Recently I read the article titled Structural Insights into the Quadruplex−Duplex 3′ Interface Formed from a Telomeric Repeat: A Potential Molecular Target by Krauss et al.. I quickly ran DSSR on the corresponding PDB entry is 5dww. Not surprisingly, DSSR can automatically identify reported key structural features (see output file 5dww.out for details), including the TAT triplet at the quadruplex−duplex junction, and the three G-quartets. Note that the result is based on biological assembly 1 in PDB file 5dww.pdb1 since the asymmetric unit contains four such molecules.
List of 4 multiplets
1 nts=3 TAT 1:A.DT17,1:A.DA19,1:B.DT7
2 nts=4 GGGG 1:A.DG1,1:A.DG5,1:A.DG9,1:A.DG14
3 nts=4 GGGG 1:A.DG2,1:A.DG6,1:A.DG10,1:A.DG15
4 nts=4 GGGG 1:A.DG3,1:A.DG7,1:A.DG11,1:A.DG16
As its title suggests, however, this blog post is about the cartoon-block representations. Four styles of such schematics are shown below, which can all be easily generated using DSSR/PyMOL.
 |
 |
| in default style |
with base-pair blocks |
 |
 |
| minor-groove highlighted |
top-face highlighted |
The cartoon-block representations possess unique features not seen elsewhere. With the help of the dssr_block in PyMOL, they are extremely easy to generate. Such schematics are likely to become popular in illustrations of nucleic acid structures.
As of today (2016-01-16), the number of registrations on the 3DNA Forum has reached 2,562. Moreover, all the members (as far as I can tell) are legitimate since the Forum has remained spam free. From the very beginning, ensuring a high information-to-noice ratio has been a top priority. The goal has been achieved by taking the following measures:
State the rules clearly in the “Registration Agreement”
This forum is dedicated to topics generally related to the 3DNA suite of software programs for the analysis, rebuilding and visualization of three-dimensional nucleic acid structures. To make the 3DNA forum a more pleasant virtual community for all of us to learn from and contribute to, please be considerate and practice good netiquette (http://www.albion.com/netiquette/).
I strive to make the forum spam free. Specifically, posts that are not 3DNA related in the broad sense are taken as spams, and are strictly forbidden. You are solely responsible for the content of your posts. We reserve the right to remove any post deemed as inappropriate, deactivate the account and ban the IP address of any abuser of the forum, WITHOUT NOTICE.
When posting on the Forum, please abide by the following rules: …
In a nutshell, you are welcome to participate and should not hesitate to ask questions, but remember to play nice and preferably share what you’ve learned! Please note that we do not tolerate spamming or off-topic trolling of any form.
Take advantage of anti-spam software
In additional to the verification of email address and check for black-listed IP addresses, the topic-specific questions have been very effective. Three examples of such questions are shown below:
What does the 'A' in 3DNA stand for? (hint: 4-char long)
How many standard bases does RNA have (hint: 1-digit number)
What is the value of the expression (3.1498 * 0 + 168)?
Overall, I do not like CAPTCHA — I’ve found the highly-distorted images in some websites especially troublesome. For the first few of years (to ~2014), the 3DNA Forum did not contain a captcha image in the registration page. Later on, however, I’ve noticed quite a few spam registrations/posts. In addition to quickly cleaning them up manually, I had refined the topic-specific questions, and turned on the visual verification image at level “Medium — Overlapping colored letters, with noise/lines”. Experience over the past couple of year has demonstrated the effectiveness of the combined strategy. As shown in the screen capture below, as of this writing, 177,562 spammers have been blocked by the anti-spam software!
Verify and approve ‘suspect’ accounts quickly
The above mentioned anti-spaming measures have blocked virtually all the “bad guys” so I do not need to waste time fighting them. I receive an email notification for each successful registration. The vast majority of registrants can then immediately access the member-only download section or post questions on the 3DNA Forum after registration. A significant portion (~1 out of 6) of the registrations, however, would be masked as suspicious and need my action. The email message for such cases reads like this:
‘xxxx’ has just signed up as a new member of your forum. Click the link below to view their profile. …
Before this member can begin posting they must first have their account approved. Click the link below to go to the approval screen. …
Wherever I have access to the Internet (including after hours with an iPad Air 2), I’ve always been quick in verifying and (mostly) approving these registrations.
Overall, since http://forum.x3dna.org was created in December 2011, the Forum has received significant attention in the field of DNA/RNA structural bioinformatics. As the community begins to appreciate and fully take advantage of what DSSR and SNAP have to offer, I have no doubt the Forum will gain even wider-spread recognition.
In recent years, reproducibility of ‘scientific’ publications has become quite a topic. See a recent essay Five selfish reasons to work reproducibly by Markowetz in Genome Biology (2015, 16:274). There are numerous reasons why reproducibility could become an issue at all in science. What I have continuously strived for in my scientific career, however, is to ensure that my published results are reproducible. As a concrete example, I created a dedicated section titled DSSR-NAR paper on the 3DNA Forum that provides full details (scripts and data files) so that any interested parties can rigorously reproduce the results reported in the DSSR Nucleic Acids Research (NAR) paper.
In my support of 3DNA for over a decade, the #1 issue I experienced is undoubtedly vague (non-reproducible) questions. For example, I have recently been asked via email why the 3DNA find_pair/analyze programs miss “some basepair … even though it is in the pdb file”. Without access to the PDB file to reproduce the problem, however, I cannot provide a concrete answer. In an effect to prevent ambiguous questions, I made the following explicit point in the “Registration Agreement” of the 3DNA Forum (no. 2 on the list):
Be specific with your questions; provide a minimal, reproducible example if possible; use attachments where appropriate.
The #2 issue is receiving 3DNA-related questions privately instead of on the intended public 3DNA Forum. I turned off “personal messaging” to receive private messages on the Forum long time ago, yet I have kept receiving questions via emails. In several locations on the 3DNA Forum, I have made this ‘public-question’ policy crystal clear:
Ask your questions in the public 3DNA forum instead of sending xiangjun emails or personal messages. (no. 1 on the ‘Registration Agreement’)
Please be aware that for the benefit of the 3DNA-user community at large, I do not provide private email/personal message support; the forum has been created specifically for open discussions of all 3DNA-related issues. In other words, any 3DNA-associated questions are welcome and should be directed here. Presumably I’ve made the message simple and clear enough to get across without further explanation. (in ‘Site announcements » Download instructions’ and ‘Downloads » 3DNA download’)
In response to the many 3DNA-related questions that still keep coming via email, I created the following entry of Canned Responses in gmail:
Thanks for your interest in using 3DNA. Please be aware that for the benefit of the 3DNA-user community at large, I do not provide private email support; the 3DNA Forum (http://forum.x3dna.org/) has been created specifically for open discussions of all 3DNA-related issues. In other words, any 3DNA-associated questions are welcome and should be directed there. I monitor the forum regularly and respond to posts promptly.
I look forward to seeing you on the 3DNA Forum (http://forum.x3dna.org/).
Overall, I’ve learned from experience that addressing reproducible questions publicly does the best for the 3DNA community. Users can register with personal (free) email address, and post simulated data to illustrate the problem at hand. Moreover, questions on the Forum have always received quick responses. Over time, the Forum has served as an archive that everyone can benefit from.
As of v2.3-2016jan01, the 3DNA analyze program outputs a list of new ‘simple’ base-pair and step parameters, by default. Shown below is a sample output for PDB entry 1xvk. This echinomycin-(GCGTACGC)2 complex has a single DNA strand as the asymmetric unit. 3DNA needs the the biological unit (1xvk.pdb1) to analyze the duplex (with the -symm option). This structure contains two Hoogsteen base pairs, and has popped up on the 3DNA Forum for the zero or negative Rise values. Note that the ‘simple’ Rise values are all positive; for the middle (#4) TA/TA step, it is now 3.09 Å instead of 0.
# find_pair -symm 1xvk.pdb1 1xvk.bps
# analyze -symm 1xvk.bps
# OR by combing the above two commands:
# find_pair -symm 1xvk.pdb1 | analyze -symm
# The output is in file '1xvk.out'
This structure contains 4 non-Watson-Crick (with leading *) base pair(s)
----------------------------------------------------------------------------
Simple base-pair parameters based on RC8--YC6 vectors
bp Shear Stretch Stagger Buckle Propeller Opening
* 1 G+C -3.07 1.55 -0.35 -6.98 0.29 67.33
2 C-G 0.27 -0.17 0.35 -22.34 3.33 -2.80
3 G-C -0.39 -0.17 0.41 22.91 1.81 -2.73
* 4 T+A -3.29 1.56 0.31 -8.03 1.59 -70.46
* 5 A+T -3.29 1.56 -0.31 -8.03 1.59 70.46
6 C-G 0.39 -0.17 0.41 -22.91 1.81 -2.72
7 G-C -0.27 -0.17 0.35 22.34 3.32 -2.80
* 8 C+G -3.07 1.55 0.35 -6.98 0.30 -67.33
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
ave. -1.59 0.69 0.19 -3.75 1.75 -1.38
s.d. 1.72 0.92 0.32 17.57 1.15 52.11
----------------------------------------------------------------------------
Simple base-pair step parameters based on consecutive C1'-C1' vectors
step Shift Slide Rise Tilt Roll Twist
* 1 GC/GC -0.55 0.39 7.41 6.40 -4.22 23.36
2 CG/CG -0.05 0.87 2.44 -0.55 3.94 -0.81
* 3 GT/AC 0.38 0.47 7.23 -8.62 3.75 25.70
* 4 TA/TA -0.00 4.73 3.09 -0.00 7.49 25.67
* 5 AC/GT -0.38 0.47 7.23 8.62 3.75 25.70
6 CG/CG 0.05 0.87 2.44 0.55 3.94 -0.82
* 7 GC/GC 0.55 0.39 7.41 -6.40 -4.22 23.36
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
ave. -0.00 1.17 5.32 -0.00 2.06 17.45
s.d. 0.39 1.59 2.50 6.21 4.49 12.52
The simple parameters are ‘intuitive’ for non-Watson-Crick base pairs and associated base-pair steps, where the existing standard-reference-frame-based 3DNA parameters may look weird. Note that these simple parameters are for structural description only, not to be fed into the ‘rebuild’ program. Overall, they complement the rigorous characterization of base-pair geometry, as demonstrated by the original analyze/rebuild pair of programs in 3DNA.
In short, the ‘simple’ base-pair parameters employ the YC6—RC8 vector as the y-axis whereas the ‘simple’ step parameters use consecutive C1’—C1’ vectors. As before, the z-axis is the average of two base normals, taking consideration of the M–N vs M+N base-pair classification. In essence, the ‘simple’ parameters make geometrical sense by introducing an ad hoc base-pair reference frame in each case. More details will be provided in a series of blog posts shortly.
Overall, this new section of ‘simple’ parameters should be taken as experimental. The output can be turned off by specifying the analyze -simple=false command-line option explicitly. As always, I greatly appreciate your feedback.
Over the years, I have played quite a few computer programming languages. ANSI C has become my top choice for ‘serious’ software projects, due to its small size, efficiency, flexibility, and ubiquitous support. Moreover, C is a mature language, with a rich ecosystem. As it turns out, C has also been consistently rated as one of the most popular computer languages (#1 or #2) over the past thirty years.
Needless to say, ANSI C has its own quirks, and it takes a steep learning curve. However, once you get over the hurdles, the language serves you. I cannot remember when, but it has been a long while that coding in ANSI C is no longer an issue. It is the understanding of scientific questions that takes most of my time, and coding helps greatly in refining my thoughts.
Not surprisingly, ANSI C was chosen as the sole language for DSSR (and SNAP, or 3DNA in general). The ensure the overall quality of the DSSR codebase, I have taken the following steps:
- The whole project is under git.
- The ANSI C source code is compiled with strict GCC options for full compliance to the standard:
-ansi -pedantic -W -Wall -Wextra -Wunused -Wshadow -Werror -O3
- The executable is checked with valgrind for any memory leak:
valgrind --leak-check=full x3dna-dssr -i=1ehz.pdb -o=1ehz.out --quiet
==19624== Memcheck, a memory error detector
==19624== Copyright (C) 2002-2013, and GNU GPL'd, by Julian Seward et al.
==19624== Using Valgrind-3.10.1 and LibVEX; rerun with -h for copyright info
==19624== Command: x3dna-dssr -i=1ehz.pdb -o=1ehz.out --quiet
==19624==
==19624==
==19624== HEAP SUMMARY:
==19624== in use at exit: 0 bytes in 0 blocks
==19624== total heap usage: 52,829 allocs, 52,829 frees, 92,878,578 bytes allocated
==19624==
==19624== All heap blocks were freed -- no leaks are possible
==19624==
==19624== For counts of detected and suppressed errors, rerun with: -v
==19624== ERROR SUMMARY: 0 errors from 0 contexts (suppressed: 0 from 0)
- Extensive tests (with the simple
diff command) to ensure the program is working as expected.
The above four measures combined allow me to add new features, refactor the code, and fix bugs, without worrying about accidentally breaking existing functionality. Reading literature (including citations to 3DNA/DSSR) and responding to user feedback on the 3DNA Forum keep me continuously improve DSSR. Some of the recent refinements to DSSR came about this way.
Recently, I became aware of the metallo-base pairs, such as T-Hg-T (PDB id: 4l24) and C-Ag-C (5ay2) from the work of Kondo et al (Pubmed: 24478025 and 26448329). As of v1.4.3-2015oct23, DSSR can detect such metallo-bps automatically, as shown below:
# x3dna-dssr -i=4l24.pdb -o=4l24.out
List of 12 base pairs
nt1 nt2 bp name Saenger LW DSSR
1 A.DC1 B.DG24 C-G WC 19-XIX cWW cW-W
2 A.DG2 B.DC23 G-C WC 19-XIX cWW cW-W
3 A.DC3 B.DG22 C-G WC 19-XIX cWW cW-W
4 A.DG4 B.DC21 G-C WC 19-XIX cWW cW-W
5 A.DA5 B.DT20 A-T WC 20-XX cWW cW-W
6 A.DT6 B.DT19 T-T Metal n/a cWW cW-W
7 A.DT7 B.DT18 T-T Metal n/a cWW cW-W
8 A.DT8 B.DA17 T-A WC 20-XX cWW cW-W
9 A.DC9 B.DG16 C-G WC 19-XIX cWW cW-W
10 A.DG10 B.DC15 G-C WC 19-XIX cWW cW-W
11 A.DC11 B.DG14 C-G WC 19-XIX cWW cW-W
12 A.DG12 B.DC13 G-C WC 19-XIX cWW cW-W
and
# x3dna-dssr -i=5ay2.pdb -o=5ay2.out
List of 24 base pairs
nt1 nt2 bp name Saenger LW DSSR
1 A.G1 B.C12 G-C WC 19-XIX cWW cW-W
2 A.G2 B.C11 G-C WC 19-XIX cWW cW-W
3 A.A3 B.U10 A-U WC 20-XX cWW cW-W
4 A.C4 B.C9 C-C Metal n/a cWW cW-W
5 A.U5 B.A8 U-A WC 20-XX cWW cW-W
6 A.CBR6 B.G7 c-G WC 19-XIX cWW cW-W
7 A.G7 B.CBR6 G-c WC 19-XIX cWW cW-W
8 A.A8 B.U5 A-U WC 20-XX cWW cW-W
9 A.C9 B.C4 C-C Metal n/a cWW cW-W
10 A.U10 B.A3 U-A WC 20-XX cWW cW-W
11 A.C11 B.G2 C-G WC 19-XIX cWW cW-W
12 A.C12 B.G1 C-G WC 19-XIX cWW cW-W
13 C.G1 D.C12 G-C WC 19-XIX cWW cW-W
14 C.G2 D.C11 G-C WC 19-XIX cWW cW-W
15 C.A3 D.U10 A-U WC 20-XX cWW cW-W
16 C.C4 D.C9 C-C Metal n/a cWW cW-W
17 C.U5 D.A8 U-A WC 20-XX cWW cW-W
18 C.CBR6 D.G7 c-G WC 19-XIX cWW cW-W
19 C.G7 D.CBR6 G-c WC 19-XIX cWW cW-W
20 C.A8 D.U5 A-U WC 20-XX cWW cW-W
21 C.C9 D.C4 C-C Metal n/a cWW cW-W
22 C.U10 D.A3 U-A WC 20-XX cWW cW-W
23 C.C11 D.G2 C-G WC 19-XIX cWW cW-W
24 C.C12 D.G1 C-G WC 19-XIX cWW cW-W
Note the name “Metal” for the metallo-bps. Moreover, the corresponding entries in the ‘dssr-pairs.pdb’ file also include the metal ions, as shown below:
It is worth noting that in a metallo-bp, the metal ion lies approximately in the bp plane. Moreover, it is in the middle of the two bases, which would otherwise not form a pair in the conventional sense.
Curves+ and 3DNA are currently the most widely used programs for analyzing nucleic acid structures (predominantly double helices). As noted in my blog post, Curves+ vs 3DNA, these two programs also complement each other in terms of features. It thus makes sense to run both to get a better understanding of the DNA/RNA structures one is interested in.
Indeed, over the past few years, I have seen quite a few articles citing both 3DNA and Curves+. Listed below are three recent examples:
The helical parameters were measured with 3DNA33 and Curves+.34 The local helical parameters are defined with regard to base steps and without regard to a global axis.
Structure analysis. Helix, base and base pair parameters were calculated with 3DNA or curve+ software packages23,24.
The major global difference between the native and mixed backbone structures is that the RNA backbone is compressed or kinked in strands containing the modified linkage (Fig. 3 B and C, by CURVES) (30). … To compare the three RNA structures at a more detailed and local level, we calculated the base pair helical and step parameters for all three structures using the 3DNA software tools (31) (Fig. 4 and Table S2). [In the Results section]
For each snapshot, the structural parameters—including six base pair parameters, six local base pair step parameters, and pseudorotation angles for each nucleotide—were calculated using 3DNA (31). The two terminal base pairs are omitted for the 3DNA analysis, because they unwind frequently in the triple 2′-5′-linked duplex. [In the Materials and Methods section]
Reading through these papers, however, it is not clear to me if the authors took advantage of the find_pair -curves+ option in 3DNA, as detailed in Building a bridge between Curves+ and 3DNA. Hopefully, this post will help draw more attention to this connection between Curves+ and 3DNA.
JSON (JavaScript Object Notation) is a simple human-readable format that expresses data objects in name-value pairs. Over the years, it has surpassed XML to become the preferred data exchange format between applications. As a result, I’ve recently added the --json command-line option to DSSR to make its numerous derived parameters easily accessible.
The DSSR JSON output is contained in a compact one-line text string that may look cryptic to the uninitiated. Yet, with commonly available JSON parsers or libraries, it is straightforward to make sense of the DSSR JSON output. In this blogpost, I am illustrating how to parse DSSR-derived .json file via two command-line tools, jq and Underscore-CLI.
jq — lightweight and flexible command-line JSON processor
According to its website,
jq is like sed for JSON data – you can use it to slice and filter and map and transform structured data with the same ease that sed, awk, grep and friends let you play with text.
Moreover, like DSSR per se, “jq is written in portable C, and it has zero runtime dependencies.” Prebuilt binaries are available for Linux, OS X and Windows. So it is trivial to get jq up and running. The current stable version is 1.5, released on August 15, 2015.
Using the crystal structure of yeast phenylalanine tRNA (1ehz) as an example, here are some sample usages with DSSR-derived JSON output:
# Pretty print JSON
x3dna-dssr -i=1ehz.pdb --json | jq .
# Extract the top-level keys, in insertion order
x3dna-dssr -i=1ehz.pdb --json | jq keys_unsorted
# Extract parameters for nucleotides
x3dna-dssr -i=1ehz.pdb --json | jq .nts
# Extract nucleotide id and its base reference frame
x3dna-dssr -i=1ehz.pdb --json | jq '.nts[] | (.nt_id, .frame)'
Underscore-CLI — command-line utility-belt for hacking JSON and Javascript.
Underscore-CLI is built upon Node.js, and can be installed using the npm package manager. It is claimed as ‘the “swiss-army-knife” tool for processing JSON data – can be used as a simple pretty-printer, or as a full-powered Javascript command-line.’
Following the above examples illustrating jq, here are the corresponding commands for Underscore-CLI:
x3dna-dssr -i=1ehz.pdb --json | underscore print --color
x3dna-dssr -i=1ehz.pdb --json | underscore keys --color
x3dna-dssr -i=1ehz.pdb --json | underscore select .nts --color
x3dna-dssr -i=1ehz.pdb --json | underscore select .nts | underscore select '.nt_id, .frame' --color
jq or Underscore-CLI — which one to use?
As always, it depends. While jq feels more like a standard Unix utility (as sed, awk, grep etc), Underscore-CLI is better integrated into the Javascript language. For simple applications such as parsing DSSR output, either jq or Underscore-CLI is more than sufficient.
I use jq most of the time, but resort to Underscore-CLI for its “smart whitespace”. Here is an example to illustrate the difference between the two:
# z-axis of A.G1 (1ehz) base reference frame
# jq output, split in 5 lines
"z_axis": [
0.799,
0.488,
-0.352
]
# Underscore-CLI, in a more-readable one line
"z_axis": [0.799, 0.488, -0.352]
From early on, the x3dna.org domain and its related sub-domains (e.g., for the forum and the web-interface to DSSR) has been served via shared hosting. By and large, this simple arrangement has worked quite well. Over the years, though, I’ve gradually realized some of its inherent limitations. One is the limited resources available to the 3DNA-related websites. Another is the accessibility issue from countries like China.
To remedy such issues, I’ve recently moved the 3DNA Forum and the web-interface to DSSR to a dedicated web server at Columbia University. Moreover, a duplicate copy of the 3DNA homepage is made available via http://home.x3dna.org hosted at Columbia. The three new websites have been verified to be accessible directly from China.
These updates on x3dna.org not only ensure global accessibility to 3DNA/DSSR, but also allow for more web services to be made available.
