The ClusPro web server is a widely used tool for protein-protein docking. It provides a simple home page for basic use, requiring only two files in Protein Data Bank format. However, ClusPro also offers advanced options to modify the search, including the removal of unstructured protein regions, applying attraction or repulsion, accounting for pairwise distance restraints, constructing homo-multimers, considering small angle X-ray scattering (SAXS) data, and finding heparin binding sites. Six different energy functions can be used depending on the type of proteins. Docking with each energy parameter set results in ten models defined by centers of highly populated clusters of low energy docked structures. This protocol describes the use of the various options, the construction of auxiliary restraints files, the selection of the energy parameters, and the analysis of the results. Although the server is heavily used, runs are generally completed in less than 4 hours. ClusPro is a web-based server for the direct docking of two interacting proteins. It was introduced in 2004 but has been substantially modified and expanded since then. The server performs three computational steps: (1) rigid body docking by sampling billions of conformations, (2) root-mean-square deviation (RMSD) based clustering of the 1000 lowest energy structures to find the largest clusters that will represent the most likely models of the complex, and (3) refinement of selected structures using energy minimization. The rigid body docking step uses PIPER, a docking program based on the Fast Fourier Transform (FFT) correlation approach. The FFT approach allows for exhaustive sampling of the conformational space on a dense grid, enabling docking of proteins without any a priori information on the structure of the complex. The PIPER docking algorithm represents the interaction energy between two proteins using an expression of the form E = w1Erep + w2Eattr + w3Eelec + w4EDARS, where Erep and Eattr denote the repulsive and attractive contributions to the van der Waals interaction energy, and Eelec is an electrostatic energy term. EDARS is a pairwise structure-based potential constructed by the Decoys as the Reference State (DARS) approach, and it primarily represents desolvation contributions. The coefficients w1, w2, w3, and w4 define the weights of the corresponding terms and are optimally selected for different types of docking problems. The server uses cluster population rather than energy value to select the most likely models of the complex. The second step of ClusPro is clustering the lowest energy 1000 docked structures using pairwise IRMSD as the distance measure. The selected structure will be defined as the center of the first cluster, and the structures within the 9Å IRMSD neighborhood of the center will constitute the first cluster. The members of this cluster are then removed, and the structure with theThe ClusPro web server is a widely used tool for protein-protein docking. It provides a simple home page for basic use, requiring only two files in Protein Data Bank format. However, ClusPro also offers advanced options to modify the search, including the removal of unstructured protein regions, applying attraction or repulsion, accounting for pairwise distance restraints, constructing homo-multimers, considering small angle X-ray scattering (SAXS) data, and finding heparin binding sites. Six different energy functions can be used depending on the type of proteins. Docking with each energy parameter set results in ten models defined by centers of highly populated clusters of low energy docked structures. This protocol describes the use of the various options, the construction of auxiliary restraints files, the selection of the energy parameters, and the analysis of the results. Although the server is heavily used, runs are generally completed in less than 4 hours. ClusPro is a web-based server for the direct docking of two interacting proteins. It was introduced in 2004 but has been substantially modified and expanded since then. The server performs three computational steps: (1) rigid body docking by sampling billions of conformations, (2) root-mean-square deviation (RMSD) based clustering of the 1000 lowest energy structures to find the largest clusters that will represent the most likely models of the complex, and (3) refinement of selected structures using energy minimization. The rigid body docking step uses PIPER, a docking program based on the Fast Fourier Transform (FFT) correlation approach. The FFT approach allows for exhaustive sampling of the conformational space on a dense grid, enabling docking of proteins without any a priori information on the structure of the complex. The PIPER docking algorithm represents the interaction energy between two proteins using an expression of the form E = w1Erep + w2Eattr + w3Eelec + w4EDARS, where Erep and Eattr denote the repulsive and attractive contributions to the van der Waals interaction energy, and Eelec is an electrostatic energy term. EDARS is a pairwise structure-based potential constructed by the Decoys as the Reference State (DARS) approach, and it primarily represents desolvation contributions. The coefficients w1, w2, w3, and w4 define the weights of the corresponding terms and are optimally selected for different types of docking problems. The server uses cluster population rather than energy value to select the most likely models of the complex. The second step of ClusPro is clustering the lowest energy 1000 docked structures using pairwise IRMSD as the distance measure. The selected structure will be defined as the center of the first cluster, and the structures within the 9Å IRMSD neighborhood of the center will constitute the first cluster. The members of this cluster are then removed, and the structure with the