Software for Deep Analysis of PDB Structures and Mechanism Discovery

 

 

 


   
   
         New Kd prediction server for PDB files Go!


                    

     
          



SEQMOL is a PDB structure analysis suite.
SEQMOL can be used to align multiple protein and DNA
sequences, compute evolutionary attributes of multiple sequence alignments
(such as sequence conservation, hydrophobicity conservation, conformational
flexibility conservation, physical covariation, protein-protein interface,
protein-RNA interface and protein-DNA interface propensity, and conservations
thereof) and to map these features onto PDB files.
Many of the features do not use multiple sequence alignment and directly analyze
PDB coordinates to yield insights that could often be valuable.


Some of the SEQMOL components:
- Maker and editor of multiple sequence alignments
- PDB surface solvation (by water) and Coulomb electrostatics analysis module
- Protein/Protein and rather accurate protein/RNA binding sites predictions module
- Binding constants prediction module for PDB complexes and crystal contacts
based on a de-novo built algorithm with sound Kd accuracy




* Kd predictions in SEQMOL are user-friendly and usually fast (seconds-minutes for
average-sized complexes on a local CPU). A
PDB file is DragDropped onto the
application. Kd along with a comprehensive interface analysis is produced in the output.
For multiple-PDB jobs, such as scoring of decoys from docking programs,
batch PDB processing is done automatically if more than one PDB was DragDropped.


** Perhaps this is where SEQMOL use is particularly appealing. If we could know Kd
for each interface in a protein crystal, we would be able to tell which crystallization
interfaces are real and biologically important and which are not.
Unfortunately, measuring individual Kd of crystal interfaces is not possible.

At present it is accepted to sometimes look at burial of accessible surface area
(ASA burial), at solvation, and sometimes at electrostatic complementarity of interfaces
to tell non-specific crystal packing contacts from true interfaces.

Yet, many stable complexes have mismatched electrostatics; some don't bury
very much ASA and some have unfavorable desolvation energy. And vice versa,
some weak crystal contacts are extensive and appear stable.


This problem is minimized by SEQMOL. It usually can identify stable or weak complexes
by calculating Kd for formation of interfaces. Except for proteins undergoing major
conformational changes, calculated Kds often agree with those
from experimental measurements.





Picture above is crystal packing of one of CDK2 kinase complexes.
Two rather large kinase-kinase interfaces can be seen in the structure.
Not much can be added to this picture, but after SEQMOL is used
additional and very non-intuitive
information is obtained:

IF1
dEsolv +8264.6 kcal/mol
dEelec -62.32 kcal/mol
dEnthalpy 13 A.U.
dASA -1684.6 (A^2)
dEntropy 119 A.U.
dGbinding -7.86 kcal/mol
Kd 1.84E-06M

IF2
dEsolv -2927.2 kcal/mol
dEelec 113.85 kcal/mol
dEnthalpy 6 A.U.
dASA -1417.6 (A^2)
dEntropy 84 A.U.
dGbinding -6.63 kcal/mol
Kd 1.45E-05M

Both interfaces are somewhat unstable (Kd ~ 1-10 uM); IF1 is more stable.
IF1 has a very unfavorable solvation energy and IF2 has a very
favorable solvation energy (yet weaker Kd!).

Electrostatics opposes the formation of the IF2
and helps the formation of the IF1. This information is useful
for designing mutations for testing the interfaces.



               
     
screen shots (click to enlarge)



Protein coloring by sequence alignment attributes example

CDK2 kinase colored by conservation, physical covariation, hydrophobicity conservation and charge conservation based on 222 non-redundant sequences of CDK2 tion based on 222 non-redundant sequences of CDK2 (click images for close view)



Download PDB  files for the above images




Predicting protein-protein interactions by two different ways

SH3 domain of Abl kinase (PDB ID 1abo). This domain binds a proline-rich peptide APTMPPPLPP.

                           Approach 1


                            Approach 2



Conservation of protein-protein interaction propensity

low
high  

peptide shown









Surface solvation energy (based on ODA method)

low
high  

peptide shown





Approach 2 summary: Binding sites on proteins sometimes employ electrostatic forces. Protein surface electrostatics can be calculated usign Poisson-Boltzmann solvers such as APBS. Often, however, hydrophobic interactions provide the binding energy and electrostatics is not involved. As of version 3.2.7, SEQMOL can evaluate this type of surface properties and calculate solvation energy of protein surfaces using a local variant of ODA approach by L. Perez-Cano et al. The process is reasonably fast and takes seconds-minutes for most proteins; the scores are mapped on the protein surace as B-factors (PyMOL->Color by B-factors). An exmaple of solvation energy calculation for MHCI:  

 




Per-atom surface solvation patch energy of dipeptidyl peptidase IV computed separately for
chains A and B (PDB 3KWH). Surface region that forms protein-protein interface stands out
as having very high desolvation energy and is seen as the red patch. Crystal structure of
the monomer thus correctly shows the location of a strong protein binding site.  


                            chain A                                  chain B

Surface colored by per-atom solvation energy. Note the red "hot spot".






                            chain A

For comparison: this surface is colored by conventional "hydrophobicity" of residues (W, F, Y, A, L, V, I etc are more blue, D, E, N, Q, R, K, S, T etc are more red). The "hot spot" is not obvious.                                 






Nucleic acid desolvation analysis example.






Predicting protein-RNA binding sites via residue interface propensity conservation with subsequent patch energy calculation





Surface electrostatics (blue-red) vs RNA binding patch energy (SRP PDB ID 1QZW).
Note that patch energy shows only correct RNA binding site (red patch) whereas
surface electrostatics commonly used to evaluate RNA binding sites is ambigous.
 



PDB structure analyses -- without sequence alignments

PDB-based calculation of surface burial, electrostatic energy and solvation energy for proteins and nucleic acids and protein-protein protein-RNA and protein-DNA complexes. The program's output is both, quantitative (a single whole-PDB value) and qualitative (PDB surface is colored by scaled per-atom values).

One unplanned but useful appliation of this module is to find pockets in PDB structures by clustering solvent-exposed atoms (read built-in help for explanations).

Computing binding constants from PDB coordinates

A dedicated module in SEQMOL can calculate binding free energy (at 1-molar reference state and 25 oC) and binding constants for protein-protein and *possibly* protein-(RNA/DNA) complexes (this method was developed explicitly for protein-protein interactions but seems to give relevant numbers for some protein-RNA complexes).

The goal of SEQMOL is to not just predict trends in binding energy, but to predict as closely as possible real Kd values one would measure in a lab. 

The procedure is fully automated and only calls for DragDropping a PDB and selecting chain/chains for virtual dissociation and Kd measurement.

Accuracy** disclaimer: in some cases binding constants may be off from the real values by orders of magnitude. However, more frequently than not, they are correct. For example, for 45 out of 59 examined complexes (76%) from the PDB database, computed binding constants were within 2.5 kcal/mol from the experimental values throughout the Kd range from subpicomoles to millimoles, and protein size range from short peptides to large proteins over 50 kD. Deviations in SEQMOL Kd may arise from several reasons: from assumptions in the code, from the fact that binding constants depend on the reaction temperature and buffers (pH, salt concentration), which differ between published Kd, whereas SEQMOL always reports a value projected for an "average" ideal buffer and standard conditions. Many PDB structures are solved at resolutions that do not allow to unmabiguously place some rotamers (HIS, ASN, GLN), resulting in PDB coordinate errors which also contribute to the calculation vs experiment scatter. 

 





**accuracy improved further in recent builds of SEQMOL
   Build 3.4.0 dG accuracy test table:
   95% predictions are under 2 kcal/mol from experiment

PDB ID dASA (A^2) dGexpmt dGcalc ddG
1e.. -766.7 -4.39 -4.36 +0.029
1d.. -1159.3 -6.80 -6.48 +0.315
1m.. -1240.2 -7.30 -6.70 +0.603
1a.. -1160.8 -8.00 -6.22 +1.781
1b.. -879.0 -8.10 -8.63 -0.527
1j.. -1237.5 -8.13 -9.53 -1.397
1b.. -1795.7 -8.90 -8.71 +0.187
3g.. -2359.0 -9.20 -8.83 +0.369
2b.. -1366.4 -9.59 -10.70 -1.113
2p.. -1198.4 -9.70 -10.59 -0.890
1b.. -1338.4 -9.70 -10.17 -0.463
1p.. -1453.8 -9.76 -8.82 +0.945
1g.. -1160.2 -9.79 -9.28 +0.509
1w.. -1205.7 -9.80 -9.16 +0.636
2e.. -1503.9 -9.80 -10.81 -1.012
1b.. -931.3 -10.10 -8.65 +1.447
1g.. -1314.5 -10.10 -9.72 +0.378
1p.. -1615.6 -10.22 -10.26 -0.040
1y.. -1533.4 -10.30 -9.15 +1.143
2n.. -1279.1 -10.30 -11.51 -1.210
1k.. -2408.3 -10.50 -10.47 +0.026
2b.. -1723.7 -10.95 -12.71 -1.768
1y.. -2906.1 -11.03 -9.29 +1.730
1s.. -1305.8 -11.40 -9.60 +1.795
1a.. -1960.7 -11.50 -11.54 -0.041
1f.. -1270.1 -11.60 -12.26 -0.675
2j.. -1540.4 -11.70 -13.09 -1.392
1m.. -1046.0 -11.70 -12.66 -0.968
1v.. -1430.1 -11.80 -14.13 -2.326
1a.. -1793.5 -11.90 -12.80 -0.901
1j.. -1931.3 -12.30 -11.34 +0.962
1f.. -2011.0 -13.00 -12.78 +0.219
1c.. -1510.3 -13.46 -14.16 -0.702
2s.. -1508.4 -13.50 -14.69 -1.188
1j.. -1295.3 -13.90 -11.81 +2.087
2s.. -1628.7 -13.90 -14.26 -0.364
1j.. -2251.1 -14.50 -12.87 +1.627
3h.. -1647.5 -15.00 -16.14 -1.141
1h.. -1690.5 -15.50 -14.89 +0.608
2s.. -1651.7 -16.00 -15.18 +0.820
1a.. -1587.9 -17.00 -16.44 +0.562
1b.. -1577.8 -17.30 -17.64 -0.344
1a.. -2753.9 -20.70 -21.02 -0.318

these PDB structures are mostly unrelated and cover a large functional
space e.g. diverse enzymes, antibodies, receptors, peptide binding motifs
and a vast range of interface sizes


A reading about performance of 9 alternative Kd prediction algorithms:

"Are scoring functions in protein-protein docking ready to predict interactomes?
Clues from a novel binding affinity benchmark."

Kastritis PL, Bonvin AM. J Proteome Res. 2010 May 7; 9(5) 2216-25
PMID: 20329755 



Beyond predicting Kd

Proteins crystallize forming several protein-protein interfaces in the crystal. Distinguishing true interfaces and crystallographic interfaces presents a challenge. With SEQMOL, it is possible to compute Kd for all observed protein-protein contacts in the crystal and obtain a good measure of the interface stability along with realistic Kds for their formation. 

This method is greatly superior to guesses based on surface conservation or accesible surface area (ASA burial), see the table above.


Finding hot spots at protein/protein interfaces

SEQMOL-Kd hosts a utility for one-click generation of PDBs with ALA-scans of any interfaces of choice.
"Hot spot" residues can be identified by a large effect on the binding free energy of the resulting ALA-permuted PDBs.
 


Protein-protein docking: SEQMOL as a decoy filtering utility

Another appliction of the Kd prediction module is to filter results of protein-protein docking programs.

In the test example below, two monomers of a known protein complex were randomly oriented in 3D space. Then they were docked back using Hex 6.0 docking utility. The docking run produced 500 solutions, top 20 of which had energy range from -560 to -470 "Hex units".

During the next step, Kd for the 20 top solutions were calulated with SEQMOL. The range of Kd was from 49000 moles (!)  to 1.0 micromole. 15 of the 20 solutions had Kd around 1 mM or weaker, and are likely to be irrelevant. The best complex according to the Hex energy was ranked #9 with Kd 1.47M (very unstable). The most stable complex according to SEQMOL (Kd 1 micromole) was similar to the one actually seen in the crystal structure:





Docking two proteins: by predicting binding constants for 20 top-ranking docking solutions
(with similar docking scores) it was possible to identify very unstable and very stable solutions.
In this example the most stable complex, as ranked by SEQMOL, was also the near-native one.



How to cite SEQMOL in a publication

SEQMOL ("sequences & molecules")
can be used "as is" with limited functionality or licensed using
built-in licensing module.

If you used the program for your paper, please cite it as
we used SEQMOL or
SEQMOL (biochemlabsolutions.com) was used.

Many of the methods used in SEQMOL have been published by different and truly great authors.
Relevant references are given either on this website or in help boxes within the program. Some
methods are new and have not been published in peer-reviewed journals yet. They will be one day.
At present it is important that they all work and help with generating testable hypotheses and
often do well at extractintg information from protein sequences and PDB structures.
 















         DOWNLOAD  Version 3.4.7
        
Many features will work as is, some will require licensing.
         Kd module, RNA binding sites module and Solvation module work on subscription basis. 
    
         Recent changes   

         -     Kd calculation module (RELEASED)    

         -     PDB structure analysis: calculate surface burial, electrostatic energiy, solvation
                      energy of proteins and nucleic acids and of protein-protein protein-RNA and
                      protein-DNA complexes
 
        -    Calculation of solvent-accessible area (ASA) of all atoms in PDB
                      (protein, RNA, DNA)

         -   Incorporation of ODA and OPRA algorithms for predicting
                      protein-RNA and protein-protein interfaces
                      (based on papers by Laura Perez-Cano and Juan Fernandez-Recio)

         -  Calculation of protein-protein, protein-RNA and protein-DNA binding sites
                      based on evolutionary conservation of residue propensities

         -  Improved surface hydrophobicity calculation routine. SEQMOL no longer
                     uses conventional hydrophobicity scales
 
         -  Computation of conformational flexibility of regions in PDB files using
                     multiple sequence alignments. Color PDB files by conservation of
                     conformational flexibility to predict rigid and dynamic parts.

         Ability to color backbone atoms separately from main chain atoms
                     using "5.1 Force backbone B-factors to" option.
                     When used, for example, during coloring by charge conservation,
                     the actual conservation of charges on the surface stands out better
                     if all backbone atoms have a neutral B-factor of 5.00.

         -  More convenient DNA sequencing tool.              

           Some uses of the program

       o  Multiple sequence alignment of proteins, DNA and RNA
           Several variants of the open source MUSCLE program are integrated
           into this distributive - courtesy of Robert C. Edgar 
       o  Browsing and
editing of multiple sequence alignments, saving results
           in several formats, including print-ready rtf (examples: rtf  pdf
  
       o  Calculating sequence alignment features such as conservation,
           hydrophobicity conservation and other types of scoring schemes;
           mapping these features onto PDB structures of proteins,
           RNA and DNA

       o  Integration of sequence alignments with PDB files to
           link residues in PDB structures with sequence alignments

       o  Exploring interresidue interactions in PDB structures to evaluate different
           conformations, structures and interfaces based on physical covariation
           analysis. Briefly, the algorithm detects 3D partners of each
           residue in a PDB file, then scores physical complementarity of resulting
           residue-residue pairs, then evaluates how well this complementarity is
           conserved between aligned sequences and outputs the resulting
           score for each residue, with the possibility to color residues in
           the PDB file according to this score.
      
o  Accessible surface area, solvation energy, surface electrostatics
           calculations for PDB
       o  Protein/protein and protein/RNA binding sites predictions
       o  Binding energy and Kd predictions for protein/protein
           and - tentatively - for protein/RNA complexes
      
o  Analysis of DNA sequencing results
       

         Manual



System requirements: .NET 3.5 framework  download
Compatible OS: Windows XP, Vista 32/64, Server 2003-2008, Windows 7; MacOS and Linux via free virtualization solution by SUN Microsystems (Licensed Windows installation CD/DVD will be needed)

In case of runtime errors during execution in Windows Vista or Windows 7 make sure that the program is not hampered by the Windows UAC. It is recommended to either disable UAC altogether or to ensure that SEQMOL is always launched with the option "Run as administrator"

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