ANDO LAB at CORNELL

Technical innovation is an important part of our lab's identity. We are passionate about developing new tools to answer questions that can't be solved with conventional approaches.

We have introduced advanced signal decomposition methods for small-angle X-ray scattering (SAXS), known as EFA (evolving factor analysis) and REGALS (regularized alternating least squares), that are now used worldwide [JACS 2016, PNAS 2018, IUCrJ 2021]. Correlated motions in protein crystals can be rigorously investigated with our macromolecular diffuse X-ray scattering (mdx) software [Nature Comm 2020]. We have also introduced a customizable, high-accuracy cryo-EM micrograph filtering tool called MIFFI (micrograph filtering utilizing Fourier space information), based on a convolutional neural network (CNN) [J Struct Bio 2024].

Finally, in addition to developing hardware for high-pressure structural biology [Protein Sci 2022], we introduced the first in-line anoxic small-angle X-ray scattering (anSAXS) system at a major national synchrotron [JBC 2023]. Ando Lab software and scripts can be found at: https://github.com/ando-lab

Obtaining molecular level insight into the correlated motions that give rise to protein allostery is a fundamental goal of structural biology. Although few techniques are sensitive to such motions, they leave distinct fingerprints in diffuse scattering, a signal that appears faintly in the background of diffraction images from protein crystals. Extracting dynamic information from this signal has long been a holy grail of crystallography [Chem Rev 2017]. In 2020, we reported the first complete description of diffuse scattering from protein crystals [Nature Comm 2020]. Since, we have continued to develop diffuse scattering analysis as a structural biology technique [Nature Comm 2023, Meth Enz 2023, Acta D 2024]. With philanthropic support from Astera Institute, we are also building diffuse scattering infrastructure for the public [The Diffuse Project]. A description for the general community can be found in ACA RefleXions 2020 ("Thinking Outside of the Lattice," by N. Ando p. 7-10).

The evolution of enzymes is of fundamental interest to us because it provides a powerful window into the relationship between protein sequence, structure, function, and dynamics. As a lab, we have a special fascination with the evolutionary significance of metalloenzymes, proteins that use metal-containing cofactors to perform challenging - and often ancient - chemistry.

One such family is the ribonucleotide reductase (RNR) family, which is used by all DNA-based life and is thought to pre-date the oxygenation of the Earth. By incorporating structural data, we were able to infer the first phylogenetic tree of the full RNR family, providing a model for the evolution of allosteric mechanisms [Nature Comm 2019, Protein Sci 2022] and for how RNRs adapted to rising oxygen levels on Earth [eLife 2022]. Surprisingly, our findings suggest that early lifeforms incorporated oxygen into their biochemistry much earlier than previously thought.

With the advent of modern cryo-electron microscopy (cryo-EM), it has become conceivable to redefine a protein’s structure as the continuum of all conformations that it can sample under a given condition. This continuum, the so-called conformational landscape, is related to the free energy landscape, which describes the probability that a protein visits certain conformations and the possible pathways accessible to the protein.

In our latest work in this area, we developed methods to construct and directly compare the 2D conformational landscapes of an enzyme during various stages of turnover [Nature Comm 2025]. This analysis revealed how the substrate directly alters the conformational landscape such that a transiently catalytic conformation becomes accessible.

Flexible, multi-domain enzymes catalyze remarkable chemical transformations by moving either their cofactor or substrate over large distances. We have studied two such enzymes: (1) one, which performs three different reactions by moving its cofactor between three different active sites [PNAS 2023] and (2) another, which performs two sequential reactions in a single active site [Nature Chem 2024]. These studies demonstrate the power of combining SAXS, which provides a holistic view of conformational ensembles, with the single-particle or single-molecule perspectives of cryo-EM and Förster resonance transfer (FRET) spectroscopy.