a software tool that is described as "the first fully automated method for detecting polyadic motifs in the three-dimensional structures of nucleic acids." I am somewhat surprised by this claim, as I believe it overlooks the 2015 DSSR paper, which includes a dedicated section on "Higher-order coplanar base associations (multiplets)" as shown below:

DSSR defines multiplets as three or more bases associated in a coplanar geometry via a network of hydrogen-bonding interactions. Multiplets are identified through inter-connected base pairs, filtered by pair-wise stacking interactions and vertical separations to ensure overall coplanarity (Supplementary Figures S1, S3, S4 and S7). The abundant A-minor motifs (33) (types I and II, Supplementary Figures S3, S4 and S7) are base triplets, the smallest multiplet. The G-tetrad motif, where four guanines are associated via four pairs in a square planar geometry, is another special case of a multiplet.

In fact, DSSR multiplets are all-encompassing, including pentads, hexads, heptads, octads, etc.

The DSSR User Manual has extensive discussions (see Section 3.2.4 "Multiplets (higher-order coplanar base associations)") and several examples of multiplets, including:

• Figure 8: The GUA triplet auto-identified by DSSR in PDB entry 1msy.
• Figure 12: Base pentad (AUAAG) auto-identified by DSSR in PDB entry 1jj2. The five nts (A306,U325,A331,A340,G345) are all within the 23S rRNA.

DSSR can successfully identify the multiplets reported in the Zurkowski et al. paper, although there may be minor differences due to variations in cutoffs and definitions. For instance, using PDB ID 6w9p (shown in Fig. 7F of the Zurkowski et al. paper), DSSR can perform the following:

x3dna-dssr -i=6w9p.pdb -o=6w9p.out
x3dna-dssr -i=dssr-multiplets.pdb --select-model=4 -o=G4T3.pdb

The relevant portions of DSSR output (6w9p.out) are shown below:

List of 4 multiplets
   1 nts=4 GGGG A.DG4,A.DG10,A.DG16,A.DG22
   2 nts=4 GGGG A.DG5,A.DG11,A.DG17,A.DG23
   3 nts=4 GGGG A.DG7,A.DG13,A.DG19,A.DG25
   4 nts=7 GTGTGTG A.DG6,A.DT9,A.DG12,A.DT15,A.DG18,A.DT21,A.DG24

...
2 dssr-multiplets.pdb -- an ensemble of multiplets

DSSR can further render the extracted G4T3.pdb into the following image using PyMOL:

DSSR has far more to offer than meets the eye. See the DSSR User Manual and the practical guide to DSSR-PyMOL integration for more details.

References

Lu,X.-J. et al. (2015) DSSR: an integrated software tool for dissecting the spatial structure of RNA. Nucleic Acids Res, gkv716.

Zurkowski,M. et al. (2025) Detecting polynucleotide motifs: Pentads, hexads, and beyond. PLoS Comput Biol, 21, e1013633.

By following citations related to 3DNA/DSSR, I recently discovered the paper “Computational Insights into the Structural and Energetic Properties of an A/C stacked Three-Way DNA Junction” by Singh et al. (2025), published in the new journal Computational and Structural Biotechnology Reports. The study investigates the conformational dynamics, stability, and compactness of a DNA three-way junction (3WJ) through molecular dynamics (MD) simulations.

3WJs are the simplest and most common branched nucleic acids, consisting of three double-helical stems (A, B, and C), connected at a junction point. Two of these stems can be coaxially stacked, forming either an A/B-stacked or A/C-stacked structure. The Singh et al. study utilized the A/C-stacked 3WJ PDB entry 1snj as the starting point for their simulations. DSSR efficiently identifies the three stems and two helices, with one helix formed by the coaxial stacking of two stems.

The entire section '2.2. Conformational Analysis of the DNA Junction' from the Singh et al. paper has been quoted below. I am delighted to see 3DNA/DSSR referenced in this manner.

In this work, 10 snapshots were gathered at periodic intervals of 100 ns from the 1000.0 ns molecular dynamics simulation trajectory. We have utilized the X3DNA program [48,49,50] to analyze, recreate, and visualize three-dimensional nucleic acid structures. X3DNA is capable of both the antiparallel and the parallel double-helices, the single-stranded structures, the triplexes, the quadruplexes, as well as the other intricate tertiary motifs which is present within the DNA and the RNA structures. This analysis procedures classify and identify all the fundamental interactions and also categorize the suitable base-pair-steps double-helical properties. This program uses a reference frame for the explanation of the nucleic acid basepair geometry as well as a rigorous matrix-based approach to compute the local conformational- parameters and also reconstruct structure using these parameters. The helicoidal parameters and the torsion-angle parameters are essential in comprehending folding and the rotation of the bases during the dynamical pathway.

Although there are specialized software tools designed for analyzing MD trajectories, the features available in DSSR appear to be adequate for applications like those described in the Singh et al. paper. DSSR has no external dependencies, is easy to use, and provides a more detailed characterization of nucleic acid structures than other tools. Furthermore, it is actively maintained and supported. If you have any questions or feature requests, feel free to reach out—I am here to help!

For completeness, I’ve included the relevant parts from the DSSR User Manual in this post.

The DSSR --nmr (or --md) option automates the analysis of an ensemble, such as NMR structures in the PDB or snapshots from MD simulations. The input coordinates file must be in either the classic PDB format where each model is delineated by MODEL/ENDMDL tags, or the mmCIF format where each ATOM/HETATM record has an associated model number.

The --json option makes it easy to parse the output of multiple models pragmatically. In addition to NMR structures, trajectories from MD simulations can also be processed. Popular MD packages (AMBER, GROMACS, CHARMM, etc.) all have their own specialized binary formats for trajectories. By design, DSSR does not work on these binary files. They must be converted to the standard PDB or mmCIF format to be analyzed by DSSR. The combination of --nmr and --json makes DSSR directly accessible to the MD community.

See also the threads "Do I need gromacs to use dnaMD for simulations?"" and "Update of do_x3dna package" on the 3DNA Forum.

References

Singh,A. et al. (2025) Computational Insights into the Structural and Energetic Properties of an A/C stacked Three-Way DNA Junction. Computational and Structural Biotechnology Reports, 100055.

Recently, I noticed that a user had uploaded a file to the website "DSSR-enabled Innovative Schematics of 3D Nucleic Acid Structures with PyMOL", which DSSR reported as 'no nucleotides found.' Upon visualizing it in PyMOL, the structure appeared to be a single-stranded RNA. Further investigation revealed that while the uploaded file was in PDB format, it did not adhere to the standard naming conventions for nucleotides typically used in RCSB PDB entries. For instance, an A nucleotide extracted from the file had its exocyclic amino group named as N553 instead of the conventional N6 (see below).

Following 3DNA, DSSR uses the atomic coordinates and standard names of base-ring atoms to identify a nucleotide. All known nucleotides share a common six-membered pyrimidine ring, with atoms named consecutively (N1, C2, N3, C4, C5, C6), and purines include three additional atoms (N7, C8, N9). See below for the standard names in Watson-Crick base pairs.

Without proper names for base ring atoms, DSSR is unable to identify nucleotides, resulting in the input structure being reported as 'no nucleotides found.' The same principle applies to amino acids in protein structures, such as specific naming conventions for amino nitrogen (N), carbonyl carbon (C), and alpha carbon (CA).

See also the blog posts "Mapping of modified nucleotides in DSSR" and "Name of base atoms in PDB formats".

By following DSSR citations, I recently noticed a bioRxiv preprint, titled "Assessment of nucleic acid structure prediction in CASP16" by Kretsch et al. The portion where DSSR is mentioned is as follows:

Secondary structures were extracted from CASP16 models with DSSR (v1.9.9-2020feb06). Some models, in particular due to large clashes, failed to run (Supplemental Table 1). The base-pair list was extracted from the table in the output file directly because the dot-bracket structure produced by DSSR, in particular for multimers, can contain errors.

While pleased to see DSSR cited in this significant study, I am concerned about the reported issues and would like to investigate the specific structures and error messages encountered. To better understand the problems and potentially find solutions, I have reached out to the authors for further details. Here is the message I sent initially:

You said DSSR failed to run on some models with large clashes. Could you please share the specific models and the error messages you encountered? I would also be interested in seeing the exact errors you observed in the DSSR-derived DBN for multi-mers. It would be a great opportunity for me to improve DSSR in this area, which would benefit both your group and the broader community. If you are willing to share them, please provide details—preferably on the **public 3DNA Forum**. Don’t hesitate to share openly any bugs or limitations you’ve encountered with DSSR.

The authors responded promptly and provided detailed information about the specific models and error messages encountered. After several iterations, I successfully resolved the issues and released an updated version of DSSR, namely v2.5.4-2025jun04. You can find the release notes here. This experience underscores the importance of proactively engaging with the community to enhance the functionality and reliability of a software tool.

In this blog post, I aim to share the specifics of these issues and the steps taken to address them. For ease of reading, I have formatted the response/feedback from the authors in red block quotes, and my enquiries/comments in blue. The beginning round of correspondence is as below.

Do note, the predictors in casp submit some truly atrocious models --- eg 14 atoms all at the exact same x-y-z coordinate. These errors would be with his v1.9.9-2020feb06 install though not your latest version. Would you still like them?

Yes, I would like to see how DSSR behaves with these models. Ideally, it should not crash, but output some warning messages. Only through such testing can we improve the robustness of DSSR. Overall, the more feedback I get, the better.

Buffer overflow bug in DSSR

Most of errors I had with dssr were due to clashes and all zero xyz predictions by predictors, for all of which dssr did not give an error message when dssr failed. There was a case where the prediction looked reasonable but dssr failed with the error message `dssr error*** buffer overflow detected ***`. Please see attached for the 2 pdbs that gave this error.

The two PDB files I received were R1283v3TS294_1o and R1283v3TS294_2o, as listed in Supplementary Table 1: "List of unscored models," with the "Reasons" column indicating a dssr error*** buffer overflow detected ***. I immediately acknowledged receipt of these files, as shown in the following message:

Thank you for sending me the two PDB files which caused DSSR to fail. I can verify the issue and will try to fix the bug ASAP. I'll keep you posted.

Using these data files, I was able to quickly fix the buffer overflow bug. The following is my response to the authors within one day after receiving the files:

With your sample PDB files, I have traced the issue that caused DSSR to fail. The bug was due to a 53-way (`R1283v3TS294_1o`) and 40-way (`R1283v3TS294_2o`) junction loops which are far from the norm. DSSR sets a default limit for the summary line for each loop which is more than sufficient for all normal PDB entries, but falls short for these unusual cases, leading to out of array boundaries. See the attached DSSR output after the bug fix for more details.

This is a clear example where user feedback is crucial for improving the software, which makes it better serve the community.

Zero xyz coordinates and large clashes

After fixing the out-of-bound bug, I also requested other problematic predicted models from the authors, as shown in the following message:

Along the line, please provide the sample PDB files:

- with zero xyz predictions -- I am curious to see what it looks like.
- where the DSSR-derived DBN is problematic for multi-mers

After solving these issues, I will release a new version of DSSR that would make your analysis more straightforward, and benefit other users as well.

The authors responded with the following message:

Thanks for looking into this. Here are some more examples with superimposed structures, large clash, and all zero xyzs in the zip file.

The ZIP file (error_examples.zip) contains three folders (all_zero_xyz, clash and superimposed), each with some problematic models in PDB format. Once again, I promptly acknowledged receipt of the files and was able to reproduce the reported issues.

Garbage in, garbage out. Given these problematic models, one should not expect DSSR to extract any meaningful information from them. Nonetheless, I am committed to enhancing the software so that it can handle such cases more effectively by providing clear error messages and terminating gracefully rather than crashing.

After several days of thinking, elaboration, intensive coding, and testing, I solved the problems. I then communicated the results to the authors in the following detailed message:

Thanks for the sample PDB files (`error_examples`) with all zero XYZ coordinates, large clashes, and superimposed structures. They helped me to understand the issues, think in context, and find solutions. Let's look them one by one:

1. `all_zero_xyz`: These two files `R1211TS159_1` and `R1211TS159_2` have identical contents, except for the MODEL IDs (1 and 2, respectively). Atoms with all-zero XYZ coordinates are a special case of duplicated coordinates. This has led me to implement a check for duplicated coordinates in an input file. The revised DSSR now reports duplicated coordinates and their corresponding atoms, and it quits if the number of duplicated atoms exceeds a certain threshold. For `R1211TS159_1`, the revised DSSR output would be as below:

   1 [e] xyz repeated 1904 times:[0.000      0.000      0.000]   1509-P@0.G1   3412-C6@0.C90
[w] no-of-repeats=1 max-freq=1904
...too many duplicates... quit!

2. `clash`: Both files `R1250TS208_1o` and `R1250TS417_1o` contain multiple models, as visible in PyMOL. Each PDB file uses a single MODEL/END pair to include all its models. This setup is akin to an NMR ensemble but without MODEL/ENDMDL delimiters, which leads to clashes when analyzed together. I have revised DSSR to explicitly check for such clashes and terminate execution if too many are detected. Using `R1250TS208_1o` as an example, the DSSR output would be as below:

[i] 0.G1 and 1.G1 in clashes: min_dist=0.57
[i] 0.G1 and 3.G1 in clashes: min_dist=0.35
[i] 0.G1 and 4.G1 in clashes: min_dist=0.41
...too many clashes... quit!

The above list contains only three of the many clashes detected in this file. One can notice immediately the G1 nucleotides from chains `0`, `1`, `3`, and `4` are in clashes (see the attached file `clashes_208.pdb`, which contains only G1 nucleotides from the four chains).

3. `superimposed`: The five example files (`R1283v3TS304_1o` ... `R1283v3TS304_5o`) have similar issues as the clash cases. Running the revised DSSR on `R1283v3TS304_1o` would produce the following output:

[i] 0.A1 and 2.A1 in clashes: min_dist=0.74
[i] 0.A1 and 3.A1 in clashes: min_dist=0.78
[i] 0.A1 and 4.A1 in clashes: min_dist=0.56
...too many clashes... quit!

Here A1 nucleotides from chains `0`, `2`, `3` , and `4` are in clashes (see the attached `superimpose-1.pdb`).

How the `clash` and `superimposed` categories are supposed to be different? They look similar to me.

Overall, the `error_examples` (in `all_zero_xyz`, `clash`, and `superimposed`) pose problems because they do not contain valid DNA/RNA structures as a whole. DSSR cannot extract meaningful information from these files. However, the revised DSSR explicitly highlights these issues, saving users from spending time on invalid data. Do these DSSR revisions make sense to you?

In the end, I am glad to receive the following feedback from the authors:

Thanks, these revisions all make sense! The examples I sent on clashes and superimposed were actually similar and I think the error output makes sense as well.

Final thoughts

This blog post offers an in-depth look at my efforts to enhance DSSR. As the developer of this software product, I am deeply committed to ensuring its quality and usability. I extend my gratitude to the authors for their valuable feedback and assistance in resolving these issues. In return, the updated version of DSSR (v2.5.4-2025jun04) should not only streamline their workflow but also benefit the broader user community.

For those who read through this lengthy post, I want to emphasize that DSSR is actively supported: I am here to listen and help. Any questions related to its use, bug reports, or feature requests are warmly welcomed on the 3DNA Forum. As I’ve mentioned before, please don’t hesitate to share any negative experiences or bugs with DSSR—just ensure to provide specific details so others can reproduce the issue. I will address these concerns as soon as I’m aware of them and will frankly acknowledge any mistakes I may have made. My goal is for DSSR to be a reliable software tool that the community can trust and build upon.

References

Kretsch,R.C. et al. (2025) Assessment of nucleic acid structure prediction in CASP16. bioRxiv; https://doi.org/10.1101/2025.05.06.652459.

In DSSR, the --frame option allows users to reorient a nucleic acid structure using the standard base reference frame (see Olson et al., 2001). This option can be applied not only to an individual base frame but also a base-pair frame, or the middle frame between two bases or base pairs. These variations facilitate the alignment of nucleic acid structures for a wide range of comparative analyses. In this blog post, I will demonstrate how to use the --frame option with concrete examples, enabling readers to apply this unique DSSR feature to their own projects.

The standard base reference frame

The standard base reference frame is derived from an idealized Watson-Crick base pairing geometry (top-left, figure below). The x-axis points in the direction of the major groove along what would be its pseudo-dyad axis—that is, the perpendicular bisector of the C1'...C1' vector spanning the base pair. The y-axis runs along the long axis of the idealized base-pair in the direction of the sequence strand, parallel to the C1'...C1' vector, and is displaced so as to pass through the intersection between the (pseudo-dyad) x-axis and the vector connecting the pyrimidine Y(C6) and purine R(C8) atoms. The z-axis is defined by the right-handed rule. For right-handed A- and B-DNA, the z-axis accordingly points along the 5' to 3' direction of the sequence strand.

Typical usages of the --frame option

Using the classic B-DNA dodecamer PDB entry 355d as an example, DSSR can be run with the --frame option as follows:

#             1...5..8....
# chain A: 5'-CGCGAATTCGCG -3'
# chain B: 3'-GCGCTTAAGCGC -5'

# reorient 355d in the reference frame of C1 on chain A
x3dna-dssr -i=355d.pdb --frame=A.1 -o=355d-b1.pdb

# reorient 355d in the frame of the Watson-Crick pair C1-G24
x3dna-dssr -i=355d.pdb --frame=A.1:wc -o=355d-bp1.pdb

#  ... with the minor-groove of pair C1-G24 facing the viewer
x3dna-dssr -i=355d.pdb --frame=A.1:wc-minor -o=355d-bp1-minor.pdb

# with the minor-groove of the middle AATT tract facing the viewer
x3dna-dssr -i=355d.pdb --frame='A.5:wc-minor A.8:wc' -o=355d-AATT-minor.pdb

# Rendered in cartoon-blocks with base-pair blocks, and black minor-groove
# Load 355d-AATT-minor.pml into PyMOL (bottom-left, figure above)
x3dna-dssr -i=355d-AATT-minor.pdb --cartoon-block --block-file=wc-minor -o=355d-AATT-minor.pml

The abbreviated notation A.1 refers to nucleotide numbered 1 (as indicated in the coordinates file) on chain A. Here, it denotes C1, as shown at the top of the listing. Similarly, A.5 and A.8 correspond to nucleotides A5 and T8 on chain A, respectively. In most cases, such as with 355d, the combination of chain identifier and residue number is sufficient to uniquely identify a nucleotide. More generally, other information such as model number or insertion code may be needed to specify a particular nucleotide.

In the above listing, wc after the colon (for example, A.1:wc) specifies the Watson-Crick base pair that the corresponding nucleotide participates in. Meanwhile, minor transforms the structure so that the minor-groove of the base (or base pair, or step) faces the viewer. The keywords wc and minor are settings that influence the construction or view of the frame. Case or order does not matter for these keywords as long as there is a match—for example, minor+wc works the same as wc-minor.

Two other examples combining the --frame option with cartoon-block representations

The intuitive geometric meaning of the standard base reference frame combined with the DSSR-enabled cartoon-block representation allows for an enhanced understanding of intricate structural features. In the top-right panel of the figure above, we see the classic yeast phenylalanine tRNA (PDB entry 1ehz) viewed into the minor-groove of the pseudo-knotted G19-C56 pair at the elbow of the L-shaped tertiary structure. The stacking interactions of the purines at the top-right of the panel are clearly visible in this view. In the bottom-right panel, an anti-parallel G-quadruplex from PDB entry 8ht7 is shown. The G-tetrads are automatically identified and rendered as square blocks, all with DSSR. This representation makes the chair conformation of the three-layered anti-parallel G-quadruplex crystal clear. The DSSR commands used are listed below:

# yeast tRNA (1ehz)
x3dna-dssr -i=1ehz.pdb --frame=A.19:wc-minor -o=1ehz-elbow.pdb
x3dna-dssr -i=1ehz-elbow.pdb --cartoon-block --block-file=wc-minor -o=1ehz-elbow.pml

# anti-parallel chair-shaped G-quadruplex (8ht7)
x3dna-dssr -i=8ht7.pdb --select=nts -o=8ht7-nts.pdb  # extract nucleotides, ignore amino acids
# reorient 8ht7 in the frame of the G-tetrad involving G1, in edge view
x3dna-dssr -i=8ht7-nts.pdb --frame=A.1:G4-minor -o=8ht7-Gtetrad.pdb
x3dna-dssr -i=8ht7-Gtetrad.pdb --block-cartoon --block-file=G4-minor -o=8ht7-Gtetrad.pml

References

Olson,W.K. et al. (2001) A standard reference frame for the description of nucleic acid base-pair geometry. Journal of Molecular Biology, 313, 229–237.

The 3DNA suite includes the mutate_bases program, which, as its name suggests, mutates bases while maintaining the backbone conformation. This feature was incorporated into the suite following user feedback and has been utilized in several studies before being formally published in the Li et al. (2019) paper. A key advantage is that the mutation process preserves both the geometry of the sugar-phosphate backbone and the base reference frame, encompassing position and orientation. Consequently, re-analyzing the mutated model yields identical base-pair and step parameters as those of the original structure.

In DSSR, the standalone mutate_bases program has become the mutate sub-command with enhanced functionality and improved usability, as documented in the User Manual. The mutate module allows users to perform base mutations efficiently and effectively by taking advantage of the powerful DSSR analysis engine.

To further expand the modeling capabilities of the DSSR, v2.5.3 introduced the --mutate-type option to allow for backbone mutations, based on the base reference frame. Furthermore, the target can be any fragment, regardless of length or composition, rather than just a single nucleotide. When combined with the rebuild module, this feature significantly enhances DSSR’s ability to model nucleic acid structures.

Here is an example of modeling PDB entry 1msy, a 27-nt structure (1msy.pdb) that mimics the sarcin/ricin loop from E. coli 23S ribosomal RNA.

x3dna-dssr analyze -i=1msy.pdb --ss --rebuild -o=1msy-expt.out
mv dssr-ssStepPars.txt 1msy-step.txt
x3dna-dssr rebuild --backbone=RNA --par-file=1msy-step.txt -o=1msy-step.pdb

x3dna-dssr -i=1msy.pdb --select-resi='A 2654' -o=1msy-A2654.pdb
x3dna-dssr -i=1msy.pdb --select-resi='A 2655' -o=1msy-G2655.pdb
x3dna-dssr -i=1msy-A2654.pdb --frame=2654 -o=frame___A.pdb
x3dna-dssr -i=1msy-G2655.pdb --frame=2655 -o=frame___G.pdb

x3dna-dssr mutate -i=1msy-step.pdb --entry='num=8 to=A; num=9 to=G' -o=1msy-C2endo.pdb --mutate-part=whole
x3dna-dssr --connect-file -i=1msy-C2endo.pdb -o=1msy-C2endo-cnt.pdb --po-bond=5.0

1. The analyze step uses options --ss and --rebuild to generate the file dssr-ssStepPars.txt (containing base-step parameters), which is then renamed to 1msy-step.txt. The rebuild step employs 1msy-step.txt to construct a structure (1msy-step.pdb) with regular C3'-endo sugar RNA backbone conformation. Note that the rebuilt structure has nucleotides numbered from 1 to 27, while in the PDB 1msy, they correspond to 2647 to 2673, respectively.
2. However, the A2654 and G2655 dinucleotides in 1msy are actually in C2'-endo sugar conformation, creating the S-shaped structure around the GpU platform. The above rebuilt structure does not reflect this distortion. So we extract A2654 and G2655 with --select-resi and then put each in its standard base reference frame, named frame___A.pdb and frame___G.pdb, respectively.
3. Now we mutate A8 and G9 in the rebuilt structure 1msy-step.pdb to A and G with option --mutate-part=backbone to ensure the backbone conformations are changed according to those in frame___A.pdb and frame___G.pdb, respectively. The resulting structure is named 1msy-C2endo.pdb. Now the S-shape around the GpU platform is preserved, even though the backbone are not always covalently connected, due to large O3'(i-1) to P(i) distances between neighboring nucleotides. The last step is to generate CONECT records with --connect-file option to connect the backbone atoms explicitly, resulting in more smooth backbone cartoon representation in PyMOL as shown below.

As noted in the Li et al. (2019) paper, users can optimize this approximate backbone connection using Phenix, while keeping the base atoms fixed. The 3DNA-Phenix combination leads to a model where the base geometry strictly follows the parameters prescribed in the user-specified file, and the backbone is regularized with improved stereochemistry and a ‘smooth’ appearance in ribbon representation.

There are other variants of the DSSR mutate module, including for building Z-DNA backbones. However, the above example is sufficient to demonstrate the power of the integrated approach enabled by DSSR for the analysis and modeling of nucleic acid structures. See the DSSR User Manual for more details.

References

Li,S. et al. (2019) Web 3DNA 2.0 for the analysis, visualization, and modeling of 3D nucleic acid structures. Nucleic Acids Res., 47, W26–W34.

Background and motivation

In late 2021, I came across the thread titled "create a 26 bp RNA from a 13 bp system" on the PyMOL mailing list. The thread began with a user asking:

I have an RNA duplex with 13 base-pairs (attached). Is it possible to duplicate this system and then fuse the two molecules to create a 26 base-pair long system using the pymol.

The message is both concise and clear. The attached 13 base-pair RNA duplex (named model.pdb) makes the task easier to understand. An expert PyMOL user responded quickly, providing a set of suggested PyMOL commands along with warnings about the complexity of the task.

No, not automatically. Your RNA is very distorted from the standard A-form. I doubt any modeling program can accurately extend such a distorted helix. Maybe someone else will prove me wrong. ... You can align the terminal base pairs manually through a series of commands. If you try by dragging one copy relative to another, you will wind up pulling out all of your hair. The commands and patience will keep you out of the mad house.

DSSR offers unique capabilities to automatically manipulate nucleic acid structures. It also enables the duplication of an RNA duplex, as specifically requested by the original poster. In my initial response to the thread, I provided a DSSR-based solution for duplicating the RNA duplex without detailed explanations, aiming to confirm whether the result met the user's needs. The feedback was positive, as indicated below:

Thanks for proving me wrong. Congratulations on your duplicated model! Please share the commands that you used with DSSR to generate the duplicated helix. --- from the PyMOL responder​

Thanks a lot for your help. The model you have duplicated is exactly what I am looking for (checked it with VMD). Unfortunately I do not have access to DSSR-Pro. Is there any way that I can reproduce your procedure with x3dna-dssr? I need to create different numbers of duplicates (2,4,6,5,8) for different systems and this will be very helpful. --- from the original poster

During that period (near the end of 2021), I was facing a funding gap. To address this challenge, we decided to license DSSR through Columbia Technology Ventures (CTV) and introduced a Pro version of DSSR for commercial users and academic institutions, providing advanced modeling features and dedicated support. Note that DSSR Pro Academic licenses entail a one-time fee of $1,020. The software can be installed on Windows, macOS, or Linux. While not explicitly included in the license agreement, I provide direct support to Pro license users via email, phone, or Zoom whatever convenient to help address their issues. I care about user experience, especially for those who invest in the Pro version.

Following user feedback, I shared detailed instructions on duplicating an RNA duplex using DSSR Pro. Gratefully, the original poster purchased a DSSR Pro Academic license and successfully duplicated the RNA helix. Later, we communicated via email to assist with other related tasks. This experience underscored the importance of engaging with the scientific community and addressing user needs to drive software development and adoption.

Detailed instructions

With funding from grant R24GM153869, I have transferred many DSSR Pro features into the free DSSR Academic version to better serve the scientific community. Included below are detailed step-by-step commands script for duplicating an RNA duplex using either DSSR Pro or the free DSSR Academic v2.5.2. The script runs instantaneously in a terminal window.

x3dna-dssr tasks -i=model.pdb --frame-pair=last -o=model1-ref-last.pdb

x3dna-dssr fiber --seq=GG --rna-duplex -o=conn.pdb
x3dna-dssr tasks -i=conn.pdb --frame-pair=first --remove-pair -o=ref-conn.pdb

x3dna-dssr tasks --merge-file='model1-ref-last.pdb ref-conn.pdb' -o=temp1.pdb

x3dna-dssr tasks -i=temp1.pdb --frame-pair=last --remove-pair -o=temp2.pdb
x3dna-dssr tasks -i=model.pdb --frame-pair=first -o=model1-ref-first.pdb

x3dna-dssr tasks --merge-file='temp2.pdb model1-ref-first.pdb' -o=duplicate-model.pdb

x3dna-dssr --order-residue -i=duplicate-model.pdb -o=temp3.pdb
x3dna-dssr --renumber-residue -i=temp3.pdb -o=temp4.pdb
x3dna-dssr --connect-file -i=temp4.pdb -o=RNA-duplicate.pdb

The procedure is essentially the same as the one used in "Building extended Z-DNA structures with backbones using DSSR". For completeness, I have included detailed explanations for each step here as well.

1. Setting Up the Reference Frame:

   • The first command places the 13 base-pair RNA duplex (model.pdb) into the reference frame of its last base pair, resulting in model1-ref-last.pdb.

2. Creating the Fiber Connector:

   • The fiber model is constructed using an RNA duplex (--rna-duplex) with the sequence GG on the leading strand (conn.pdb). This connector is oriented into the reference frame of its first base pair.
   • The first base pair is removed. Thus, the resulting coordinate file, ref-conn.pdb, contains only one pair.
   • Note: The sequence GG serves as a placeholder. It can be replaced with any other two bases: for instance, changing --seq=GG to --seq=AA. Moreover, using --seq=GA10G allows for creating a linker with 10 adenines.

3. Merging PDB Files:

   • The two PDB files, model1-ref-last.pdb and ref-conn.pdb, share a common reference frame and are merged into a single file named temp1.pdb.

4. Adjusting the Reference Frame:

   • The merged file (temp1.pdb) is then aligned with the last base pair, which is subsequently removed to produce temp2.pdb. This completes the role of the GG fiber connector.

5. Reorienting the RNA Duplex:

   • The original 13 base-pair RNA duplex (model.pdb) is reoriented into the reference frame of its first base pair, generating model1-ref-first.pdb.

6. Final Merging:

   • The two PDB files, temp2.pdb and model1-ref-first.pdb, contain identical 13 base-pair RNA duplexes but in different orientations. They are merged into a single file (duplicate-model), establishing the final duplicated RNA structure.
7. Bookkeeping for Visualization:
   • The last three commands utilize options (--order-residue, --renumber-residue, --connect-file) to prepare the structure for visualizing backbone connectivity in PyMOL. Specifically, it solves the reported issue that "PyMOL does not generate the cartoon representation for the backbones of your duplicate helix." in my solution posted on the PyMOL mailing list.

The duplicated RNA helix is illustrated in the image below.

Some caveats

The original 13 base-pair RNA duplex (model.pdb) contains three main PDB format inconsistencies:

• Missing Chain Identifiers: The two strands lack proper chain identifiers in column 22.
• Incorrect Covalent Bond Distance: Nucleotides RU25 and RC26 are not covalently linked. Specifically, the distance between O3' of RU25 and P of RC26 is 3.5 Å, exceeding the expected 1.6 Å for a proper covalent bond.
• Misclassified Ligand Record: The ligand (LIG27) is incorrectly designated as ATOM instead of the appropriate HETATM record.