X3DNA-DSSR: a resource for structural bioinformatics of nucleic acids
(An NIGMS National Resource supported by NIH grant R24GM153869)

DSSR produces RNA secondary structures in connect table (.ct) format. According to "RNAstructure Command Line Help: File Formats" (with slight editing):

CT File Format

A CT (Connectivity Table) file contains secondary structure information for a sequence. These files are saved with a CT extension. When entering a structure to calculate the free energy, the following format must be followed.

1. Start of first line: number of bases in the sequence
2. End of first line: title of the structure
3. Each of the following lines provides information about a given base in the sequence. Each base has its own line, with these elements in order:
   • Base number: index n
   • Base (A, C, G, T, U, X)
   • Index n-1
   • Index n+1
   • Number of the base to which n is paired. No pairing is indicated by 0 (zero).
   • Natural numbering. RNAstructure ignores the actual value given in natural numbering, so it is easiest to repeat n here.

Using PDB entry 1msy as an example (see Figure 1 below):

Figure 1. 3D and 2D structures of PDB entry 1msy. (A) 3D schematic auto-created via the DSSR-PyMOL integration. The labeled residues follow PDB coordinates. (B) 2D diagram rendered with VARNA using DSSR-derived 2D structural information in the .ct format. This figure was annotated using Inkscape.

With commands:

x3dna-dssr -i=1msy.pdb
cp dssr-2ndstrs.ct 1msy-dssr-default.ct

The file 1msy-dssr-default.ct has the following contents:

   27 ENERGY = 0.0 [1msy] -- secondary structure derived by DSSR
    1 U     0     2     0  2647
    2 G     1     3    26  2648
    3 C     2     4    25  2649
    4 U     3     5    24  2650
    5 C     4     6    23  2651
    6 C     5     7    22  2652
    7 U     6     8     0  2653
    8 A     7     9     0  2654
    9 G     8    10     0  2655
   10 U     9    11     0  2656
   11 A    10    12     0  2657
   12 C    11    13    17  2658
   13 G    12    14     0  2659
   14 U    13    15     0  2660
   15 A    14    16     0  2661
   16 A    15    17     0  2662
   17 G    16    18    12  2663
   18 G    17    19     0  2664
   19 A    18    20     0  2665
   20 C    19    21     0  2666
   21 C    20    22     0  2667
   22 G    21    23     6  2668
   23 G    22    24     5  2669
   24 A    23    25     4  2670
   25 G    24    26     3  2671
   26 U    25    27     2  2672
   27 G    26     0     0  2673

Here the first line contains 27 (as the number of bases) and ENERGY = 0.0 [1msy] -- secondary structure derived by DSSR (as the title). While RNAstructure ignores the actual values given in natural numbering, DSSR outputs the residue numbers of the nucleotides (e.g. U2467 and G2673) in the PDB file.

With the DSSR option --structure-title (or --str-title, actually via regex "^-?-?str(ucture)?[-_]?title"), users can set the title for the derived .ct file, as shown below:

x3dna-dssr -I=1msy.pdb --structure-title='CT file derived from DSSR'
cp dssr-2ndstrs.ct 1msy-dssr-title.ct

   27 CT file derived from DSSR
    1 U     0     2     0  2647
    2 G     1     3    26  2648
......
   26 U    25    27     2  2672
   27 G    26     0     0  2673

One can also remove the title, by using an empty string "" (i.e., --str-title="") or simply --str-title (or --str-title=).

x3dna-dssr -I=1msy.pdb --structure-title=""
cp dssr-2ndstrs.ct 1msy-dssr-notitle.ct

   27
    1 U     0     2     0  2647
    2 G     1     3    26  2648
......

With the --more option, DSSR also outputs additional info that can be used to easily identify a nucleotide and its pairing partner.

x3dna-dssr -I=1msy.pdb --more --structure-title="1msy with extra info"
cp dssr-2ndstrs.ct 1msy-dssr-extra.ct

   27 1msy with extra info
    1 U     0     2     0  2647 # name=A.U2647
    2 G     1     3    26  2648 # name=A.G2648, pairedNt=A.U2672
    3 C     2     4    25  2649 # name=A.C2649, pairedNt=A.G2671
......

Note that unlike for the .bpseq format with extra info which cannot be fed directly into VARNA, the extra info for the .ct format causes no troubles for VARNA to visualize the 2d structure.

The --structure-title option is another small feature implemented in DSSR. It is currently not documented in the DSSR User Manual since this feature is unlikely of general interest.

DSSR commands used, and the output .ct files:

x3dna-dssr -i=1msy.pdb
cp dssr-2ndstrs.ct 1msy-dssr-default.ct

x3dna-dssr -I=1msy.pdb --structure-title='CT file derived from DSSR'
cp dssr-2ndstrs.ct 1msy-dssr-title.ct

x3dna-dssr -I=1msy.pdb --structure-title=""
cp dssr-2ndstrs.ct 1msy-dssr-notitle.ct

x3dna-dssr -I=1msy.pdb --more --structure-title="1msy with extra info"
cp dssr-2ndstrs.ct 1msy-dssr-extra.ct

• 1msy-dssr-default.ct
• 1msy-dssr-title.ct
• 1msy-dssr-notitle.ct
• 1msy-dssr-extra.ct

By default, DSSR produces RNA secondary structures in three commonly used file formats––ViennaRNA package dbn, Mfold connect table (.ct), and CRW bpseq––that can be fed directly into visualization tools such as VARNA. In this blog post, I want to dig deeper into the bpseq format, and show the variations available from DSSR.

According to "RNA STRAND v2.0 - The RNA secondary STRucture and statistical ANalysis Database" (with slight editing):

BPSEQ format:The file name should end with the suffix ".bpseq", as in "mystr.bpseq". The bpseq format is a simple text format in which there is one line per base in the molecule, listing the position of the base (leftmost position is 1), the base name (A,C,G,U, or other alphabetical characters), and the position number of the base to which it is paired, with a 0 denoting that the base is unpaired. For more information, see the Comparative RNA Web Site. An example is as follows:

1 G 8 
2 G 7 
3 C 0 
4 A 0 
5 U 0  
6 U 0 
7 C 2 
8 C 1 

For complexes with more than one molecule, the molecules are listed in sequence, with the base pairs numbers of each successive molecule following in order from the previous molecule.

The bases in bpseq format are identified by position numbers starting from 1 for the leftmost position. That is the convention DSSR follows by default in its .bpseq output. For example, for the PDB entry 1msy, which contains 27 nucleotides, the command x3dna-dssr -i=1msy.pdb will generate a file named dssr-2ndstrs.bpseq with the following contents (abbreviated):

    1 U     0
    2 G    26
    3 C    25
......
   25 G     3
   26 U     2
   27 G     0

However, according to PDB atomic coordinates, the nucleotides are numbered from U2647 (#1) to G2673 (#27) as shown in the Figure 1 below:

Figure 1. 3D and 2D structures of PDB entry 1msy. (A) 3D schematic auto-created via the DSSR-PyMOL integration. The labeled residues follow PDB coordinates. (B) 2D diagram rendered with VARNA using DSSR-derived 2D structural information in the .ct format. This figure was annotated using Inkscape.

It makes sense that the labelling of bases in the 2D bpseq format follows those from the 3D atomic coordinates in the PDB. Thus instead of starting from position 1 as shown above, the bpseq file would start with 2647. That's exactly what the DSSR --bpseq option is created for. Thus, with the command x3dna-dssr -i=1msy.pdb --bpseq, the output file dssr-2ndstrs.bpseq now has the following contents (abbreviated):

  2647 U      0
  2648 G   2672
  2649 C   2671
......
  2671 G   2649
  2672 U   2648
  2673 G      0

This .bpseq file can be read by VARNA (tested with VARNAv3-93.jar) to generate a 2D image as shown in Figure 1(B) above.

Moreover, with the command x3dna-dssr -i=1msy.pdb --bpseq=extra, the output file dssr-2ndstrs.bpseq now contains additional info to easily identify a nucleotide and its pairing partner:

  2647 U      0 # name=A.U2647
  2648 G   2672 # name=A.G2648, pairedNt=A.U2672
  2649 C   2671 # name=A.C2649, pairedNt=A.G2671
......
  2671 G   2649 # name=A.G2671, pairedNt=A.C2649
  2672 U   2648 # name=A.U2672, pairedNt=A.G2648
  2673 G      0 # name=A.G2673

It should be noted that this .bpseq output file is no longer compliant to the standard, and can not be fed into VARNA for visualization.

The --bpseq option has been added upon users' request. The --bpseq=extra variation was implemented recently to ensure that the --bpseq option by itself produce a valid .bpseq file without extra info (e.g., enabled with the global --more option). Now the extra info for .bpseq output is enabled only by setting --bpseq=extra explicitly.

This --bpseq option and its evolution is a good example of how DSSR responds to community requests. I'm here to listen and I'm always willing to improve DSSR that better fit users' needs. If you make use of DSSR in your pipeline and need some adaptions, please do not hesitate to contact me. I may consider adding a new option or revising the code otherwise that would streamline the integration of DSSR into your project.

DSSR commands used, and the output .bpseq files:

x3dna-dssr -i=1msy.pdb
cp dssr-2ndstrs.bpseq 1msy-dssr-default.bpseq

x3dna-dssr -i=1msy.pdb --bpseq
cp dssr-2ndstrs.bpseq 1msy-dssr-bpseq.bpseq

x3dna-dssr -i=1msy.pdb --bpseq=extra
cp dssr-2ndstrs.bpseq 1msy-dssr-bpseq-extra.bpseq

• 1msy-dssr-default.bpseq
• 1msy-dssr-bpseq.bpseq
• 1msy-dssr-bpseq-extra

By following citations to 3DNA/DSSR, I recently came across the paper "RNAtango: Analysing and comparing RNA 3D structures via torsional angles" in PLOS Computational Biology by Mackowiak M, Adamczyk B, Szachniuk M, and Zok T. This work provides a nice summary of definitions of torsion and pseudo-torsion angles in RNA structure, and an angular metrics (MCQ, Mean of Circular Quantities) to score structure similarity. The RNAtango web application allows user to explore the distribution of torsion angles in a single structure/fragment (Single model), compare RNA models with a native structure (Models vs Target), or perform a comparative analysis in a set of models (Model vs Model).

In the Introduction section, 3DNA/DSSR are mentioned along with other related tools, as below:

Several bioinformatics tools have been designed for analyzing torsion and pseudotorsion angles, each with its own strengths and limitations. 3DNA, an open-source toolkit, provides comprehensive functionality, including torsion and pseudotorsion angle calculations [27], but lacks support for the current standard PDBx/mmCIF file format. DSSR, the successor to 3DNA, overcomes this limitation by supporting both PDB and PDBx/mmCIF files. However, it is a closed-source, commercial application that requires licensing, even for research purposes [28]. Curves+, another tool used for torsion angle analysis, is currently inaccessible due to the unavailability of its webpage and source code hosting [29]. Barnaba, a Python library and toolset for analyzing single structures or trajectories, supports torsion angle calculations but, like 3DNA, does not support the PDBx/mmCIF format [30]. For users seeking a more user friendly option, AMIGOS III offers a PyMOL plugin that calculates pseudotorsion angles and presents them in Ramachandran-like plots [17].

Every bioinformatic software has been developed for a specific purpose, and no two such tools can be identical. It is a good thing that the community has a choice for RNA backbone analysis. Indeed, 3DNA has been superseded by DSSR, which is licensed by Columbia Technology Ventures (CTV) to ensure its continuous development and availability. However, DSSR remain competitive due to its unmatched functionality, usability, and support: it saves users a substantial amount of time and effort when compared to other options.

From the very beginning, it has been my dream to make DSSR stand out for its quality and value, and be widely accessible. The CTV DSSR distribution by no means follow typical commercial license for a software product: specifically, it does not include a license key to limit DSSR's usage to a specific machine and operating system, and there is no expire date for the software either. Moreover, the Basic Academic license was free of charge when DSSR was initially licensed by the CTV in August 2020, and remained so until around end of 2021 when the web-based "Express Licenses" functionality no longer worked. Manually handling the large number of requests for free academic licenses was not sustainable, and that was when the DSSR Basic Academic free license was removed. Upon user requests, we late on re-introduced DSSR Basic Academic license, but with a one-time fee of $200 to cover the running cost. That may be reason for the remark in the RNAtango paper that DSSR "requires licensing, even for research purposes".

With the recent NIH R24 funding support on "X3DNA-DSSR: a resource for structural bioinformatics of nucleic acids", we are providing DSSR Basic free of charge to the academic community. Academic Users may submit a license request for DSSR Basic or DSSR Pro by clicking "Express Licensing". Checking the list of licensees, I am thrilled to see the many new DSSR users from leading institutions around the world, including Stockholm University, Ghent University, Universitaet Heidelberg, University of Palermo, CSSB-Hamburg, Nicolaus Copernicus University, NIH, Harvard, ... Clearly, DSSR fills a niche, and the demands for it remain strong!

Back to torsion angles, it is safe to say that DSSR has unique features not available or easily accessible elsewhere. Here are some use cases using tRNA PDB entry 1ehz as an example:

x3dna-dssr -i=1ehz.cif # generate dssr-torsions.txt among other output files
x3dna-dssr -i=1ehz.cif --torsion-file -o=1ehz-torsions.txt # just the torsion file 1ehz-torsions.txt
x3dna-dssr -i=1ehz.cif --json | jq .nts[54] > 1ehz-PSU55.txt # DSSR-derived features for nucleotide PSU55

Users can easily run the DSSR commands listed above and get the results in human-readable text and machine-friendly JSON formats. For verification, the contents of 1ehz-torsions.txt and 1ehz-PSU55.txt are available by clicking the links.

It is worth noting that DSSR has the --nmr option for the analysis of an ensemble of NMR structures, in .pdb or .cif format, as deposited in the PDB. The combination of --nmr and --json renders DSSR easily accessible to the molecular dynamics (MD) community.

In principle, calculating torsion angles is a straightforward process. In reality, factors such as modified nucleotides (especially pseudouridine), missing atoms, NMR ensembles or MD trajectories, PDB vs mmCIF formats, etc. make the implementation complicated. Without paying great attention to details, it is easy to make subtle mistakes. For example, with RNAtango the chi (χ) torsion angle for A.PSU55 of 1ehz is listed as -152.42°, which is wrong. The correct value should be -147.0° as reported by DSSR (see below and the link 1ehz-PSU55.txt above).

DSSR provides a comprehensive list of backbone parameters (as listed below for 1ehz). The program is efficient and robust, and has been battle tested. I am always quick to fix any bugs once verified, and am willing to add new features once thoroughly studied. In short, DSSR has been designed to be a reliable tool that the community can trust and build upon.

DSSR-derived backbone features for tRNA 1ehz:

         Output of DNA/RNA backbone conformational parameters
             DSSR v2.4.5-2024sep24 by xiangjun@x3dna.org
******************************************************************************************
Main chain conformational parameters:

  alpha:   O3'(i-1)-P-O5'-C5'
  beta:    P-O5'-C5'-C4'
  gamma:   O5'-C5'-C4'-C3'
  delta:   C5'-C4'-C3'-O3'
  epsilon: C4'-C3'-O3'-P(i+1)
  zeta:    C3'-O3'-P(i+1)-O5'(i+1)
  e-z:     epsilon-zeta (BI/BII backbone classification)

  chi for pyrimidines(Y): O4'-C1'-N1-C2; purines(R): O4'-C1'-N9-C4
    Range [170, -50(310)] is assigned to anti, and [50, 90] to syn

  phase-angle: the phase angle of pseudorotation and puckering
  sugar-type: ~C2'-endo for C2'-endo like conformation, or
               ~C3'-endo for C3'-endo like conformation
              Note the ONE column offset (for easy visual distinction)

ssZp: single-stranded Zp, defined as the z-coordinate of the 3' phosphorus atom
      (P) expressed in the standard reference frame of the 5' base; the value is
      POSITIVE when P lies on the +z-axis side (base in anti conformation);
      NEGATIVE if P is on the -z-axis side (base in syn conformation)
  Dp: perpendicular distance of the 3' P atom to the glycosidic bond
      [Ref: Chen et al. (2010): "MolProbity: all-atom structure
            validation for macromolecular crystallography."
            Acta Crystallogr D Biol Crystallogr, 66(1):12-21]
splay: angle between the bridging P to the two base-origins of a dinucleotide.

          nt               alpha    beta   gamma   delta  epsilon   zeta     e-z        chi            phase-angle   sugar-type    ssZp     Dp    splay
 1     G A.G1                ---  -128.1    67.8    82.9  -155.6   -68.6    -87(BI)   -167.8(anti)    16.1(C3'-endo)  ~C3'-endo    4.59    4.57   24.92
 2     C A.C2              -67.4  -178.4    53.8    83.4  -145.1   -76.8    -68(BI)   -163.8(anti)    16.1(C3'-endo)  ~C3'-endo    4.52    4.63   21.15
 3     G A.G3              -74.5   169.7    59.5    80.7  -148.3   -80.0    -68(BI)   -161.9(anti)    14.6(C3'-endo)  ~C3'-endo    4.75    4.69   22.28
 4     G A.G4              -64.4   162.2    60.7    82.2  -157.4   -68.7    -89(BI)   -168.7(anti)    20.8(C3'-endo)  ~C3'-endo    4.68    4.57   25.22
 5     A A.A5              -74.7  -176.5    53.4    84.9  -137.5   -81.7    -56(BI)   -162.9(anti)     4.8(C3'-endo)  ~C3'-endo    4.49    4.76   22.04
 6     U A.U6              -48.8   157.6    55.3    81.3  -151.0   -77.0    -74(BI)   -160.0(anti)    18.2(C3'-endo)  ~C3'-endo    4.31    4.51   22.89
 7     U A.U7              -59.5  -178.7    62.5   137.3  -105.9   -52.0    -54(--)   -133.1(anti)   156.1(C2'-endo) ~C2'-endo     1.55    1.41  126.99
 8     U A.U8              -83.8  -145.6    55.4    78.6  -142.8  -118.6    -24(--)   -161.5(anti)    10.5(C3'-endo)  ~C3'-endo    4.60    4.76   62.37
 9     A A.A9              -69.7  -141.7    52.3   147.8  -106.2   -77.3    -29(--)    -70.5(anti)   149.8(C2'-endo) ~C2'-endo     1.00    1.14   57.38
 10    g A.2MG10           177.8   147.2    60.1    89.3  -126.2   -88.7    -37(--)    169.6(anti)     6.6(C3'-endo)  ~C3'-endo    4.68    4.63   23.87
 11    C A.C11             -56.1   167.9    48.2    87.2  -150.5   -69.9    -81(BI)   -160.9(anti)    16.8(C3'-endo)  ~C3'-endo    4.28    4.46   21.20
 12    U A.U12             -67.8   172.9    51.8    80.7  -158.5   -65.2    -93(BI)   -158.3(anti)    25.2(C3'-endo)  ~C3'-endo    4.29    4.45   21.01
 13    C A.C13             166.6  -169.9   178.6    82.5  -153.1   -97.4    -56(BI)   -168.3(anti)    23.7(C3'-endo)  ~C3'-endo    4.28    4.36   31.59
 14    A A.A14              83.4  -158.3  -114.6    92.0  -125.5   -57.3    -68(--)   -170.7(anti)   358.9(C2'-exo)   ~C3'-endo    4.67    4.74   38.01
 15    G A.G15             -55.1   162.5    51.9    79.8  -136.3  -143.9      8(--)   -164.5(anti)    16.0(C3'-endo)  ~C3'-endo    4.72    4.74   26.17
 16    u A.H2U16            -6.1    91.2    76.8    96.8   -61.8  -131.2     69(--)    -85.8(anti)    18.8(C3'-endo)  ~C3'-endo   -0.71    3.38  145.77
 17    u A.H2U17            27.8   107.7   174.1    94.8   178.0    76.2    102(--)   -142.5(anti)   341.4(C2'-exo)   ~C3'-endo   -0.90    4.20  105.55
 18    G A.G18              45.4  -159.4    59.0   150.6   -95.2  -179.1     84(BII)   -99.5(anti)   154.3(C2'-endo) ~C2'-endo     1.60    1.09   51.64
 19    G A.G19             -71.4  -178.9    53.8   153.8   -91.6   -83.7     -8(--)    -80.3(anti)   167.6(C2'-endo) ~C2'-endo    -1.14    0.48  130.30
 20    G A.G20             -81.3  -150.7    47.8    89.9  -122.3   -54.1    -68(--)    177.8(anti)     8.7(C3'-endo)  ~C3'-endo    4.90    4.76   57.04
 21    A A.A21             -75.6   148.6  -176.6    78.2  -168.9   -75.6    -93(BI)   -160.2(anti)    13.0(C3'-endo)  ~C3'-endo    4.00    4.26   40.66
 22    G A.G22             158.8   153.5   179.3    82.0  -145.0   -80.4    -65(BI)   -175.5(anti)   353.8(C2'-exo)   ~C3'-endo    4.60    4.73   25.62
 23    A A.A23             -53.3   174.8    52.5    82.3  -155.3   -66.4    -89(BI)   -158.0(anti)    12.6(C3'-endo)  ~C3'-endo    4.18    4.61   22.96
 24    G A.G24             -68.8   178.2    46.8    83.6  -144.3   -72.8    -71(BI)   -160.7(anti)    13.4(C3'-endo)  ~C3'-endo    4.63    4.74   20.51
 25    C A.C25             -65.1   168.9    53.9    83.3  -145.1   -68.4    -77(BI)   -160.3(anti)    17.4(C3'-endo)  ~C3'-endo    4.56    4.70   30.70
 26    g A.M2G26           -53.8   170.8    47.7    86.0  -136.3   -76.9    -59(BI)   -163.4(anti)     9.3(C3'-endo)  ~C3'-endo    4.57    4.67   27.36
 27    C A.C27             -53.0   166.9    43.6    83.4  -148.5   -73.4    -75(BI)   -168.2(anti)    18.3(C3'-endo)  ~C3'-endo    4.53    4.62   23.07
 28    C A.C28             -72.4   178.3    49.3    80.1  -152.1   -67.0    -85(BI)   -160.6(anti)     9.2(C3'-endo)  ~C3'-endo    4.55    4.73   21.61
 29    A A.A29             -66.6   174.0    55.6    81.4  -155.5   -78.3    -77(BI)   -165.9(anti)    13.7(C3'-endo)  ~C3'-endo    4.73    4.65   26.96
 30    G A.G30             -54.0   165.9    56.9    83.6  -144.7   -62.3    -82(BI)   -171.7(anti)    14.5(C3'-endo)  ~C3'-endo    4.67    4.65   25.72
 31    A A.A31             -69.9   177.8    52.3    83.7  -137.0   -75.5    -61(BI)   -156.7(anti)    14.6(C3'-endo)  ~C3'-endo    4.24    4.72   21.52
 32    c A.OMC32           -52.7   161.4    49.3    80.1  -145.9   -71.2    -75(BI)   -149.9(anti)    20.4(C3'-endo)  ~C3'-endo    4.16    4.63   25.94
 33    U A.U33             -67.7  -177.0    47.0    82.1  -148.0   -53.7    -94(BI)   -148.2(anti)    13.3(C3'-endo)  ~C3'-endo    4.19    4.64   75.47
 34    g A.OMG34           171.1   148.1    52.5    83.4  -132.5   -71.8    -61(BI)   -171.2(anti)    12.2(C3'-endo)  ~C3'-endo    4.15    4.58   22.09
 35    A A.A35             -47.7   163.7    40.2    80.9  -143.7   -59.5    -84(BI)   -154.4(anti)    21.9(C3'-endo)  ~C3'-endo    4.20    4.54   20.57
 36    A A.A36             -52.4   165.7    51.3    72.2  -160.4   -85.2    -75(BI)   -158.4(anti)    45.8(C4'-exo)   ~C3'-endo    4.49    4.31   24.48
 37    g A.YYG37           -57.5   163.0    47.8    81.1  -148.1   -67.0    -81(BI)   -168.8(anti)    15.4(C3'-endo)  ~C3'-endo    4.63    4.65   32.08
 38    A A.A38             -61.8  -180.0    46.9    82.5  -136.8   -76.4    -60(BI)   -169.4(anti)     2.4(C3'-endo)  ~C3'-endo    4.63    4.78   23.75
 39    P A.PSU39           -47.7   160.4    53.3    79.3  -140.1   -68.6    -72(BI)   -165.6(anti)    15.8(C3'-endo)  ~C3'-endo    4.55    4.68   26.68
 40    c A.5MC40           -67.4   172.0    56.2    83.2  -154.2   -74.9    -79(BI)   -162.6(anti)    17.3(C3'-endo)  ~C3'-endo    4.52    4.60   27.71
 41    U A.U41             -68.2  -179.4    52.4    78.9  -137.3   -84.7    -53(BI)   -169.0(anti)    13.4(C3'-endo)  ~C3'-endo    4.54    4.75   24.14
 42    G A.G42             -47.9   158.7    55.6    79.8  -160.3   -70.3    -90(BI)   -169.0(anti)    20.9(C3'-endo)  ~C3'-endo    4.43    4.51   23.54
 43    G A.G43             -67.0  -178.3    55.6    81.6  -154.9   -76.4    -78(BI)   -160.2(anti)    12.6(C3'-endo)  ~C3'-endo    4.24    4.61   20.95
 44    A A.A44             -59.7   162.1    60.0    85.3  -142.8   -57.2    -86(BI)   -159.4(anti)    16.9(C3'-endo)  ~C3'-endo    4.25    4.61   31.07
 45    G A.G45             -71.9  -176.9    51.0    87.6  -135.1   -78.7    -56(BI)   -149.3(anti)    15.4(C3'-endo)  ~C3'-endo    4.01    4.58   40.27
 46    g A.7MG46           -56.8  -146.5    48.4   141.6  -102.7  -137.9     35(--)    -65.8(anti)   154.5(C2'-endo) ~C2'-endo     0.21    0.96  139.04
 47    U A.U47              62.4  -164.0    44.4   146.1   -93.7   -78.0    -16(--)   -112.0(anti)   164.9(C2'-endo) ~C2'-endo     0.26    0.39  157.37
 48    C A.C48             -73.5  -174.3   161.5   145.6  -143.5    75.6    141(--)   -140.1(anti)   158.2(C2'-endo) ~C2'-endo     1.92    1.80  147.54
 49    c A.5MC49            50.7   168.5    42.2    84.3  -145.0   -82.1    -63(BI)   -173.6(anti)    10.1(C3'-endo)  ~C3'-endo    4.77    4.75   25.83
 50    U A.U50             -51.7   177.2    42.1    80.4  -150.6   -67.8    -83(BI)   -165.3(anti)     5.6(C3'-endo)  ~C3'-endo    4.38    4.75   23.15
 51    G A.G51             -63.9   176.8    52.8    79.4  -150.4   -71.3    -79(BI)   -156.6(anti)    11.5(C3'-endo)  ~C3'-endo    4.44    4.67   21.28
 52    U A.U52             -64.7   173.6    48.5    80.3  -156.5   -69.4    -87(BI)   -164.0(anti)    14.1(C3'-endo)  ~C3'-endo    4.64    4.74   25.47
 53    G A.G53             -56.9   171.5    56.2    83.9  -159.4   -64.9    -95(BI)   -169.2(anti)    19.8(C3'-endo)  ~C3'-endo    4.59    4.57   24.53
 54    t A.5MU54           -79.7  -172.8    57.7    77.6  -128.6   -70.7    -58(BI)   -161.5(anti)    20.6(C3'-endo)  ~C3'-endo    4.56    4.80   30.73
 55    P A.PSU55           -49.7   168.8    44.1    76.6  -140.8   -69.9    -71(BI)   -147.0(anti)    10.1(C3'-endo)  ~C3'-endo    4.15    4.74   71.28
 56    C A.C56             166.4   171.8    53.3    83.4  -132.7   -70.6    -62(BI)   -161.5(anti)    12.6(C3'-endo)  ~C3'-endo    4.37    4.76   28.07
 57    G A.G57             -65.7   167.1    57.5    81.7  -145.2   -67.6    -78(BI)   -159.3(anti)    12.8(C3'-endo)  ~C3'-endo    4.36    4.65   42.47
 58    a A.1MA58           -60.8  -146.1    71.8   156.7   -78.3  -169.3     91(BII)   -86.3(anti)   161.1(C2'-endo) ~C2'-endo     0.48    0.68   73.92
 59    U A.U59              72.6  -158.8    63.7    84.6  -148.8   -53.7    -95(BI)   -165.6(anti)    25.8(C3'-endo)  ~C3'-endo    4.67    4.42   27.88
 60    C A.C60             -72.2   179.5    66.0   148.3   -97.1   -66.4    -31(--)   -117.8(anti)   154.8(C2'-endo) ~C2'-endo     0.99    0.86   90.64
 61    C A.C61             -84.3   179.8    38.2    83.0  -152.3   -74.5    -78(BI)   -166.7(anti)    14.8(C3'-endo)  ~C3'-endo    4.45    4.52   25.80
 62    A A.A62             -60.1   179.6    46.9    80.5  -145.6   -74.1    -71(BI)   -158.7(anti)     9.9(C3'-endo)  ~C3'-endo    4.18    4.66   19.23
 63    C A.C63             -62.0   167.3    50.9    80.7  -152.3   -70.7    -82(BI)   -152.6(anti)    10.7(C3'-endo)  ~C3'-endo    4.32    4.62   23.62
 64    A A.A64             -66.9   180.0    44.1    75.8  -147.5   -76.5    -71(BI)   -161.8(anti)    12.9(C3'-endo)  ~C3'-endo    4.68    4.86   25.64
 65    G A.G65             -44.0   164.2    49.9    79.8  -152.0   -73.3    -79(BI)   -172.8(anti)    16.5(C3'-endo)  ~C3'-endo    4.92    4.76   25.20
 66    A A.A66             -57.9   178.5    52.0    81.7  -151.0   -73.5    -77(BI)   -164.9(anti)    22.5(C3'-endo)  ~C3'-endo    4.56    4.60   22.73
 67    A A.A67             -62.0   164.1    54.2    83.2  -152.2   -78.3    -74(BI)   -162.8(anti)    15.0(C3'-endo)  ~C3'-endo    4.71    4.67   23.30
 68    U A.U68             -59.8   175.3    47.3    82.2  -152.9   -65.4    -88(BI)   -160.1(anti)    11.2(C3'-endo)  ~C3'-endo    4.30    4.60   24.35
 69    U A.U69             -63.8   168.1    55.1    79.1  -155.4   -85.6    -70(BI)   -161.4(anti)    14.7(C3'-endo)  ~C3'-endo    4.55    4.61   19.23
 70    C A.C70             -61.7   164.6    53.1    79.0  -158.5   -64.5    -94(BI)   -152.0(anti)    15.0(C3'-endo)  ~C3'-endo    4.20    4.56   20.96
 71    G A.G71             -78.4   173.6    60.3    80.3  -149.6   -68.4    -81(BI)   -162.8(anti)    13.5(C3'-endo)  ~C3'-endo    4.50    4.71   22.80
 72    C A.C72             -73.2   176.2    62.1    83.0  -152.3   -67.9    -84(BI)   -161.6(anti)    19.5(C3'-endo)  ~C3'-endo    4.56    4.63   26.14
 73    A A.A73             -63.3   177.7    50.4    81.6  -148.2   -66.1    -82(BI)   -167.4(anti)    15.0(C3'-endo)  ~C3'-endo    4.65    4.71   26.33
 74    C A.C74             -66.9  -174.9    50.7    85.9  -145.0   -58.8    -86(BI)   -153.1(anti)    11.8(C3'-endo)  ~C3'-endo    4.22    4.61   33.45
 75    C A.C75             -52.3   175.7    42.3    85.6  -131.9   163.9     64(BII)  -151.7(anti)    15.1(C3'-endo)  ~C3'-endo    3.96    4.60  159.78
 76    A A.A76             -71.0   130.2   164.6   160.9     ---     ---     ---       138.5(anti)   176.1(C2'-endo) ~C2'-endo      ---     ---     ---
******************************************************************************************
Virtual eta/theta torsion angles:

  eta:    C4'(i-1)-P(i)-C4'(i)-P(i+1)
  theta:  P(i)-C4'(i)-P(i+1)-C4'(i+1)
    [Ref: Olson (1980): "Configurational statistics of polynucleotide chains.
          An updated virtual bond model to treat effects of base stacking."
          Macromolecules, 13(3):721-728]

  eta':   C1'(i-1)-P(i)-C1'(i)-P(i+1)
  theta': P(i)-C1'(i)-P(i+1)-C1'(i+1)
    [Ref: Keating et al. (2011): "A new way to see RNA." Quarterly Reviews
          of Biophysics, 44(4):433-466]

  eta":   base(i-1)-P(i)-base(i)-P(i+1)
  theta": P(i)-base(i)-P(i+1)-base(i+1)

          nt                eta   theta     eta'  theta'    eta"  theta"
 1     G A.G1                ---  -139.3     ---  -136.5     ---  -110.8
 2     C A.C2              171.9  -144.6  -175.5  -144.1  -136.1  -118.1
 3     G A.G3              160.2  -151.4   173.9  -153.9  -145.0  -143.7
 4     G A.G4              164.3  -144.6   177.7  -144.1  -154.8   -98.7
 5     A A.A5              166.9  -138.1  -178.3  -135.8  -116.3  -111.6
 6     U A.U6              172.1  -149.7  -170.8  -143.9  -130.1  -126.5
 7     U A.U7             -158.0   -42.7  -138.7   -60.7  -120.5   -31.5
 8     U A.U8              162.7   160.7  -159.9  -163.8  -142.6   176.2
 9     A A.A9             -140.6   -38.9  -159.3  -112.7   157.1  -105.5
 10    g A.2MG10            27.8  -130.3    97.2  -130.1   134.8  -110.3
 11    C A.C11             170.3  -135.8  -175.7  -136.7  -137.8  -119.9
 12    U A.U12             159.9  -121.6   176.5  -130.6  -148.5  -101.4
 13    C A.C13             178.1  -179.1  -166.8   176.7  -118.5   178.4
 14    A A.A14             171.9  -146.5   172.1  -133.4  -179.7   -74.6
 15    G A.G15             164.3  -177.9  -166.6  -161.0   -92.6  -101.8
 16    u A.H2U16          -124.1   -77.5  -114.2  -108.3   -72.5  -127.0
 17    u A.H2U17           -10.5   -64.3     7.7   -94.7    17.3  -125.4
 18    G A.G18             -21.0  -167.4    45.3  -160.9    61.3  -124.2
 19    G A.G19            -127.4   -43.3  -122.0   -72.9  -105.8    -7.8
 20    G A.G20             165.3  -100.4  -160.4  -101.1  -177.9  -115.4
 21    A A.A21             -78.3   152.7   -68.0   155.1   -61.1   154.8
 22    G A.G22             159.5   167.6   156.6   178.8   157.1  -162.6
 23    A A.A23             178.4  -141.8  -173.5  -141.2  -156.1  -112.0
 24    G A.G24             163.7  -139.5   177.7  -137.6  -137.6  -103.8
 25    C A.C25             161.4  -132.6   179.2  -131.0  -128.2   -89.0
 26    g A.M2G26           173.0  -133.0  -167.7  -130.4  -106.9   -93.6
 27    C A.C27             163.5  -142.3  -178.0  -141.5  -123.6  -105.6
 28    C A.C28             157.5  -143.8   171.1  -144.3  -136.3  -125.5
 29    A A.A29             163.5  -152.9   179.0  -150.8  -142.9  -124.7
 30    G A.G30             178.3  -127.8  -167.7  -126.5  -128.2   -72.5
 31    A A.A31             165.4  -133.9  -174.3  -131.0  -101.0   -93.9
 32    c A.OMC32           164.5  -139.2  -175.9  -138.0  -122.3  -108.9
 33    U A.U33             165.1  -114.0   177.8  -158.5  -141.1   138.3
 34    g A.OMG34            27.3  -121.7    50.5  -123.7    22.7   -84.4
 35    A A.A35             162.5  -127.7  -177.7  -128.5  -116.8  -113.4
 36    A A.A36             164.9  -172.7  -174.4  -169.2  -142.3  -115.1
 37    g A.YYG37           163.1  -135.2   174.1  -131.3  -119.8   -79.8
 38    A A.A38             170.2  -133.9  -173.3  -129.0  -104.3  -105.5
 39    P A.PSU39           174.0  -132.6  -168.6  -131.2  -127.5   -89.6
 40    c A.5MC40           163.1  -148.5  -177.6  -149.3  -115.9  -131.7
 41    U A.U41             169.4  -148.8   177.2  -144.0  -152.9  -120.5
 42    G A.G42             171.2  -150.4  -171.5  -151.6  -133.9  -124.5
 43    G A.G43             174.2  -151.6  -174.4  -150.0  -134.0  -124.5
 44    A A.A44             173.2  -120.4  -171.8  -120.0  -133.3   -72.6
 45    G A.G45             168.6  -141.6  -168.3  -128.4  -103.4  -133.4
 46    g A.7MG46          -143.2  -107.3  -133.6  -149.6  -148.2  -162.7
 47    U A.U47             -31.5   -56.8     4.8   -91.0    24.9  -110.7
 48    C A.C48             -82.5    53.9   -29.3    17.5     1.5  -107.6
 49    c A.5MC49           -56.7  -145.3   -36.6  -142.8   103.2  -130.2
 50    U A.U50             174.8  -146.6  -176.9  -142.8  -153.6  -113.8
 51    G A.G51             170.3  -147.3  -175.5  -148.2  -134.2  -122.1
 52    U A.U52             160.3  -145.8   173.9  -144.3  -141.8  -119.6
 53    G A.G53             174.9  -141.5  -167.2  -142.4  -124.7  -111.6
 54    t A.5MU54           171.1  -129.2  -177.4  -122.6  -133.3   -76.4
 55    P A.PSU55           165.3  -115.2  -173.6  -155.4  -112.1   145.1
 56    C A.C56              31.4  -126.9    51.6  -124.1    25.3   -87.4
 57    G A.G57             164.3  -142.5  -174.1  -131.9  -119.2  -113.8
 58    a A.1MA58          -131.5  -108.7  -105.3  -171.2  -104.2   159.8
 59    U A.U59               1.8  -119.4    26.8  -109.9    49.0   -56.9
 60    C A.C60            -171.8   -40.7  -130.1   -68.5   -70.2   -35.8
 61    C A.C61             122.4  -148.3   168.6  -144.1  -158.2  -117.4
 62    A A.A62             173.0  -146.6  -176.9  -144.9  -142.0  -119.6
 63    C A.C63             164.5  -148.3   177.9  -149.6  -143.9  -128.6
 64    A A.A64             158.4  -151.0   168.5  -148.2  -154.8  -122.8
 65    G A.G65             173.6  -147.3  -172.0  -145.4  -130.5  -121.2
 66    A A.A66             177.6  -145.4  -170.1  -142.7  -133.5  -111.9
 67    A A.A67             165.6  -149.3  -176.9  -149.8  -129.8  -126.7
 68    U A.U68             168.9  -138.2   179.4  -136.1  -143.2   -96.5
 69    U A.U69             165.6  -160.5  -176.0  -161.2  -118.8  -156.9
 70    C A.C70             166.7  -146.2   173.6  -149.0  -171.6  -127.0
 71    G A.G71             161.0  -143.0   174.0  -142.3  -146.3  -113.4
 72    C A.C72             166.1  -141.5  -177.5  -141.9  -131.5  -110.2
 73    A A.A73             167.6  -137.8  -177.2  -133.3  -127.1   -89.8
 74    C A.C74             171.2  -122.1  -172.8  -116.5  -116.2   -72.1
 75    C A.C75             174.9   106.5  -161.9   109.8  -102.9  -139.3
 76    A A.A76               ---     ---     ---     ---     ---     ---
******************************************************************************************
Sugar conformational parameters:

  v0: C4'-O4'-C1'-C2'
  v1: O4'-C1'-C2'-C3'
  v2: C1'-C2'-C3'-C4'
  v3: C2'-C3'-C4'-O4'
  v4: C3'-C4'-O4'-C1'

  tm: the amplitude of pucker
  P:  the phase angle of pseudorotation
    [Ref: Altona & Sundaralingam (1972): "Conformational analysis
          of the sugar ring in nucleosides and nucleotides. A new
          description using the concept of pseudorotation."
          J Am Chem Soc, 94(23):8205-8212]

          nt                 v0      v1      v2      v3      v4      tm      P   Puckering
 1     G A.G1                1.7   -23.4    35.1   -35.2    21.1    36.5    16.1  C3'-endo
 2     C A.C2                1.6   -23.2    34.8   -34.8    20.9    36.2    16.1  C3'-endo
 3     G A.G3                2.7   -25.1    36.8   -36.1    21.2    38.1    14.6  C3'-endo
 4     G A.G4               -1.6   -22.3    36.3   -38.2    25.0    38.8    20.8  C3'-endo
 5     A A.A5               10.1   -32.6    41.5   -36.6    16.7    41.7     4.8  C3'-endo
 6     U A.U6                0.3   -24.0    37.3   -38.1    23.9    39.2    18.2  C3'-endo
 7     U A.U7              -24.4    35.4   -32.4    18.9     3.3    35.4   156.1  C2'-endo
 8     U A.U8                5.8   -28.7    39.7   -37.2    19.7    40.4    10.5  C3'-endo
 9     A A.A9              -31.7    41.8   -35.6    18.1     8.4    41.2   149.8  C2'-endo
 10    g A.2MG10             7.8   -28.0    36.7   -33.0    15.9    37.0     6.6  C3'-endo
 11    C A.C11               1.2   -21.2    32.1   -32.5    19.8    33.5    16.8  C3'-endo
 12    U A.U12              -4.6   -19.3    34.5   -37.9    26.7    38.1    25.2  C3'-endo
 13    C A.C13              -3.4   -19.4    33.8   -36.4    25.1    36.9    23.7  C3'-endo
 14    A A.A14              12.6   -30.8    36.8   -30.2    11.0    36.8   358.9   C2'-exo
 15    G A.G15               1.9   -24.6    36.8   -36.8    22.2    38.3    16.0  C3'-endo
 16    u A.H2U16             0.0   -18.7    29.2   -30.2    19.2    30.9    18.8  C3'-endo
 17    u A.H2U17            23.0   -36.7    35.1   -23.2     0.2    37.0   341.4   C2'-exo
 18    G A.G18             -27.9    39.5   -35.0    20.2     4.8    38.9   154.3  C2'-endo
 19    G A.G19             -17.6    31.0   -31.9    23.1    -3.8    32.7   167.6  C2'-endo
 20    G A.G20               6.6   -27.8    36.6   -34.2    17.5    37.0     8.7  C3'-endo
 21    A A.A21               3.8   -25.0    35.1   -34.4    19.4    36.0    13.0  C3'-endo
 22    G A.G22              16.4   -34.1    38.1   -29.5     8.3    38.3   353.8   C2'-exo
 23    A A.A23               4.2   -26.6    37.4   -36.5    20.1    38.3    12.6  C3'-endo
 24    G A.G24               3.9   -28.4    40.3   -39.3    22.4    41.5    13.4  C3'-endo
 25    C A.C25               0.6   -24.5    37.8   -38.0    23.6    39.6    17.4  C3'-endo
 26    g A.M2G26             6.3   -27.5    37.1   -34.7    17.9    37.6     9.3  C3'-endo
 27    C A.C27               0.2   -23.5    36.5   -37.2    23.6    38.4    18.3  C3'-endo
 28    C A.C28               6.6   -29.0    39.1   -36.3    18.8    39.6     9.2  C3'-endo
 29    A A.A29               3.4   -26.6    38.4   -37.4    21.4    39.5    13.7  C3'-endo
 30    G A.G30               2.6   -24.2    35.7   -34.9    20.4    36.9    14.5  C3'-endo
 31    A A.A31               2.6   -24.0    35.0   -34.6    20.2    36.2    14.6  C3'-endo
 32    c A.OMC32            -1.2   -21.7    35.1   -36.7    23.9    37.4    20.4  C3'-endo
 33    U A.U33               3.5   -25.4    36.5   -35.3    20.1    37.5    13.3  C3'-endo
 34    g A.OMG34             3.9   -22.7    32.2   -30.8    17.1    32.9    12.2  C3'-endo
 35    A A.A35              -2.0   -19.9    32.7   -34.9    23.4    35.2    21.9  C3'-endo
 36    A A.A36             -20.6    -7.3    30.6   -43.2    40.5    43.9    45.8   C4'-exo
 37    g A.YYG37             2.1   -24.1    36.0   -35.6    21.0    37.4    15.4  C3'-endo
 38    A A.A38              10.9   -30.3    37.6   -32.5    13.6    37.7     2.4  C3'-endo
 39    P A.PSU39             2.1   -25.6    38.5   -38.4    22.8    40.0    15.8  C3'-endo
 40    c A.5MC40             0.8   -22.5    34.6   -35.0    21.5    36.3    17.3  C3'-endo
 41    U A.U41               3.8   -27.7    39.9   -38.6    22.0    41.0    13.4  C3'-endo
 42    G A.G42              -1.7   -22.4    36.8   -38.6    25.4    39.4    20.9  C3'-endo
 43    G A.G43               4.3   -27.6    39.1   -37.6    21.1    40.1    12.6  C3'-endo
 44    A A.A44               1.0   -23.0    35.2   -35.4    21.6    36.8    16.9  C3'-endo
 45    G A.G45               2.1   -24.3    35.7   -35.4    21.2    37.0    15.4  C3'-endo
 46    g A.7MG46           -27.4    38.6   -34.7    19.7     4.7    38.5   154.5  C2'-endo
 47    U A.U47             -20.9    34.8   -35.1    24.3    -2.2    36.4   164.9  C2'-endo
 48    C A.C48             -25.6    38.4   -35.6    22.1     2.1    38.4   158.2  C2'-endo
 49    c A.5MC49             5.8   -28.1    38.7   -36.0    19.1    39.3    10.1  C3'-endo
 50    U A.U50               9.4   -32.2    41.0   -36.4    17.6    41.2     5.6  C3'-endo
 51    G A.G51               4.9   -27.9    38.9   -36.8    20.3    39.7    11.5  C3'-endo
 52    U A.U52               3.2   -28.5    41.4   -40.1    23.6    42.7    14.1  C3'-endo
 53    G A.G53              -1.0   -23.1    37.0   -38.3    24.9    39.4    19.8  C3'-endo
 54    t A.5MU54            -1.4   -22.2    35.9   -37.7    24.8    38.3    20.6  C3'-endo
 55    P A.PSU55             6.2   -29.9    40.9   -38.3    20.4    41.5    10.1  C3'-endo
 56    C A.C56               3.8   -25.3    35.7   -34.5    19.2    36.6    12.6  C3'-endo
 57    G A.G57               4.0   -26.7    37.9   -36.5    20.6    38.9    12.8  C3'-endo
 58    a A.1MA58           -24.3    38.4   -36.9    23.9     0.2    39.0   161.1  C2'-endo
 59    U A.U59              -4.4   -18.3    31.8   -35.7    25.4    35.3    25.8  C3'-endo
 60    C A.C60             -28.8    40.5   -36.4    21.2     4.7    40.3   154.8  C2'-endo
 61    C A.C61               2.6   -25.5    36.8   -36.6    21.5    38.1    14.8  C3'-endo
 62    A A.A62               5.9   -27.8    38.1   -35.4    18.8    38.7     9.9  C3'-endo
 63    C A.C63               5.4   -27.3    37.5   -35.5    19.1    38.1    10.7  C3'-endo
 64    A A.A64               4.1   -28.6    40.2   -38.8    22.2    41.2    12.9  C3'-endo
 65    G A.G65               1.5   -26.6    39.5   -39.9    24.3    41.2    16.5  C3'-endo
 66    A A.A66              -2.9   -21.6    36.5   -38.8    26.5    39.5    22.5  C3'-endo
 67    A A.A67               2.4   -24.9    36.5   -36.1    21.4    37.8    15.0  C3'-endo
 68    U A.U68               5.3   -28.4    39.5   -37.5    20.3    40.3    11.2  C3'-endo
 69    U A.U69               2.9   -26.3    38.3   -37.9    22.3    39.6    14.7  C3'-endo
 70    C A.C70               2.4   -25.9    38.7   -37.9    22.4    40.1    15.0  C3'-endo
 71    G A.G71               3.7   -27.4    39.2   -38.3    21.8    40.3    13.5  C3'-endo
 72    C A.C72              -0.6   -21.9    34.9   -36.2    23.1    37.0    19.5  C3'-endo
 73    A A.A73               2.4   -25.4    37.3   -36.9    21.8    38.6    15.0  C3'-endo
 74    C A.C74               4.4   -25.4    35.6   -34.0    18.6    36.4    11.8  C3'-endo
 75    C A.C75               2.3   -22.5    33.1   -33.0    19.2    34.3    15.1  C3'-endo
 76    A A.A76             -13.6    30.5   -34.8    27.7    -9.1    34.8   176.1  C2'-endo
******************************************************************************************
Assignment of sugar-phosphate backbone suites

  bin: name of the 12 bins based on [delta(i-1), delta, gamma], where
       delta(i-1) and delta can be either 3 (for C3'-endo sugar) or 2
       (for C2'-endo) and gamma can be p/t/m (for gauche+/trans/gauche-
       conformations, respectively) (2x2x3=12 combinations: 33p, 33t,
       ... 22m); 'inc' refers to incomplete cases (i.e., with missing
       torsions), and 'trig' to triages (i.e., with torsion angle
       outliers)
  cluster: 2-char suite name, for one of 53 reported clusters (46
           certain and 7 wannabes), '__' for incomplete cases, and
           '!!' for outliers
  suiteness: measure of conformer-match quality (low to high in range 0 to 1)

    [Ref: Richardson et al. (2008): "RNA backbone: consensus all-angle
          conformers and modular string nomenclature (an RNA Ontology
          Consortium contribution)." RNA, 14(3):465-481]

          nt             bin    cluster   suiteness
 1     G A.G1            inc      __       0
 2     C A.C2            33p      1a       0.935
 3     G A.G3            33p      1a       0.868
 4     G A.G4            33p      1a       0.842
 5     A A.A5            33p      1a       0.847
 6     U A.U6            33p      1a       0.664
 7     U A.U7            32p      1b       0.803
 8     U A.U8            23p      2a       0.509
 9     A A.A9            32p      1[       0.046
 10    g A.2MG10         23p      2g       0.640
 11    C A.C11           33p      1a       0.507
 12    U A.U12           33p      1a       0.898
 13    C A.C13           33t      1c       0.824
 14    A A.A14           trig     !!       0
 15    G A.G15           33p      1a       0.484
 16    u A.H2U16         trig     !!       0
 17    u A.H2U17         33t      !!       0
 18    G A.G18           32p      5p       0.026
 19    G A.G19           22p      4b       0.512
 20    G A.G20           23p      2a       0.623
 21    A A.A21           33t      !!       0
 22    G A.G22           33t      1f       0.714
 23    A A.A23           33p      1a       0.840
 24    G A.G24           33p      1a       0.881
 25    C A.C25           33p      1a       0.967
 26    g A.M2G26         33p      1a       0.819
 27    C A.C27           33p      1a       0.698
 28    C A.C28           33p      1a       0.923
 29    A A.A29           33p      1a       0.973
 30    G A.G30           33p      1a       0.838
 31    A A.A31           33p      1a       0.914
 32    c A.OMC32         33p      1a       0.782
 33    U A.U33           33p      1a       0.897
 34    g A.OMG34         33p      1g       0.784
 35    A A.A35           33p      1a       0.517
 36    A A.A36           33p      1a       0.670
 37    g A.YYG37         33p      1a       0.625
 38    A A.A38           33p      1a       0.903
 39    P A.PSU39         33p      1a       0.680
 40    c A.5MC40         33p      1a       0.942
 41    U A.U41           33p      1a       0.945
 42    G A.G42           33p      1a       0.630
 43    G A.G43           33p      1a       0.882
 44    A A.A44           33p      1a       0.837
 45    G A.G45           33p      1a       0.749
 46    g A.7MG46         32p      1[       0.849
 47    U A.U47           22p      4p       0.589
 48    C A.C48           22t      2u       0.283
 49    c A.5MC49         23p      6d       0.520
 50    U A.U50           33p      1a       0.656
 51    G A.G51           33p      1a       0.981
 52    U A.U52           33p      1a       0.945
 53    G A.G53           33p      1a       0.896
 54    t A.5MU54         33p      1a       0.720
 55    P A.PSU55         33p      1a       0.586
 56    C A.C56           33p      1g       0.894
 57    G A.G57           33p      1a       0.837
 58    a A.1MA58         32p      1[       0.332
 59    U A.U59           23p      4d       0.411
 60    C A.C60           32p      1b       0.662
 61    C A.C61           23p      2a       0.553
 62    A A.A62           33p      1a       0.895
 63    C A.C63           33p      1a       0.964
 64    A A.A64           33p      1a       0.791
 65    G A.G65           33p      1a       0.586
 66    A A.A66           33p      1a       0.940
 67    A A.A67           33p      1a       0.941
 68    U A.U68           33p      1a       0.891
 69    U A.U69           33p      1a       0.951
 70    C A.C70           33p      1a       0.809
 71    G A.G71           33p      1a       0.761
 72    C A.C72           33p      1a       0.832
 73    A A.A73           33p      1a       0.965
 74    C A.C74           33p      1a       0.886
 75    C A.C75           33p      1a       0.639
 76    A A.A76           32t      !!       0

Concatenated suite string per chain. To avoid confusion of lower case
modified nucleotide name (e.g., 'a') with suite cluster (e.g., '1a'),
use --suite-delimiter to add delimiters (matched '()' by default).

1   A RNA nts=76  G1aC1aG1aG1aA1aU1bU2aU1[A2gg1aC1aU1cC!!A1aG!!u!!u5pG4bG2aG!!A1fG1aA1aG1aC1ag1aC1aC1aA1aG1aA1ac1aU1gg1aA1aA1ag1aA1aP1ac1aU1aG1aG1aA1aG1[g4pU2uC6dc1aU1aG1aU1aG1at1aP1gC1aG1[a4dU1bC2aC1aA1aC1aA1aG1aA1aA1aU1aU1aC1aG1aC1aA1aC1aC!!A

The DSSR-PyMOL schematics have been featured in all 12 cover images (January to December) of the RNA Journal in 2021. Moreover, the January 2022 issue of RNA continues to highlight DSSR-enabled schematics (see the note below). In the current Covid-19 pandemic, this cover seems to be a fit for the upcoming Christmas holiday season.

Ebola virus matrix protein octameric ring (PDB id: 7K5L; Landeras-Bueno S, Wasserman H, Oliveira G, VanAernum ZL, Busch F, Salie ZL, Wysocki VH, Andersen K, Saphire EO. 2021. Cellular mRNA triggers structural transformation of Ebola virus matrix protein VP40 to its essential regulatory form. Cell Rep 35: 108986). The Ebola virus matrix protein (VP40) forms distinct structures linked to distinct functions in the virus life cycle. VP40 forms an octameric ring-shaped (D4 symmetry) assembly upon binding of RNA and is associated with transcriptional control. RNA backbone is displayed as a red ribbon; block bases use NDB colors: A—red, G—green, U—cyan; protein is displayed as a gold ribbon. Cover image provided by the Nucleic Acid Database (ndbserver.rutgers.edu). Image generated using DSSR and PyMOL (Lu XJ. 2020. Nucleic Acids Res 48: e74).

Thanks to Dr. Cathy Lawson at the NDB for generating these cover images using DSSR and PyMOL for the RNA Journal. I’m gratified that the 2020 NAR paper is explicitly acknowledged: it’s the first time I’ve published as a single author in my scientific career.

Did you know that you can easily generate similar DSSR-PyMOL schematics via the http://skmatic.x3dna.org/ website? It is “simple and effective”, “good for teaching”, and has been highly recommended by Dr. Quentin Vicens (CU Denver) in FacultyOpinions.com.

The 12 PDB structures illustrated in the 2021 covers are:

1. January 2021 “iMango-III fluorescent aptamer (PDB id: 6PQ7; Trachman III RJ, Stagno JR, Conrad C, Jones CP, Fischer P, Meents A, Wang YX, Ferre-D’Amare AR. 2019. Co-crystal structure of the iMango-III fluorescent RNA aptamer using an X-ray free-electron laser. Acta Cryst F 75: 547). Upon binding TO1-biotin, the iMango-III aptamer achieves the largest fluorescence enhancement reported for turn-on aptamers (over 5000-fold).”
2. February 2021 “Human adenosine deaminase (E488Q mutant) acting on dsRNA (PDB id: 6VFF; Thuy-Boun AS, Thomas JM, Grajo HL, Palumbo CM, Park S, Nguyen LT, Fisher AJ, Beal PA. 2020. Asymmetric dimerization of adenosine deaminase acting on RNA facilitates substrate recognition. Nucleic Acids Res. https://doi.org/10.1093/nar/gkaa532). Adenosine deaminase enzymes convert adenosine to inosine in duplex RNA, a modification that strongly affects RNA structure and function in multiple ways.”
3. March 2021 “Hepatitis A virus IRES domain V in complex with Fab (PDB id: 6MWN; Koirala D, Shao Y, Koldobskaya Y, Fuller JR, Watkins AM, Shelke SA, Pilipenko EV, Das R, Rice PA, Piccirilli JA. 2019. A conserved RNA structural motif for organizing topology within picornaviral internal ribosome entry sites. Nat Commun 10: 3629).”
4. April 2021 “Mouse endonuclease V in complex with 23mer RNA (PDB id: 6OZO; Wu J, Samara NL, Kuraoka I, Yang W. 2019. Evolution of inosine-specific endonuclease V from bacterial DNase to eukaryotic RNase. Mol Cell 76: 44). Endonuclease V cleaves the second phosphodiester bond 3′ to a deaminated adenosine (inosine). Although highly conserved, EndoV change substrate preference from DNA in bacteria to RNA in eukaryotes.”
5. May 2021 “Manganese riboswitch from Xanthmonas oryzae (PDB id: 6N2V; Suddala KC, Price IR, Dandpat SS, Janeček M, Kührová P, Šponer J, Banáš P, Ke A, Walter NG. 2019. Local-to-global signal transduction at the core of a Mn2+ sensing riboswitch. Nat Commun 10: 4304). Bacterial manganese riboswitches control the expression of Mn2+ homeostasis genes. Using FRET, it was shown that an extended 4-way-junction samples transient docked states in the presence of Mg2+ but can only dock stably upon addition of submillimolar Mn2+.”
6. June 2021 “Sulfolobus islandicus Csx1 RNase in complex with cyclic RNA activator (PDB id: 6R9R; Molina R, Stella S, Feng M, Sofos N, Jauniskis V, Pozdnyakova I, Lopez-Mendez B, She Q, Montoya G. 2019. Structure of Csx1-cOA4 complex reveals the basis of RNA decay in Type III-B CRISPR-Cas. Nat Commun 10: 4302). CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA, promoting the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases. The Csx1 RNase dimer is shown with cyclic (A4) RNA bound.”
7. July 2021 “M. tuberculosis ileS T-box riboswitch in complex with tRNA (PDB id: 6UFG; Battaglia RA, Grigg JC, Ke A. 2019. Structural basis for tRNA decoding and aminoacylation sensing by T-box riboregulators. Nat Struct Mol Biol 26: 1106). T-box riboregulators are a class of cis-regulatory RNAs that govern the bacterial response to amino acid starvation by binding, decoding, and reading the aminoacylation status of specific transfer RNAs.”
8. August 2021 “CAG repeats recognized by cyclic mismatch binding ligand (PDB id: 6QIV; Mukherjee S, Blaszczyk L, Rypniewski W, Falschlunger C, Micura R, Murata A, Dohno C, Nakatan K, Kiliszek A. 2019. Structural insights into synthetic ligands targeting A–A pairs in disease-related CAG RNA repeats. Nucleic Acids Res 47:10906). A large number of hereditary neurodegenerative human diseases are associated with abnormal expansion of repeated sequences. RNA containing CAG repeats can be recognized by synthetic cyclic mismatch-binding ligands such as the structure shown.”
9. September 2021 “Corn aptamer complex with fluorophore Thioflavin T (PDB id: 6E81; Sjekloca L, Ferre-D’Amare AR. 2019. Binding between G quadruplexes at the homodimer interface of the Corn RNA aptamer strongly activates Thioflavin T fluorescence. Cell Chem Biol 26: 1159). The fluorescent compound Thioflavin T, widely used for the detection of amyloids, is bound at the dimer interface of the homodimeric G-quadruplex-containing RNA Corn aptamer.”
10. October 2021 “Cas9 nuclease-sgRNA complex with anti-CRISPR protein inhibitor (PDB id: 6JE9; Sun W, Yang J, Cheng Z, Amrani N, Liu C, Wang K, Ibraheim R, Edraki A, Huang X, Wang M, et al. 2019. Structures of Neisseria meningitidis Cas9 complexes in catalytically poised and anti-CRISPR-inhibited states. Mol Cell 76: 938­–952.e5). Nme1Cas9, a compact nuclease for in vivo genome editing. AcrIIC3 is an anti-CRISPR protein inhibitor.”
11. November 2021 “Two-quartet RNA parallel G-quadruplex complexed with porphyrin (PDB id: 6JJI; Zhang Y, Omari KE, Duman R, Liu S, Haider S, Wagner A, Parkinson GN, Wei D. 2020. Native de novo structural determinations of non-canonical nucleic acid motifs by X-ray crystallography at long wavelengths. Nucleic Acids Res 48: 9886–9898).”
12. December 2021 “Structure of S. pombe Lsm1–7 with RNA, polyuridine with 3’ guanosine (PDB id: 6PPV; Montemayor EJ, Virta JM, Hayes SM, Nomura Y, Brow DA, Butcher SE. 2020. Molecular basis for the distinct cellular functions of the Lsm1–7 and Lsm2–8 complexes. RNA 26: 1400–1413). Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1–7 complex initiates mRNA decay, while the nuclear Lsm2–8 complex acts as a chaperone for U6 spliceosomal RNA.”

On December 9, 2021, at 15:00 CET, I will present a BioExcel webinar titled “X3DNA-DSSR, a resource for structural bioinformatics of nucleic acids.”

• Here is the announcement link.
• Here is the registration URL.

Today, I released a video overview of DSSR (http://docs.x3dna.org/dssr-overview/).

DSSR has a sizable user base. However, in my opinion, DSSR is still underutilized for what it has to offer. This overview video is intended not only to attract new DSSR users, but also to highlight features that even experienced users may overlook.

As documented in the Overview PDF, DSSR can be easily incorporated into other structural bioinformatics pipelines. Working with Robert Hanson and Thomas Holder respectively, I initiated the integrations of DSSR into Jmol and PyMOL, two of the most popular molecular viewers. The DSSR-Jmol and DSSR-PyMOL integrations lead to unparalleled search capabilities and innovative visualization styles of 3D nucleic acid structures. They also exemplify the critical roles that a domain-specific analysis engine may play in general-purpose molecular visualization tools.

On January 27, 2016, I wrote the blogpost Integrating DSSR into Jmol and PyMOL. Four years later, these integrations have led to two peer-reviewed articles, both published in Nucleic Acids Research (NAR). This blogpost (dated 2020-09-15) highlights key features in each case and reflects on my experience in these two exciting collaborations.

The DSSR-Jmol integration

Hanson RM and Lu XJ (2017). DSSR-enhanced visualization of nucleic acid structures in Jmol. The DSSR-Jmol integration excels in its SQL-like, flexible searching capability of structural features, as demonstrated at the website http://jmol.x3dna.org. This work fills a gap in RNA structural bioinformatics by enabling deep analyses and SQL-like queries of RNA structural characteristics, interactively. Here are some simple examples:

SELECT WITHIN(dssr, "nts WHERE is_modified = true") # modified nucleotides
SELECT pairs # all pairs
Select WITHIN(dssr, "pairs WHERE name = 'Hoogsteen'") # Hoogsteen pairs
SELECT WITHIN(dssr, "pairs WHERE name != 'WC'") # non-Watson-Crick pairs
SELECT junctions # all junctions loops
select within(dssr, "junctions WHERE num_stems = 4") # four-way junction loops

In a recently email communication, Bob wrote:

How are you doing? I’m smiling, because I am remembering our incredible, animated discussions and how fun it was to work together with you on Jmol and DSSR.

The DSSR-PyMOL integration

Lu XJ (2020). DSSR-enabled innovative schematics of 3D nucleic acid structures with PyMOL. The DSSR-PyMOL integration brings unprecedented visual clarity to 3D nucleic acid structures, especially for G-quadruplexes. The four interfaces cover virtually all conceivable use cases. The easiest way to get started and quickly benefit from this work is via the web application at http://skmatic.x3dna.org.

I approached Thomas to write the DSSR-PyMOL manuscript together, in a similar way as the DSSR-Jmol paper. He wrote back, saying “I’m not interesting in being co-author of the paper”, adding:

But, if there is anything I can help you with, like revising the `dssr_block.py` script, or proof-reading the PyMOL related parts of the manuscript, I’ll be happy to do so.

Indeed, Thomas helped in several aspects of the DSSR-PyMOL project, as acknowledged in the paper:

I appreciate Thomas Holder (PyMOL Principal Developer, Schrödinger, Inc.) for writing the DSSR plugin for PyMOL, and for providing insightful comments on the manuscript and the web application interface.

Enhanced vs Innovative

Some viewers may noticed the difference in titles of the two NAR papers: “DSSR-enhanced visualization of nucleic acid structures in Jmol” vs. “DSSR-enabled innovative schematics of 3D nucleic acid structures with PyMOL”. As a matter of fact, the initial title of the DSSR-PyMOL paper was DSSR-enhanced visualization of nucleic acid structures in PyMOL, as shown in the December 02, 2019 announcement post on the 3DNA Forum.

In an era where reproducibility of “scientific” publications has become an issue and “break-throughs” are often broken or hardly held, I hesitate to use phrases such as “innovative”, “novel”, “paradigm shift” etc. Instead, I often use the modest words “refinement”, “enhance”, “improved”, “revised” etc, and try to deliver more than claimed. However, reviewers may take solid work but modest writing as “incremental” or “unexciting”. Before submitting the DSSR-PyMOL paper, I changed the title to DSSR-enabled innovative schematics of 3D nucleic acid structures with PyMOL. Does it mean that the DSSR-PyMOL integration is more innovative than the DSSR-Jmol case? Not necessarily. I do have a paper with “innovative” in its title.

Recently, while reading the Miskiewicz et al. review article How bioinformatics resources work with G4 RNAs, I noticed the term DSSR-G4DB under the category Databases with G4-related data. It refers to the website http://G4.x3dna.org (or g4.x3dna.org) that has been there since 2017 and weekly updated with new G-quadruplexes from the PDB. The DSSR-G4 resource, DSSR-Enabled Automatic Identification and Annotation of G-quadruplexes in the PDB, has already been cited several times in literature. However, I have not written up a paper on it yet, and thus have never thought carefully on a name for the resource. The term DSSR-G4DB sounds good to me, and I may well use it in the future.

Given below are the relevant quotations on DSSR and the DSSR-G4DB resource in the Miskiewicz et al. review article and my notes. The underlined headings (e.g., “Conclusion”) are those of the Miskiewicz et al. review article.

Methods: Databases with G4-related data

Currently, there exist 16 databases, which store information concerning quadruplexes. They fall into three categories: databases that collect primary or tertiary structures with experimentally verified G4s (DSSR-G4DB, G4IPDB, G4LDB, G4RNA, Lit392 and Lit638); databases storing data from high-throughput sequencing with mapped quadruplexes (GSE63874, GSE77282, GSE110582 and GSE129281); and databases of sequences with G4s identified in silico (Greglist, GRSDB2, G4-virus, Non-B DB v2.0, Plant-GQ and QuadBase2)

DSSR-G4DB [38] contains quadruplex nucleic acid structures found by DSSR in the Protein Data Bank [30], currently 354 entries. The data are annotated. Users can find information about G-tetrads, G4 helices and G4-stems and visualize the 3D models of G4 structures. Availability: webserver (http://g4.x3 dna.org). Recent update: 5 June 2020.

Note: DSSR-G4DB is updated weekly. The latest update is on 2020-09-09, with 362 G-quadruplexes auto-curated with DSSR from the PDB.

Methods: Tools that analyze and visualize 2D and 3D structure

Currently, four tools can analyze and visualize G4 structures. DSSR [38] … ElTetrado [31] … RNApdbee [66, 69] … 3D-NuS [65]

DSSR [38] processes the 3D structure of the RNA molecule and annotates its secondary structure. It is a part of the 3DNA suite [67] designed to work with the structures of nucleic acids. DSSR identifies, classifies and describes base pairs, multiplets and characteristic motifs of the secondary structure; helices, stems, hairpin loops, bulges, internal loops, junctions and others. It can also detect modules and tertiary structure patterns, includ- ing pseudoknots and kink-turns. The recent extension, DSSR- PyMOL [68], allows drawing cartoon-block schemes of the 3D structure and responds to the need for simplified visualization of quadruplexes. Input data formats: PDB, mmCIF and PDB ID. Availability: standalone program, web application (http://dssr.x3 dna.org/, http://skmatic.x3dna.org/).

Note: The other three tools all depend on or make use of DSSR and 3DNA:

• ElTetrado “ElTetrado depends on DSSR (Lu, Bussemaker and Olson, 2015) in terms of detection of base pairing and stacking.”
• RNApdbee uses 3DNA/DSSR as the default to identify base pairs.
• 3D-NuS employs 3DNA for structural analysis and model building.
  “These filtrated structures (225 DNA and 166 RNA structures) have been used to derive the local base pair step and base pair parameters (Table S2 for DNA and Table S3 for RNA) using 3DNA software package [35] and are stored in the server for 3D-NuS modeling.”
  “Soon after the user submits input for sequence-specific modeling, the server fetches the appropriate base pair step and base pair parameters from the database and creates a 3DNA style input file. Subsequently, the template model is built using the rebuild module of 3DNA software package and subjected to energy optimization using X-plor [56] to remove steric hindrance, specifically in the mismatch- containing duplexes (Fig. 1).”

Results: Computational experiments with structure-based tools

DSSR and ElTetrado identified quadruplexes in the input PDB files. Both programs focused on structural aspects of the input molecule, explicitly informing about quadruplexes and tetrads within the structure. DSSR provided an extensive analysis of 3D structures and output the data about G-tetrads, G-helices and G4-stems. It computed planarity for each G-tetrad and gave the sections area, rise and twist parameters for G4-helix and G4-stems. The program automatically assigned loop topologies according to the predefined types (P—parallel, D—diagonal and L—lateral) and their orientation (+/−). DSSR-PyMOL generated block schemes of both quadruplexes (Figure 4A3 and B3). ElTetrado also calculated planarity, rise and twist parameters and identified strand directions for both quadruplexes. It classified the quadruplexes and their component tetrads to ONZ classes. Finally, it generated the arc diagram (Figure 4A1 and B1) and two-line dot-bracket encoding of every quadruplex.

Note: DSSR contains an undocumented option --G4. With the ONZ variant, i.e., --g4=onz (case does not matter), DSSR also outputs the ONZ classification of G-tetrads from the same chain.

Conclusion

DSSR comprehensively examines the G4 structure, determines a variety of its parameters and provides the schematic 3D view.

It is worth noting that DSSR has been categorized under “Databases with G4-related data” and “Tools that analyze and visualize 2D and 3D structure” of the Methods section. It is not a tool that predicts G4 location in the sequence. There are 14 tools listed in “Table 2. Selected features of PQS prediction tools”, including G4Hunter and QGRS Mapper etc.

Recently, while visiting the NAR website on DSSR-enabled innovative schematics of 3D nucleic acid structures with PyMOL, I noticed a big red circle ① near “View Metrics”. I was quite curious to see what it meant. After a few clicks, I was delighted to read the following recommendation in Faculty Opinions by Quentin Vicens:

I really enjoyed “playing” with the revised and expanded version of Dissecting the Spatial Structure of RNA (DSSR) described by Xiang-Jun Lu in this July issue of NAR. The software is known to generate ‘block view’ representations of nucleic acids that make many parameters more immediately visible, such as base composition, stacking, and groove depth. This new version includes Watson-Crick pairs shown as single rectangles, and G quadruplexes as large squares, making such regions more quickly distinguishable from other regions within an overall tertiary structure. I was amazed at how simple and effective the web interface was, and I liked the possibility to download a PyMOL session to look at molecules under different angles. If need be, blocks can be further edited in PyMOL using the provided plugin (see on page 35). I highly recommend it!

The DSSR-PyMOL schematics paper/website has been rated “Very Good”, and classified as “Good for Teaching”. See Vicens Q: Faculty Opinions Recommendation of [Lu XJ, Nucleic Acids Res 2020 48(13):e74]. In Faculty Opinions, 14 Aug 2020; 10.3410/f.738001682.793577327.