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For simple and automated computation and analysis of low-frequency normal modes for proteins. The computation is to understand whether the protein undergo large amplitude movements, so we can decide if it is worthwile performing a complete study with thorough analysis.

Reference

Hollup SM, Slensminde G, Reuter N.

Two types of analysis Single structure: Calculates the lowest frequency normal modes of the protein and gives different calculations to analyse the modes that were calculated. Comparative

Single structure analysis Deformation energies Normalized squared atomic displacements. Interactive mode visualization using JMol: vibrations associated with the lowerfrequency modes and vector field representation. Overlap between normal modes and a structure difference vector.

Comparative analysis(Beta version)These tools allow the comparative analysis of multiple structures. Deformation Energy profiles Atomic Fluctuation profiles Conformational Overlap Comparison Still under devolopment

PDB format The Protein Data Bank (PDB) format provides a standard representation for macromolecular structure data derived from X-ray diffraction and NMR studies. This representation was created in the 1970's and a large amount of software using it has been written. Documentation describing the PDB file format is available from the wwPDB at http://www.wwpdb.org/docs.html. Historical copies of the PDB file format from 1992 and 1996 are available.

PDB Data The primary information stored in the PDB archive consists of co-ordinate files for biological molecules. These files list the atoms in each protein, and their 3D location in space. These files are available in several formats (PDB, mmCIF, XML). A typical PDB formatted file includes a large "header" section of text that summarizes the protein, citation information, and the details of str solution, followed by the sequence and a long list of the atoms and their coordinates. The archive also contains the experimental observations that are used to determine these atomic coordinates.

webmn@ page

Results PDB structure file used was 3Q3J Classification :Membrane Protein Binding: Structure Weight :37644.10

Deformation Energies(3Q3J)

Atomic displacement analysis The square of the displacement of each C-alpha atom (for modes 7 to 12), normalized so that the sum over all residues is equal to 100. Highest values correspond to the most displaced regions. On the plots, one should look for cluster of peaks, those identify significantly big regions. Isolated peaks reflect local flexibility and are not relevant. Results are retrieved as plots at the PNG format or raw data (first column: resid, second column: normalized squared atomic displacement)

Obtained results

The calculation of the dot product (overlap) between the difference vector and the full set of normal modes is complete. Results are provided as: PDF and PNG files of the plots. Squared overlap and cumulative squared overlap are plotted against mode numbers. Raw data files containing the modes and the difference vector.

Linear interpolation b/w 2 structures OVERLAP

Cumulative OVERLAP

HINGE-PROT hinge motions are similar to rotations around an articulated joint and therefore can be very large. Hinge motion is characterized by large changes in main-chain torsional angles occurring at a localized region, which is called a hinge. Hinge motions usually occur upon binding to another molecule, or upon activation/deactivation of the protein.

hinge regions are the mechanistically informative regions of the structure and are of great importance in mediating cooperative motions that have functional importance. HingeProt is a web server for predicting rigid protein parts and the flexible hinge regions connecting them in the native topology of protein chains by employing elastic network (EN) models. HingeProt makes use of both Gaussian Network Model (GNM) [2, 3] and Anisotropic Network models (ANM)

Method GNM predicts the relative magnitudes of the fluctuations, whereas ANM predicts the directionalities of the collective motions in addition to their magnitudes. GNM results are more robust, and thus are preferentially used for evaluating square displacements in low frequency modes. Here GNM is used to calculate mean-square fluctuations and correlation between the fluctuations of residues ANM to generate the conformations that describe the fluctuations of residues from the average, X-ray, structure in the principal directions of motion.

First GNM decomposes the fluctuations of N residues of a structure into a series of N-1 nonzero modes, given the Cartesian coordinates of Ca atoms. The eigenvectors corresponding to the slowest first and second modes are extracted. The square of these vectors describes the mean-square fluctuations of residues from equilibrium positions along the principal coordinates. Minima of mean square fluctuations at a given mode describe the flexible joints of the structure,

Input a PDB code or PDB format file. If the input structure includes more than one chain, the user will be requested to select one of the chains in the next step The largest number of residues allowed is 999.

Output Rigid parts:The server provides a table in which the rigid parts and the hinge residues calculated using the slowest first and second modes are given. The output files contain three models of the protein chain. The first model is the original chain (x-ray or NMR structure or a model structure), the second and the third models are predicted chain conformations obtained by adding the fluctuation vectors (+/-) in the respective modes to the equilibrium position vectors (average structure).

Output files: The output files contain three models of the protein chain. The first model is the original chain (x-ray or NMR structure or a model structure). second and the third models are predicted chain conformations obtained by adding the fluctuation vectors (+/-) in the respective modes to the equilibrium position vectors (average structure).

Visualization: Rasmol is suitable for the specific purpose.

Eigen values for GNM

Eigen values for ANM

Slowest mode1 atomic representation

Slowest mode2 atomic representation

ANM uses Elastic Network (EN) methodology and represents the system in the residue level. The macromolecule is thus represented as a network, or graph. Each node is the C atom of a residue and the overall potential is simply the sum of harmonic potentials between interacting nodes. The network includes all interactions within a cutoff distance which is the only predetermined parameter in the model.

Information about the orientation of each interaction with respect to the global coordinates system is considered within the Force constant matrix The force constant of the system can be described by a Hessian matrix

Each element Hi,j is a 33 matrix which holds the anisotropic informations regarding the orientation of nodes i,j.

applications About to add options for analysis of nucleic acids in the near future. For full functionality, the size of the protein should be up to 1500 residues. For larger proteins some problems with the graphics are expected. The calculation of the bfactors might take longer. The dominant modes calculation, however, will be performed and text files with the results will still be available within few minutes.

This site uses BLZpack for extracting a subset of eigen modes. Currently we calculate and report the slowest 20 modes. BLZPACK is a Fortran 77 implementation of the block Lanczos algorithm or the solution of the eigen value problems. Jmol is the the molecular graphics tool.

Other softwares used Matlab is used for matrix manipulations and a fast calculation of theoretical B-factors based on all the modes using the PowerB method. Perl modules are applied for web programming and preparations of graphs and figures. C code is used for parsing of structures and calculation of the Hessian matrix.

Modes: Visualization of a mode of the 20 modes calculated. The fluctuations of the selected mode appear and the molecule is colored according to the self-fluctuations. Red colors correspond to large fluctuations and blue colors to small fluctuations. By default, the vibrations in the first mode are animated and the residues are colored accordingly. Frequencies of vibrations : The default state of the analysis page presents the vibration frequencies of the different modes in the same proportions as the real frequencies. The user, however, can change the frequency for each mode using the pulldown menu for the purpose of visualization or analysis. In this case the proportions between the vibrational frequencies of the different modes are no realistic any more. The visualization frequencies (in Hertz units) are, of course, the vibrational frequencies as appear on the screen and not the real vibrational frequencies of the molecule.

Amplitude of vibrations : The amplitude of the vibrations can be changed by the user to emphasize the motion or to observe minor fluctuations that can not be distinguished otherwise. It is important to note the model does not always accurately represents the real size of the fluctuations but only the relative amplitude between the residues. Vectors : Vectors represent the direction of motion of each residue in each vibrational mode can be shown or hidden by checking on the vectors check box. The user can change the color of the vectors, their width and their length.

Display : The size of individual atoms (space fill) and the width of the bonds (wire frame) can be controlled by the pulldown menus in the graphical user interface, in addition to the available built in options in Jmol. If for some reason the desired action is not performed, clicking on other value in the same pulldown menu and then pressing again on the initial one does. Another possibility is that not all atoms are selected. From the Jmol menu (right click on the applet area) choose "select all" and then try again to perform the action. Labels : Residues can be labeled by their type and their number in the coordinates part of the PDB input file. Individual atoms can be labeled, as well as all atoms. The clear option in the menu remove all the labels. Note that the regular labeling options of Jmol will not be functional with this file format.

Colors : The protein can be colored by the polypeptide chains, by the experimental temperature (b) factors and by the fluctuations in each individual mode. The coloring scale is red-white-blue, where red indicates larger fluctuations. The coloring mode chosen from this pulldown menu is disconnected of the display method or the vibrational mode, so potentially we can have the residues vibrate according to one mode but colored according to a different mode. ANM model : Shows the network of the interactions for 10.0 cutoff. Chain connectivity : Color each polypeptide chain in different color and shows only the trace of the chains with no other non bonded interactions between sequentially distal residues. Because of the poor annotation in this file format, the Jmol built-in menu does not support equivalent options. The chain connectivity option may take some time to be executed for large proteins.

Flexserv

the study of flexibility is mostly a task for theoretical methods. The most powerful of them is here FlexServ, an integrated tool that tries to link most important coarse-grained based methods, structural databases, and atomistic models with a powerful analysis platform. FlexServ incorporates three coarse-grained algorithms for the representation of protein flexibility: Discrete dynamics (DMD), Normal mode analysis (NMA) based on different types of elastic networks Brownian dynamics (BD).atomistic molecular dynamics

FlexServ incorporates a large variety of methods to characterize flexibility. Thus, the server performs basic analysis like structural oscillation using root mean square deviation (RMSd) or a gaussian RMSd (gRMSd) algorithm analysis data are presented as plain text, or 2D plots or shown on the 3D structure when appropriate.

Advanced features Advanced capabilities of FlexServ include calculation of the apparent stiffness between interacting residues and the determination of residue-correlations using different algorithms that allow the user to filter non-relevant or spurious correlations. determination of dynamic domains and hinge points using a variety of techniques

For 3Q3J protein results are ParameterValue Most similar protein1m7b_A Number of chains2 (A,B) Number of residues278 First residueLEU A1490 Last residueASN B189 Analysis type NMA Algorithm Kovacs Force constant (kcal/mol*) 40 Requested accuracy (%) 90