3DNA is a versatile, integrated software system for the analysis, rebuilding and visualization of three-dimensional nucleic-acid-containing structures. The software is applicable not only to DNA (as the name 3DNA may imply), but also to complicated RNA structures and DNA-protein complexes. In 3DNA, structural analysis and model rebuilding are two sides of the same coin: the description of structure is rigorous and reversible, thus allowing for its exact reconstruction based on the derived parameters. 3DNA automatically detects all non-cannonical base pairs, base triplets and higher-order associations, and coaxially stacked helices; provides a comprehensive collection of fiber models of regular DNA and RNA helices; generates highly effective schematic presentations that reveal key features of nucleic-acid structures; performs undisturbed base mutations, and have facilities for the analysis of molecular dynamics simulation trajectories. The recently added DSSR program has been designed from the ground up to make RNA structure analyses straightforward, and it has a decent user manual!

3DNA is under active development. In particular, any 3DNA-related questions are welcome and should be directed to the 3DNA forum — I strive to provide a prompt and concrete response to each and every question posted there.

More info · Seeing is believing · What’s new · 3DNA forum · Download


Exterior loop in RNA secondary structure

A single-stranded RNA molecule can fold back onto itself to form various loops delineated by double helical stems, as shown in the figure below [taken from the Nearest Neighbor Database website from the Turner group].

Various loops in RNA secondary structure

Of special note is the exterior loop (at the bottom) which includes the 5′ and 3′ ends of the sequence. The Mfold User Manual defines the exterior loop as such:

The collection of bases and base pairs not accessible from any base pair is called the exterior (or external) loop … . It is worth noting that if we imagine adding a 0th and an (n + 1)st base to the RNA, and a base pair 0.(n+1), then the exterior loop becomes the loop closed by this imaginary base pair. … The exterior loop exists only in linear RNA.

While each of the other loops (hairpin, bulge, internal or junction) forms a closed ‘circle’ with two neighboring bases connected by either a canonical pair or backbone covalent bond, the ‘exterior loop’ has only an imaginary pair to close the 5′ and 3′ ends of the sequence. Moreover, the two ends of an RNA molecule are not necessarily close in three-dimenional space, as may be implied in the above secondary structure diagram. For example, in the H-type pseudoknotted structure 1ymo from human telomerase RNA, the 5′ and 3′ ends are on the opposite sides.

DSSR does not has the concept of an ‘exterior loop’ due to its lack of a closing pair to form a ‘circle’. Instead, each of the 5′ and 3′ dangling ends is taken as a ‘non-loop single-stranded segment’, if applicable. For the crystal structure of yeast phenylalanine tRNA (1ehz, see the figure at the bottom), the relevant portion of DSSR output is as below. Note that since the 5′ end is paired, only the single-stranded region at the 3′ end is listed. Presumably, the ‘exterior loop’ in this case would also include the G1—C72 pair, with the imaginary closing pair connecting G1 and A76.

List of 1 non-loop single-stranded segment
   1 nts=4 ACCA A.A73,A.C74,A.C75,A.A76

yeast phenylalanine tRNA



DSSR-derived DBN for an input entry with multiple RNA molecules

Dot bracket notation (dbn) is a popular format to represent RNA secondary structures. Initially introduced by the ViennaRNA package, dbn uses dots (.) for unpaired bases, and matched parentheses () for the canonical Watson-Crick A-T and G-C or the wobble G-U pairs. This compact representation was designed for fully nested (i.e., pseudoknot free) RNA secondary structures in a single RNA molecule. Over the years, it has been extended to cover pseudoknots (of possibly higher orders) using matched pairs of [], {}, and <> etc.

To derive dbn from three-dimensional atomic coordinates with DSSR, I was faced with an issue on how to represent multiple RNA chains (molecules). A closely related yet practical problem is chain breaks, as in x-ray crystal structures where disordered regions may not have fitted coordinates. I searched but failed to find any ‘standard’ way to account for chain breaks or multiple molecules in dbn. The commonly used programs for visualizing RNA secondary structure diagrams that I tested at that time did not take such cases into consideration — they simply showed all bases as if they were from a single continous RNA chain.

I discussed the issue with Dr. Yann Ponty, the maintainer of the popular VARNA program. After a few around of email exchanges, we introduced an extra symbol (&) in both sequence and dbn to designate multiple chains or breaks within a chain to communicate between DSSR and VARNA.

As an example, the DSSR-derived dbn for the double-stranded DNA structure 355d (the famous Dickerson dodecamer) is as below:

Secondary structures in dot-bracket notation (dbn) as a whole and per chain
>355d nts=24 [whole]
>355d-A #1 nts=12 [chain] DNA
>355d-B #2 nts=12 [chain] DNA

As another example, the PDB entry 2fk6 contains a tRNA with chain breaks — nucleotides 26 to 45 are missing from the structure (see figure below). The DSSR-derived dbn is as follows — note the * at the end of the header line.

>2fk6-R #1 nts=53 [chain] RNA*

2FK6: RNAse Z/tRNA(Thr) complex with chain break

It is worth mentioning a subtle point in DSSR-derived dbn with multiple chains, i.e., the order of the chains may make a difference! The point is best illustrated with a concrete example — here, 4un3, the crystal structure of Cas9 bound to PAM-containing DNA target. Based on the data file downloaded directly from the PDB (4un3.pdb), the relevant portions of DSSR output are:

Special notes:
   o Cross-paired segments in separate chains, be *careful* with .dbn

This structure contains *1-order pseudoknot
   o You may want to run DSSR again with the '--nested' option which removes
     pseudoknots to get a fully nested secondary structure representation.
   o The DSSR-derived dbn may be problematic (see notes above).

Secondary structures in dot-bracket notation (dbn) as a whole and per chain
>4un3 nts=120 [whole]
>4un3-A #1 nts=81 [chain] RNA
>4un3-C #2 nts=28 [chain] DNA
>4un3-D #3 nts=11 [chain] DNA

The notes in the DSSR output is worth paying attention to. Specifically, it reports a “*1-order pseudoknot” — note also the *! Here the target DNA chain C comes before DNA chain D in the PDB file. The 5′-end bases in chain C pair with bases in D, and the 3′-end bases in C pair with RNA bases in chain A. There exist pairs crossing along the ‘linear’ sequence position-wise, hence the reported “pseudoknot”. However, simply reverse DNA chains C and D, i.e., moving chain D before C (in file 4un3-ADC.pdb), the “pseudoknot” will be gone, as shown below:

Secondary structures in dot-bracket notation (dbn) as a whole and per chain
>4un3-ADC nts=120 [whole]
>4un3-ADC-A #1 nts=81 [chain] RNA
>4un3-ADC-D #2 nts=11 [chain] DNA
>4un3-ADC-C #3 nts=28 [chain] DNA



The Biophysical Society (BPS) 59th annual meeting at Baltimore

I’m going to attend the Biophysical Society (BPS) 59th Annual Meeting to be held during February 7-11 at Baltimore, Maryland. In last year’s BPS annual meeting (San Francisco, California), I was delighted to come across a few 3DNA users at poster sessions. I thought this post may help to connect me with some DSSR/3DNA users in the coming meeting.

Want to have a meetup at Baltimore? Please drop me a message!



Weird atom names of ligand thiamine pyrophosphate (TPP)

Recently I came across the ligand thiamine pyrophosphate (TPP) in some RNA riboswitch structures. I was a bit surprised by the atom names adopted for the ligand by the PDB. See figures below for the chemical structure of TPP from the RCSB PDB website (first), and the three-dimensional structure of the ligand from the riboswitch 2gdi (second).

Chemical structure of ligand thiamine pyrophosphate

Ligand thiamine pyrophosphate in PDB entry 2gdi

Specifically, the planar base-like moiety at the right has atom names ending with prime. To my knowledge, only sugar atom names of DNA and RNA nucleotides have the prime suffix, such as the 2′-hydroxyl group in RNA.

The RCSB webpage for TPP shows that currently there are 107 entries in the PDB, among which 100 are from proteins, 6 from RNA, and one in a RNA-protein complex. It is not clear to me whether the prime-bearing names in TPP are following any documented ‘standard’ or convention. DSSR is nevertheless taking a note of such ‘weird’ cases.



The 3DNA Forum registered users have reached 2000

As of today, the number of registered users on the 3DNA Forum has reached 2000. Over the past three years, the annual average of resignations is 650, corresponding to approximately 1.8 per day. While many registrations use free email services (gmail, hotmail or yahoo, etc), a significant portion (especially more recent ones) employs their job email (e.g., .edu). This is clear sign of increasing trust the community puts in the Forum.

To ensure the 3DNA Forum spam-free, I’ve adhered a zero-tolerance policy of any trolling or suspicion activities. The anti-spam software has played a big role in making this clean status feasible, as is evident from the note: “120,933 Spammers blocked up until today”.

From a scientific perspective, all posted questions have been addressed promptly, normally within hours. Instead of feeling like a burden, maintaining the Forum and answering user questions have been a pleasure. I’d love to see more questions or posts on the Forum.



Characterization of H-type pseudoknots with DSSR

The v1.2.1 (2015feb01) release of DSSR contains a new functionality to characterize the so-called H-type pseudoknots. In this classical and most common type of pseudoknots, nucleotides from a hairpin loop form Watson-Crick base pairs with a single-stranded region outside of the hairpin to create another (adjacent) stem, as shown in the following illustration (taken from the Huang et al. paper A heuristic approach for detecting RNA H-type pseudoknots).

Schematic diagram the H-type pseudoknot

Normally, L2 is absent (i.e., with zero nucleotides) due to direct coaxial stacking of the two stems. An example output of DSSR on 1ymo (a human telomerase RNA pseudoknot) is shown below:

3D and secondary structures of an H-type pseudoknot (1ymo)

The corresponding sections from DSSR output are:

List of 3 H-type pseudoknot loop segments
   1 stem#1(hairpin#1) vs stem#2(hairpin#2) L1 groove=MAJOR nts=8 UUUUUCUC U7,U8,U9,U10,U11,C12,U13,C14
   2 stem#1(hairpin#1) vs stem#2(hairpin#2) L2 groove=----- nts=0
   3 stem#1(hairpin#1) vs stem#2(hairpin#2) L3 groove=minor nts=8 CAAACAAA C30,A31,A32,A33,C34,A35,A36,A37

Secondary structures in dot-bracket notation (dbn) as a whole and per chain
>1ymo-1-A #1 nts=47 [chain] RNA

Checking against the three-dimensional image and the secondary structure in linear form shown above, the meaning of the new section should be obvious. If you want to see more details, click the link to the DSSR-output file on 1ymo.



Two more citations to DSSR

Recently I came across the following two citations to DSSR:

Base pair types were annotated with RNAview (45,46). Hydrogen bonds were annotated manually and with the help of DSSR of the 3DNA package (47,48). Helix parameters were obtained using the Curves+ web server (49). Structural figures were prepared using PyMol (50).

It is interesting to note that DSSR is cited here for its identification of hydrogen bonds, not its annotation of base pairs, among many other features. The simple geometry-based H-bonding identification algorithm, originally implemented in find_pair/analyze of 3DNA (and adopted by RNAView) and highly refined in DSSR, works well for nucleic acid structures. With the --get-hbonds option, users can now use DSSR as a tool just for its list of H-bonds outside of the program.

All figures were generated using PyMOL (60) or Chimera (48). The secondary structure diagram of the human mitoribosomal RNA was prepared by extracting base pairs from the model using DSSR (61). The secondary structure diagram was drawn in VARNA (62) and finalized in Inkscape.

I am very pleased to see that DSSR was cited for its ‘intended’ use in this important piece of work from a leading laboratory in structural biology. In the middle of last November (2013), I was approached by the lead author for proper citation of DSSR, and I suggested the two 3DNA papers. As far as I can remember, this was the first time I received such a question on DSSR citation. It prompted to write a FAQ entry in the DSSR User Manual, titled “How to cite DSSR?”. Hopefully, this citation issue will be gone in the near future.

Over the past two years, I’ve devoted significant efforts to make DSSR a handy tool for RNA structural bioinformatics; it certainly represents my view as to what a scientific software program should be like. As time passes by, DSSR is becoming increasingly sophisticated and citations to DSSR can only be higher.



Processing large structures in mmCIF format

Recently, PDB begins to release atomic coordinates of large (ribosomal) structures in mmCIF format. For nucleic-acid-containing structures, the largest one so far is 4v4g, the crystal structure of five 70S ribosomes from Escherichia coli in complex with protein Y. It is assembled from ten PDB entries (1voq, 1vor, 1vos, 1vou, 1vov, 1vow, 1vox, 1voy, 1voz, 1vp0), consisting of 22,345 nucleotides, and a total of 717,805 atoms.

This humongous structure poses no problems to DSSR at all, as shown below.

Command: x3dna-dssr -i=4v4g.cif -o=4v4g.out
Processing file '4v4g.cif' [4v4g]

total number of base pairs: 9277
total number of multiplets: 918
total number of helices: 1099
total number of stems: 1221
total number of isolated WC/wobble pairs: 603
total number of atom-base stacking interactions: 1736
total number of hairpin loops: 504
total number of bulges: 170
total number of internal loops: 775
total number of junctions: 214
total number of non-loop single-stranded segments: 429
total number of kissing loops: 5
total number of A-minor (type I and II) motifs: 100
total number of ribose zippers: 58 (1159)
total number of kink turns: 39

Time used: 00:00:10:45

It took less than 11 minutes to run on an iMac (and nearly 14 minutes on a Ubuntu Linux machine). Given the



DNA/RNA molecular dynamics trajectory analysis with do_x3dna

With great pleasure, I read the following annoancement from Rajendra Kumar on the 3DNA Forum:

Re: do_x3dna: a tool to analyze DNA/RNA in molecular dynamics trajectories 
« Reply #1 on: Today at 10:53:31 AM »


I have now made a new website for do_x3dna
(http://rjdkmr.github.io/do_x3dna). This website contains detailed
documentation for do_x3dna program and Python APIs.

Documentation for Python API is now available

Few tutorials about the Python APIs are also now available


With best regards,

Browsing through the do_x3dna website, I am impressed by the extensive documentation and tutorial. Clearly, do_x3dna has pushed the boundaries (in applicability and documentation) of the x3dna_ensemble Ruby script distributed with 3DNA v2.1.

As noted in GitHub page, do_x3dna has been developed to analyze fluctuations in DNA or RNA structures in molecular dynamics (MD) trajectories. It can be used for GROMACS MD trajectories, as well as those from NAMD and AMBER. It leaves no doubt that do_x3dna will boost 3DNA’s applications in the increasingly active field of DNA/RNA MD simulations.



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