PACUPP: Pockets And Cavities Using Pseudoatoms in Proteins

PACUPP is a free, open-source program for identifying and visualizing cavities in macromolecules. It is a Jmol script that fills pockets, cavities, tunnels/channels in proteins with pseudoatoms. PACUPP provides tools to isolate cavities of interest and display their contents, their linings (and list the lining atoms, spreadsheet-ready), entrances (mouths), volumes, shapes, and dimensions. PACUPP adds the pseudoatoms (holmium, Ho, think "holes") to an output PDB file, which can then be viewed in Jmol with PACUPP commands, or in the software of your choice (e.g. PyMOL, Chimera, VMD, Cn3D, etc.). PACUPP (Jmol) is Java-based, and thus works in Windows, macOS, and linux. PACUPP Version 1.0 was released December 9, 2020. The PACUPP program and documentation can be downloaded at molviz.org/pacupp. There you will also find a YouTube Video demonstrating how to use PACUPP with several examples, and a slideshow also providing examples and some guidance in how to use the program. Example We'll explore a (alpha-N-acetylgalactosaminidase) 2ixb. It has a tunnel containing NAD that includes, at one entrance, a pocket where substrate can access the catalytic site, which includes the NAD. With PACUPP's default settings (cavity detail: fine; cavity size: small), the two entrances to the channel containing NAD are not quite connected. With cavity detail set to very fine, the channel connects. having 5 or more pseudoatoms/cavity. All but one of these are shallow pockets on the surface. In contrast, the pink cavity penetrates into the protein. Color by depth and isolate This is easier to see when the (PACUPP command d; also the backbone trace was toggled off with the command b). Only the formerly pink (largest) cavity has blue (deep) pseudoatoms. In PACUPP, . Inside the tunnel , the NAD is visible near substrate (GalNAc). The PACUPP views command v evokes a menu offering this view. This representation of the tunnel has several "wings", that is, shallow surface grooves that happen to be connected to the main tunnel, but appear not to be important parts of it. Trimming the tunnel and its volume PACUPP offers two methods of trimming unwanted "wings" off of clusters of pseudoatoms representing cavities. These methods are demonstrated in the YouTube video (available at molviz.org/pacupp). . PACUPP reports its volume as 2,516 cubic Å, which is 14% less than the volume before it was trimmed. Tunnel lining PACUPP's views menu offers to show the protein atoms within 5 Å of the pseudoatoms representing the tunnel. as balls (sticks are farther away and are shown for context): nitrogen and oxygen. PACUPP can list these atoms in a spreadsheet-ready text file, and also offers the same options for apolar lining atoms (carbons and sulfurs). Tunnel dimensions PACUPP's views menu also offers a view suitable for measuring cavities. . These are pseudoatom center-to-center distances. To get the outside dimension, add one pseudoatom diameter (reported by PACUPP as 2.4 Å for "very fine" definition). Further Examples These examples are shown in the YouTube video: Coronoavirus SARS-CoV-2 spike protein cavity that is a potential drug target Ribosome nascent peptide exit channel Proteasome HIV protease (detailed demonstration of PACUPP commands) Acetylcholinesterase catalytic pocket with inhibitor DNA, major and minor grooves Some of the above, plus these examples are shown in the slideshow: Orthogonal tunnels accessing the heme in protoglobin Oligopeptide-binding protein A with an octapeptide in a buried cavity (detailed demonstration of PACUPP commands) Pros and Cons Here are some advantages of PACUPP: Provides easy tools to see what is inside a cavity, see the mouths of cavities, see and list (spreadsheet ready) atoms lining a cavity, and measure cavity dimensions. Also, cavity volumes are reported. You see the actual size and shape of each pocket, tunnel or cavity -- not an idealized radially-symmetric representation. Output PDB files include pseudoatoms (holmium, Ho, think "holes") and can be explored in software of the user’s choice, such as PyMOL or Chimera. Animations of the types in the slides are easy to make using the animation kit that is included. Provides easy tools to limit display to one or a few cavities of interest, and trim off fenestrations or “wings” if desired. Two thirds of entries in the Protein Data Bank process in < 15 sec each (with cavity detail at the default setting of fine). Very large models (proteasomes, ribosomes, etc.) can be set up as batch jobs that process unattended. The output PDB files can be visualized later in Jmol with all PACUPP commands available. Most small to medium-sized cavities are shown automatically with no adjustments to settings. There are simple adjustments for large cavities or cavity detail. Handles multiple models and alternate locations by ignoring them automatically. No knowledge of Jmol command language is needed. Here are some limitations of PACUPP: Does not provide electrostatic potential maps of cavity linings. The method is inherently slightly “hit or miss” regarding fine details of cavity shape, volume, or detection of very small cavities. Pseudoatoms are placed where they do not clash with the macromolecule. Clash details depend on how the 3D grid of possible pseudoatoms aligns with the cavity boundaries. An optional offset of the grid origin is provided to assess such issues. PACUPP is limited to PDB files. It does not process mmCIF files (the largest models, ~1% of entries in the Protein Data Bank). However, mmCIF files are generally available as a bundle of PDB files: see the Ribosome example. Does not animate cavities over ensembles of models such as molecular dynamic simulations.

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