Biology subjects

Miller, M. D.

Publications and source records attributed to Miller, M. D..

6 recordsLinked to original sources

bioRxiv2026

Structure of influenza D virus NS1 reveals a novel Zn-binding site and ssRNA-binding activity

Influenza D virus (IDV) is an emerging pathogen associated with bovine respiratory disease and has potential for zoonotic transmission. Although nonstructural protein 1 (NS1) is a major virulence factor in influenza A and B viruses, the structure and function of IDV NS1 remain poorly defined. Here, we determined crystal structures of the IDV NS1 N-terminal RNA-binding domain (NTD) and C-terminal effector domain (CTD). The NTD retains the conserved homodimeric six-helix fold, whereas the CTD adopts a distinct architecture containing a novel Zn2+-binding site. IDV NS1 CTD preferentially binds single-stranded RNA, and mutagenesis identified lysine and arginine residues critical for this activity. Single-residue substitutions disrupting either the Zn2+-binding site or RNA-binding interface prevented recovery of infectious recombinant IDV. These findings reveal distinct structural and functional features of IDV NS1 and establish the importance of CTD integrity for viral replication. Conservation of key CTD residues in influenza C virus NS1 suggests shared structural features that distinguish influenza C and D virus NS1 proteins from their influenza A and B counterparts.

biochemistry↗

bioRxiv2025

Confocal-Compatible Workflow for Sectioning, Staining, and Imaging Serial Vibratome Sections for 3D Anatomical Reconstruction of the Lymph Node

3D anatomical reconstruction provides quantitative insight into tissue architecture, variability, and function. However, existing whole-organ imaging methods, such as light-sheet microscopy and micro-CT, are often costly, slow, and inaccessible for many laboratories. We present an accessible, confocal-compatible workflow for harvesting, sectioning, and staining murine lymph nodes for high-fidelity 3D reconstruction. The protocol preserves Z-plane resolution through vibratome sectioning and immunostaining, enabling detailed visualization of lymph node lobules and vasculature using standard confocal microscopes. The resulting image stacks can be used for quantitative morphometry and spatiotemporal pharmacokinetic modeling. This reproducible workflow expands access to spatial and structural studies of the lymph node and can be adapted to other small, heterogeneous organs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/685350v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1e16022org.highwire.dtl.DTLVardef@16e6366org.highwire.dtl.DTLVardef@3ffee6org.highwire.dtl.DTLVardef@177c2af_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Heterotrimeric Collagen Helix with High Specificity of Assembly Results in a Rapid Rate of Folding

The most abundant natural collagens form heterotrimeric triple helices. Synthetic mimics of collagen heterotrimers have been found to fold slowly, even compared to the already slow rates of homotrimeric helices. These prolonged folding rates are not understood and have not been studied. This work compares three heterotrimeric collagen mimics stabilities, specificities and folding rates. One of these was designed through a computational-assisted approach, resulting in a well-controlled composition and register, in addition to providing increased amino acid diversity and excellent specificity. The crystal structure of this heterotrimer elucidates the composition, register and geometry of pairwise cation-{pi} and axial and lateral salt bridges. Complementary experimental methods of circular dichroism and NMR suggest the folding paradigm is frustrated by unproductive, competing heterotrimer species and these species must completely unwind to the monomeric state before refolding into the thermodynamically favored assembly. This collagen heterotrimer, which displays the best reported thermal specificity, was also found to fold much faster (hours vs days) than comparable, well-designed systems. The heterotrimeric collagen folding rate was observed to be both concentration and temperature-independent, suggesting a complex, multi-step mechanism. These results suggest heterotrimer folding kinetics are dominated by frustration of the energy landscape caused by competing triple helices.

biophysics↗

bioRxiv2020

Direct Observation of the Mechanism of Antibiotic Resistance by Mix-and-Inject at the European XFEL

In this study, we follow the diffusion and buildup of occupancy of the substrate ceftriaxone in M. tuberculosis {beta}-lactamase BlaC microcrystals by structural analysis of the enzyme substrate complex at single millisecond time resolution. We also show the binding and the reaction of an inhibitor, sulbactam, on a slower millisecond time scale. We use the mix-and-inject technique to initiate these reactions by diffusion, and determine the resulting structures by serial crystallography using ultrafast, intense X-ray pulses from the European XFEL (EuXFEL) arriving at MHz repetition rates. Here, we show how to use the EuXFEL pulse structure to dramatically increase the size of the data set and thereby the quality and time resolution of "molecular movies" which unravel ligand binding and enzymatically catalyzed reactions. This shows the great potential for the EuXFEL as a tool for biomedically relevant research, particularly, as shown here, for investigating bacterial antibiotic resistance. One Sentence SummaryDirect observation of fast ligand binding in a biomedically relevant enzyme at near atomic resolution with MHz X-ray pulses at the European XFEL.

biophysics↗

bioRxiv2020

Prochlorococcus phage ferredoxin: structural characterization and interactions with cyanobacterial sulfite reductases

Marine cyanobacteria are infected by phage whose genomes encode ferredoxin (Fd) electron carriers. While these Fds are thought to redirect the energy harvested from light to phage-encoded oxidoreductases that enhance viral fitness, it is not clear how the biophysical properties and partner specificities of phage Fds relate to those in photosynthetic organisms. Bioinformatic analysis using a sequence similarity network revealed that phage Fds are most closely related to cyanobacterial Fds that transfer electrons from photosystems to oxidoreductases involved in nutrient assimilation. Structural analysis of myovirus P-SSM2 Fd (pssm2-Fd), which infects Prochlorococcus marinus, revealed high similarity to cyanobacterial Fds ([≤]0.5 [A] RMSD). Additionally, pssm2-Fd exhibits a low midpoint reduction potential (-336 mV vs. SHE) similar to other photosynthetic Fds, albeit lower thermostability (Tm = 28{degrees}C) than many Fds. When expressed in an Escherichia coli strain with a sulfite assimilation defect, pssm2-Fd complemented growth when coexpressed with a Prochlorococcus marinus sulfite reductase, revealing that pssm2-Fd can transfer electrons to a host protein involved in nutrient assimilation. The high structural similarity with cyanobacterial Fds and reactivity with a host sulfite reductase suggest that phage Fds evolved to transfer electrons to cyanobacterial-encoded oxidoreductases.

biochemistry↗

bioRxiv2017

Authentic Enzyme Intermediates Captured "on-the-fly" by Mix-and-Inject Serial Crystallography

Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Here, we demonstrate a general method for capturing enzyme catalysis in-action by mix-and-inject serial crystallography. Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis -lactamase with the 3rd generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation on the millisecond to second time scales including the crossover from transition state kinetics to steady-state kinetics.\n\nSynopsisAn enzymatically catalyzed reaction is initiated by diffusion based mixing of substrate and followed at runtime by time-resolved serial crystallography using a free electron laser.

biophysics↗