My lab's MolProbity web service for diagnosing and guiding correction of local modeling errors in macromolecular crystal structures has proven highly effective and is used by most of that research community worldwide. However, as urgent need has recently arisen for new methods that can extend MolProbity's reach to poorer resolutions in the 2.5-4 range. That applies to the exciting, big molecular machines solved by crystallography, and especially to the surge of high resolution cryoEM structures now accessible since the revolution in detector and image collection technology. For several reasons, our existing diagnostic criteria (and everyone else's) break down at resolutions where peptide orientation is no longer visible in the map density. We have developed a new tool called CaBLAM at a multi-residue length scale, which proved extremely effective in our assessments for the CryoEM Model Challenge. That experience also gave us ideas for several other criteria that could diagnose errors common at 2.5-4, such as flipped-over peptides and local sequence misalignments. This proposal aims to create a unified toolkit for these resolutions in MolProbity, an urgently needed functionality which does not currently exist anywhere. The more biomedically important these new structures are, the more crucial it is not to model the wrong amino acid at the critical site for catalysis, dynamics, drug design, or other specific binding. When developing the original MolProbity site we found that such new tools never act quite as one expects, and so extensive testing in real, varied production use is necessary. For this proposal, we will make those tests immediately valuable to the end-users of structures by a workflow called GEMS. We will identify individual x-ray or cryoEM structures that are a most-used archetype for their molecule but which contain local misfittings that affect their functional interpretation. If the depositor-of-record is willing, we will collaborate on healing those problems using the new tools. An important change this year to facilitate this goal is that the wwPDB is implementing an archival versioning system to enable coordinate updates without changing the PDB code, which makes structural biologists much more willing to deposit a corrected GEM structure. The lessons from creating these GEMS will be a major guide in tuning the new toolkit for maximum effectiveness, ease of use, and a minimum of false changes at the lower resolutions. There is now a new threat to MolProbity's ability to provide complex, dynamic services open to the worldwide scientific community in an increasingly hostile internet environment. MolProbity has recently been the target of extremely sophisticated attacks, which have required very significant personnel effort including near-constant monitoring, reinstallations, and close collaboration with medical center security experts. We have plugged some vulnerabilities but more work is needed, such as patches to our current version, perhaps a new web deployment modality, and laying the groundwork for a major redesign.
Most traditional structure-validation criteria break down at 2.5 to 4 resolution, where a majority of the most biologically and medically relevant big protein and nucleic acid structures are being solved by crystallography or, now, cryoEM; the PI has developed one new method and proposes to develop more which can robustly improve and attest to their local accuracy crucial to drug design. Additionally, important individual structures with local errors that affect their biological interpretation will be worked over and re-versioned as 'GEMS' in the worldwide database.
