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Input File Types:
  1. Files containing coordinates of the atoms in PDB format (XXXX.pdb)
  2. Files containing coordinates of the atoms in mmCIF format (XXXX.cif)
  3. Files containing coordinates of atoms obtained from NMR or MD simulation, with many models in the same file.
  4. In case of RNA or DNA oligonucleotide structure, whose coordinates for single strands are distributed by PDB or NDB, one needs to download its Biological Assembly file in the users machine. It is found that both RCSB and NDB use ENDMDL to denote (i) end of asymmetric unit, i.e., coordinates of first strand and before the coordinates of symmetry related second strand in a biological assembly as well as to denote (ii) ends of different NMR models in the NMR derived structures. Users are recommended to edit the biological assembly file to remove the ENDMDL line before uploading a biological assembly.
  5. Confusion arises when multiple atomic positions of same atoms are found in a PDB file, which have different Occupancy values. The program choses those atoms whose Occupancies are greater or equal to 0.5. In case of two alternate positions both with 0.5 Occupancy, the first positions are considered. In cases where three positions are given, but none with Occupancy larger than 0.5, the atoms are not considered.
  6. When the key atoms of a nucleotide base, such as C1', N3, N1, etc., are not given for a residue or these atoms are discarded due to Occupancy problem while other atoms are given, the residue is not considered for analysis.

Different Options for BPFIND:
  1. Hydrogen bond distance cutoff: To set default hydrogen bond distance cutoff (default = 3.8)
  2. Pseudo angle cutoff: To set default pseudo angle cutoff (default = 120.0)
  3. E-value cutoff: E-value is calculated on the basis of distance and angle (Follow our paper BPFIND(2006) )
  4. Select desired chain identifier in PDB entry: To select desired chain identifier in the cif/pdb File. (default = all chains)
  5. Select desired NMR model in the File: For NMR model number.
  6. Include HETATM entries in PDB : To include HETATM entries.
  7. Avoid identification of base pairs stabilized by C-H...O/N H-bonds : To avoid identification of base pairs stabilized by C-H...O/N H-bonds
  8. Avoid identification of base pairs involving sugar O2' atoms : To avoid identification of base pairs involvingsugar O2' atoms
  9. Avoid base pairing between residue no. i and i+1 : To avoid base pairing between two consequtive residues.
  10. Avoid printing base pairing information w.r.t. the second strand. : This is suitable for simple oligonucleotides .

Different Options for NUPARM:
  1. Do not use default cross-product method: Finding the vectors normal to each base is vital for calculation of most parameters and none of the methods are perfect. By default the NUPARM program calculates all atom-triads (three atoms at a time) to find out normals to them and calculates average of these normals. Clicking this box forces the program to use Least-Squares method for finding mean base perpendicular.
  2. Use C1' - C1' as Y-axis: Traditionally Dickerson's FREEDNA program used to consider the vector joining two C1' atoms of the two paired bases as base pair Y-axis. This has some other limitation as the mid-point between two C1' atoms is far from the center of mass of the base pair. Choice of C8—C6 joining line as base pair Y-axis is better in that respect and that is the default. The user can click this button to compare results with FREEDNA parameters.
  3. Do not use C6-C8 midpoint as BP center: One can select this button to use center of mass of a base pair instead of C7—C8 midpoint.
  4. Required Single stranded parameters: This is often required for complete analysis of DNA/RNA complete double helical molecules but for functional RNA such calculation may not be required.
  5. Consider all base pairs as Watson-crick type: NUPARM, by default, calculates all intra-base pair parameters, (Propeller, Buckle, etc.,) considering hydrogen bonding edge specific axis system. As a result parameters of a good non-canonical base pair also are calculated near zero (except for Stretch, which is true distance between the two bases and is around 2.8). This option has been kept to compare NUPARM results, especially for intra-base pair parameters, with parameters calculated by CURVES or 3DNA packages.

Output File Descriptions:
  1. XXXX.cor : Contains coordinates of the nucleic acids only considered for searching H-bonds. Base moieties, not having all the purine/pyrimidine ring atoms and C1' atoms, are not considered for basepairing. Therefore, they are not included in XXXX.cor files.
  2. XXXX.out : Contains detailed descriptions of the basepairing patterns present within the RNA molecule. This file also contains information about base triples or multiplets, i.e. when a base pairs with more than one bases. This file can be consulted to know PDB residue number of the residue serial numbers given in XXXX.cor, XXXX.nup or XXXX.hlx files
  3. XXXX.nup : Contains basepairing information required to run the associated basepair parameter calculation program NUPARM. The NUPARM software can also be downloaded from the same site. [It is planned that in near future NUPARM would read the information from the XXXX.out file].
  4. XXXX.dat : Contains secondary structure information of each nucleotide residue in simple fasta format, such as H for Watson-Crick basepairs, N for non-canonical basepairs, T for base triples and L for unpaired bases. Different chains are separately mentioned in this file. XXXX.dbn - Contains base pairing information in Dot-Bracket format suitable
  5. XXXX.dbn : Contains secondary structure information of each nucleotide residue in simple fasta format, such as H for Watson-Crick basepairs, N for non-canonical basepairs, T for base triples and L for unpaired bases. Different chains are separately mentioned in this file.
  6. XXXX.hlx : Contains base pairing information of only the double helices, ignoring the unpaired as well as isolated pairs.
  7. XXXX_pairing.csv : Information similar to XXXX.out file but in a format suitable to load in databases
  8. XXXX_structure.csv : Information similar to XXXX.dat file but in a format suitable to load in databases
  9. XXXX.prm : All the parameter information like open, buckle etc are stored in this file.
  10. Other files: Other than these files, some other image files are generated by the program.

Base Pair Edge Descriptions:
  1. W - Watson-Crick edge (Capital W).
  2. H - Hoogsteen edge (Capital H).
  3. S - Sugar edge (Capital S).
  4. w - Watson-Crick edge with one or more C-H...O/N type of hydrogen bond (Small w).
  5. h - Hoogsteen edge with one or more C-H...O/N type of hydrogen bond (Small h).
  6. s - Sugar edge with one or more C-H...O/N type of hydrogen bond (Small s).
  7. + - Protonated Watson-Crick edge.
  8. z - Protonated Sugar edge.
  9. g - Protonated Hoogsteen edge (rarely found though).

Base Pair Orientations:
  1. C - Cis Orientation.
  2. T - Trans Orientation.

Base Pair Types:
  1. BP - Normal base pair.
  2. TP - Tartiary pair.
  3. BF - Bifurcated pair (Follow our paper BPFIND(2006) )

Rules for Selection and Rejection of Atoms:
  For Selection and Rejection of atoms, BPFIND uses the following rules.

  1. The software considers an atom when its Occupancy value given by PDB is more than 0.5.
  2. It, however, considers the atom if its Occupancy value is 0.0, as in model structures.
  3. If there is a single Occupancy with value less than 0.5, the atom is Poorly Defined and therefore Rejected.
  4. If there are multiple positions for an atom and Occupancy values of all of these are less than 0.5, then all the atomic positions are Poorly Defined and hence are Rejected.
  5. If there are multiple positions of an atom and Occupancies of some of them are greater or equal to 0.5, then atomic position with highest Occupancy value is Selected and rest all atomic positions are Rejected.
  6. If for an atom, two positions are given with Occupancy values 0.5 for both, then the first atomic position is selected and the second one is Rejected.
  7. For a nucleotide base, if three vital atoms, such as C1', N1(for pyrimidine)/N9(of purine) and N3 could not be selected, then the entire residue is Rejected.

Basepairing Criteria:
  1. Bases not having coordinates of all the ring atoms (purine/pyrimidine) and C1' atoms are not considered for detecting hydrogen bonds.
  2. At least two hydrogen bonds must exist between two bases, at least one of which must be between the base moieties. Basepairs involving both 2'-OH mediated hydrogen bonds are not detected by BPFind. It considers basepairs for which both the H-bonds are of C-H...O/N type, as long as one H-bond is found between the base rings.
  3. BPFind considers a range of modified bases which are usually under the 'HETATM' lines in a PDB file. They are listed in the four files:
    You may keep these files in any directory of your choice but indicate the Unix shell the locations of these files. Please see Section 1 (Installation) of this documentation to know how to declare that.

    Any new modified residue name can be included in one of these files accordingly in order to be recognized by BPFind. You would need to keep in mind the similarity of the modified base with the natural ones in terms of positions of hydrogen bonding donor and acceptor atoms.
  4. BPFind uses a cutoff distance of 3.8 angstrom between the acceptor and donor atoms, a cutoff angle of 120.0 degree for checking planarity of precursor atoms (designed carefully to accommodate more labile basepairs) and a cutoff E-value of 1.8 to signify the overall distortion and maintain a good basepairing geometry. This file contains several columns, their descriptions are given on top header starting "#"
  5. BPFind uses a more stringent option for protonated basepair options in order to avoid false positives. It considers cutoff angle of 150.0 degree for protonated basepairs.
  6. BPFind allows more flexibility if 2'-OH of the ribose sugar moiety is involved in hydrogen bonding, the cutoff angle considered is 100.0 degree.
  7. In case of structures with multiple Occupancy of some atoms, the positions with highest Occupancy is considered. If the positions of highest Occupancy is less than 0.5, the atomic position is ignored. When two atoms have identical Occupancy of 0.5, the first atomic position is considered.