Recently, I read carefully the two papers by Farag et al. on the ASC-G4 algorithm to calculate "advanced structural characteristics of G-quadruplexes" (2023), and the comprehensive analysis results of intramolecular G4 structures in the PDB (2024). By developing a convention to orient and number the four strands, ASC-G4 allows for unambiguous determination of the intramolecular G4 topology. It also has an in-depth discussion on assigning syn or anti glycosidic configuration of guanosines, and categorizes four different types of snapbacks.

I am glad to see that DSSR is cited in these two papers, as quoted below:

X3dna-DSSR (19) (http://x3dna.org) is a website that was created to calculate nucleic acid structural parameters, like the local base-pair parameters, local step base-pair parameters, torsion angles, etc, but not the special characteristics of G4. A subdomain dedicated to G4, DSSR-G4DB (Dissecting the Spatial Structure of RNA – G4 Data Base) (http://g4.x3dna.org) emanated from this website. It is a database that gathers and calculates some specific structural information about published G4s, like the topology, the rise, the helical twist, etc, but not the groove widths or the presence of snapbacks. -- Farag et al. (2023) 

Indeed, DSSR-G4DB dose not classify snapbacks. I was aware of such non-canonical G4s when I first developed the G4 module in DSSR around 2017-2018, and the V-shaped loops was derived to reflect the peculiarity of snapbacks.

DSSR classifies groove widths as medium, wide, or narrow, based on the glycosidic angles of neighboring guanosines in a G-tetrad, following the G4 literature. Using PDB entry 2lod as an example, the relevant part of the DSSR output is shown below. The groove widths of the three G-tetrads in the G4-stem have the same pattern of groove=--wn, standing for medium, medium, wide, and narrow, respectively. Note that the medium groove is represented by a dash instead of m because --wn stands out more clearly than mmwn (similar idea applies to glycosidic bond, e.g., sss-).

 1  glyco-bond=sss- sugar=---- groove=--wn Major-->WC N- nts=4 GGGG A.DG1,A.DG6,A.DG20,A.DG16
 2  glyco-bond=---s sugar=---- groove=--wn WC-->Major N+ nts=4 GGGG A.DG2,A.DG7,A.DG21,A.DG15
 3  glyco-bond=---s sugar=---- groove=--wn WC-->Major N+ nts=4 GGGG A.DG3,A.DG8,A.DG22,A.DG14

Since DSSR-G4DB is a database, the user cannot provide his own G4 structure, to obtain structural information. Hence the necessity of developing a website where the user uploads his G4 structure file to obtain all its important and specific structural characteristics (like the topology, the groove width, the tilt and twist angles, etc.). This can be very useful, not only for the analysis of published PDB structures but also for structures in refinement or obtained from MD simulations, to evaluate their quality. To our knowledge, there is no website dedicated to G4 to do such calculations in real-time. Therefore, we developed the algorithm ASC-G4 (advanced structural characteristics of G4) and deployed it as a user-friendly website at the following address: http://tiny.cc/ASC-G4. -- Farag et al. (2023)

Thanks to the NIH R24GM153869 grant support, the http://g4.x3dna.org website now allows users to upload their own atomic structures in PDB for mmCIF format for the identification, annotation, and visualization of G4s. See the example of uploading PDB coordinate file 2lod.pdb.

As background, I had long aspired to develop a dynamic website for on-demand G4 structural analysis but was unable to pursue this goal until recently. During the 4-year funding gap, I still managed to maintain the website g4.x3dna.org, which provides DSSR results for G4 structures in the PDB (a resource now known as the DSSR-G4DB database). To date, the only published work related to G4s is my 2020 paper on the integration of DSSR with PyMOL. Clearly, a dedicated method paper detailing the G4 module in DSSR and the g4.x3dna.org website has been long overdue.

As an initial step toward addressing this gap, I have recently revised the G4-related code in DSSR, fixed existing bugs, and added new features. The g4.x3dna.org website has undergone a complete overhaul, enabling users to upload their own structures for dynamic G4 analysis. Additionally, the DSSR-G4DB database is being actively updated on a weekly basis as new PDB entries are added.

Calculation of the twist and tilt angles. In G4, the helix twist is the rotation of a tetrad relative to its successive one. To measure the twist angle, the most spread method is that described by Lu and Olson (2003) (32) and Reshetnikov et al. (2010) (33). In this method, the angle is calculated from the dot product between two C1’–C1’ vectors from two successive tetrads, i and i + 1, the C1’ atoms of each vector belonging to two adjacent guanosines of a Hbp. The issue with this method is that it does not allow access to the sign of the angle, which defines the direction of the G4 helix, viz. right-handed or left-handed. -- Farag et al. (2023)

There is clearly a misunderstanding in the above text. 3DNA/DSSR can handle left-handed Z-DNA without any issues. DSSR also reports negative twist angles for left-handed G4s, as shown clearly for PDB entry 7d5e, for example.

3DNA/DSSR derives a complete of set of six base-pair parameters (including shear and opening), six step parameters (including twist and rise), and six helical parameters, using a rigorously defined and completely reversible algorithm (CEHS) and the standard base-reference frame. See section "3.2.3 Base pairs" in DSSR User Manual for more details. The DSSR output for G4s (as in DSSR-G4DB) reports only twist and rise, along with overlapped areas, simply because these are the most important parameters and easily interpretable.

The list of the resolved G4 structures was downloaded from the ONQUADRO website (https://onquadro.cs.put.poznan.pl/) (39) at about the end of October 2023. It consisted of 291 intramolecular structures (named unimolecular in the website) and 154 intermolecular G4s (96 bimolecular and 58 tetramolecular). Only the intramolecular structures were kept for this study. To this list, we added 55 missing intramolecular structures that were found on the website of DSSR-G4DB (http://g4.x3dna.org) (40). From the merged list, 345 structures were downloaded from the Protein Data Bank (PDB) (http://www.rscb.org/pdb/) (41) because one structure had no available coordinates in the PDB format (7ZJ5 (42)). -- Farag and Mouawad (2024)

DSSR adopts the frame of reference of Webba da Silva, designating the four strands and grooves of G4-stem as shown below using PDB entries 8ht7 (G1 in syc) and 5ua3 (G1 in anti) as example for the syn or anti glycosidic bond of the 5'-guanosine, respectively.

In DSSR, the first strand (#1) is always upward (U) from 5' to 3'-end, and the polarity of the other three strands is determined by its orientation relative to #1: U if parallel, or D if antiparallel. There are a total of 2x2x2=8 possible combinations of U and D for the three strands, which define parallel (U4: UUUU), antiparallel (U2D2: UDDU, UDUD, UUDD), or hybrid (UD3: UDDD; U3D: UDUU, UUDU, UUUD). For example, the PDB entry 2lod is characterized by DSSR as:  "hybrid-(mixed), UUUD, U3D(3+1)", and PDB entry 8ht7 as:  "anti-parallel, UDUD, chair(2+2)". This notation is topologically equivalent to the one adopted by ASC-G4 but with opposite orientation of the strands.

Overall, DSSR and ASC-G4 provide different perspectives on G4 structures. It is to the user to decide which one is more suitable for their needs.

References

Farag,M. et al. (2023) ASC-G4, an algorithm to calculate advanced structural characteristics of G-quadruplexes. Nucleic Acids Res., 51, 2087–2107.

Farag,M. and Mouawad,L. (2024) Comprehensive analysis of intramolecular G-quadruplex structures: furthering the understanding of their formalism. Nucleic Acids Res., gkae182.