Niko Vlahakis

All Publications

• Combining MicroED and native mass spectrometry for structural discovery of enzyme-small molecule complexes. Proceedings of the National Academy of Sciences of the United States of America Vlahakis, N. W., Flowers, C. W., Liu, M., Agdanowski, M. P., Johnson, S., Summers, J. A., Jacobs, L. M., Keyser, C., Russell, P., Rose, S. L., Orlans, J., Adhami, N., Chen, Y., Sawaya, M. R., Basu, S., de Sanctis, D., Chen, Y., Wakatsuki, S., Nelson, H. M., Loo, J. A., Tang, Y., Rodriguez, J. A. 2025; 122 (31): e2503780122

  Abstract

  With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event-based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach, which we term electron diffraction with native mass spectrometry (ED-MS), allows assignment of protein target structures bound to ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors and crude biosynthetic reactions. This extends to libraries of printed ligands dispensed directly onto TEM grids for later soaking with microcrystal slurries, and complexes with noncovalent ligands. ED-MS resolves structures of the natural product, epoxide-based cysteine protease inhibitor E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. It further identifies papain binding to its preferred natural products, by showing that two analogs of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analog from crude biosynthetic reactions, without purification. ED-MS also resolves binding of the CTX-M-14 β-lactamase, a target of active drug development, to the non-β-lactam inhibitor, avibactam, alone or in a cocktail of unrelated compounds. These results illustrate the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets, promising utility for drug discovery.

  View details for DOI 10.1073/pnas.2503780122

  View details for PubMedID 40720654

• Accounting for electron-beam-induced warping of molecular nanocrystals in MicroED structure determination IUCRJ Vlahakis, N., Clauss, A., Rodriguez, J. A. 2025; 12: 223-238

  Abstract

  High-energy electrons induce sample damage and motion at the nanoscale to fundamentally limit the determination of molecular structures by electron diffraction. Using a fast event-based electron counting (EBEC) detector, we characterize beam-induced, dynamic, molecular crystal lattice reorientations (BIRs). These changes are sufficiently large to bring reciprocal lattice points entirely in or out of intersection with the sphere of reflection, occur as early events in the decay of diffracted signal due to radiolytic damage, and coincide with beam-induced migrations of crystal bend contours within the same fluence regime and at the same illuminated location on a crystal. These effects are observed in crystals of biotin, a series of amino acid metal chelates, and a six-residue peptide, suggesting that incident electrons inevitably warp molecular lattices. The precise orientation changes experienced by a given microcrystal are unpredictable but are measurable by indexing individual diffraction patterns during beam-induced decay. Reorientations can often tilt a crystal lattice several degrees away from its initial position before irradiation, and for an especially beam-sensitive Zn(II)-methionine chelate, are associated with dramatic crystal quakes prior to 1 e- Å-2 electron beam fluence accumulates. Since BIR coincides with the early stages of beam-induced damage, it echoes the beam-induced motion observed in single-particle cryoEM. As with motion correction for cryoEM imaging experiments, accounting for BIR-induced errors during data processing could improve the accuracy of MicroED data.

  View details for DOI 10.1107/S2052252524012132

  View details for Web of Science ID 001437426800009

  View details for PubMedID 39927752

  View details for PubMedCentralID PMC11878443

• Fast event-based electron counting for small-molecule structure determination by MicroED ACTA CRYSTALLOGRAPHICA SECTION C-STRUCTURAL CHEMISTRY Vlahakis, N., Qu, S., Richards, L. S., de Moraes, L., Cascio, D., Nelson, H. M., Rodriguez, J. A. 2025; 81: 116-+

  Abstract

  Electron counting helped realize the resolution revolution in single-particle cryoEM and is now accelerating the determination of MicroED structures. Its advantages are best demonstrated by new direct electron detectors capable of fast (kilohertz) event-based electron counting (EBEC). This strategy minimizes the inaccuracies introduced by coincidence loss (CL) and promises rapid determination of accurate structures. We used the Direct Electron Apollo camera to leverage EBEC technology for MicroED data collection. Given its ability to count single electrons, the Apollo collects high-quality MicroED data from organic small-molecule crystals illuminated with incident electron beam flux densities as low as 0.01-0.045 e-/Å2/s. Under even the lowest flux density (0.01 e-/Å2/s) condition, fast EBEC data produced ab initio structures of a salen ligand (268 Da) and biotin (244 Da). Each structure was determined from a 100° wedge of data collected from a single crystal in as few as 50 s, with a delivered fluence of only ∼0.5 e-/Å2. Fast EBEC data collected with a fluence of 2.25 or 3.33 e-/Å2 also facilitated a 1.5 Å structure of thiostrepton (1665 Da). While refinement of these structures appeared unaffected by CL, a CL adjustment applied to EBEC data further improved the distribution of intensities measured from the salen ligand and biotin crystals. However, CL adjustment only marginally improved the refinement of their corresponding structures, signaling the already high counting accuracy of detectors with counting rates in the kilohertz range. Overall, by delivering low-dose structure-worthy data, fast EBEC collection strategies open new possibilities for high-throughput MicroED.

  View details for DOI 10.1107/S2053229624012300

  View details for Web of Science ID 001437433100002

  View details for PubMedID 39982366

  View details for PubMedCentralID PMC11881165

• Combining MicroED and native mass spectrometry for structural discovery of enzyme-biosynthetic inhibitor complexes. bioRxiv : the preprint server for biology Vlahakis, N. W., Flowers, C. W., Liu, M., Agdanowski, M., Johnson, S., Summers, J. A., Keyser, C., Russell, P., Rose, S., Orlans, J., Adhami, N., Chen, Y., Sawaya, M. R., Basu, S., de Sanctis, D., Wakatsuki, S., Nelson, H. M., Loo, J. A., Tang, Y., Rodriguez, J. A. 2025

  Abstract

  With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach resolves structures of the epoxide-based cysteine protease inhibitor, and natural product, E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. The combined structural power of MicroED and the analytical capabilities of native mass spectrometry (ED-MS) allows assignment of papain structures bound to E-64-like ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors, and crude biosynthetic reactions. ED-MS further discriminates the highest-affinity ligand soaked into microcrystals from a broad inhibitor cocktail, and identifies multiple similarly high-affinity ligands soaked into microcrystals simultaneously. This extends to libraries of printed ligands dispensed directly onto TEM grids and later soaked with papain microcrystal slurries. ED-MS identifies papain binding to its preferred natural products, by showing that two analogues of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analogue from crude biosynthetic reactions, without purification. This illustrates the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets.

  View details for DOI 10.1101/2025.02.20.638743

  View details for PubMedID 40060639

  View details for PubMedCentralID PMC11888187

• 3D Nanocrystallography and the Imperfect Molecular Lattice ANNUAL REVIEW OF PHYSICAL CHEMISTRY Vlahakis, N., Holton, J., Sauter, N. K., Ercius, P., Brewster, A. S., Rodriguez, J. A. 2024; 75: 483-508

  Abstract

  Crystallographic analysis relies on the scattering of quanta from arrays of atoms that populate a repeating lattice. While large crystals built of lattices that appear ideal are sought after by crystallographers, imperfections are the norm for molecular crystals. Additionally, advanced X-ray and electron diffraction techniques, used for crystallography, have opened the possibility of interrogating micro- and nanoscale crystals, with edges only millions or even thousands of molecules long. These crystals exist in a size regime that approximates the lower bounds for traditional models of crystal nonuniformity and imperfection. Accordingly, data generated by diffraction from both X-rays and electrons show increased complexity and are more challenging to conventionally model. New approaches in serial crystallography and spatially resolved electron diffraction mapping are changing this paradigm by better accounting for variability within and between crystals. The intersection of these methods presents an opportunity for a more comprehensive understanding of the structure and properties of nanocrystalline materials.

  View details for DOI 10.1146/annurev-physchem-083122-105226

  View details for Web of Science ID 001273689900021

  View details for PubMedID 38941528

  View details for PubMedCentralID PMC11801403