Biology subjects

Bui, K. H.

Publications and source records attributed to Bui, K. H..

At least 19 recordsLinked to original sources

bioRxiv2026

CiliaBuilder2: a flexible ChimeraX bundle to visualize cilia and centriole structures in 3D

Centrioles and cilia are large organelles essential for cell division, motility, and signalling. They are characterized by distinctive, nine-fold symmetric microtubule doublets or triplets decorated with periodic protein complexes. While cilia and centrioles are highly conserved across eukaryotes, their composition and structure vary considerably between species and tissue types. Advances in cryo-electron tomography and single-particle cryo-electron microscopy have recently led to a surge in structural data for ciliary components. However, visualizing these components within the ciliary context remains challenging due to their scale and the diverse data formats involved, such as density maps, atomic models, and 3D geometric objects. Here, we introduce CiliaBuilder2, a toolset integrated into UCSF ChimeraX that enables simple, flexible visualization of complete ciliary structures from diverse data types using parametric calculations.

biophysics↗

bioRxiv2026

CsMT: a robust and streamlined CryoSPARC workflow for cryo-EM reconstruction of microtubules

Microtubules are cytoskeletal filaments that are involved in intracellular transport, cell division, and motility. Despite their biological importance, determining their high-resolution structures via cryo-electron microscopy remains a significant technical challenge due to their polymorphisms and pseudo-helical assembly. Current processing workflows are complex, often requiring the integration of multiple software packages and custom scripts, which creates a steep learning curve for many research groups. To address these limitations, we introduce CsMT, a streamlined workflow implemented entirely within the CryoSPARC environment and using synthetic references. CsMT simplifies microtubule reconstruction by utilizing a novel protofilament-pair classification approach, which effectively handles the inherent pseudo-symmetry and structural heterogeneity of microtubules with minimal manual intervention. Our workflow is versatile, capable of processing both undecorated and decorated microtubules while accurately determining seams and performing high-resolution refinement. We demonstrate the efficacy of this workflow by achieving a 2.3 and 2.7 [A] resolution reconstruction of homotypic and heterotypic maps of undecorated microtubules, matching the best-resolved microtubule structures in the field. By unifying the pipeline into a single and portable workflow, CsMT enhances reproducibility and accessibility, empowering more laboratories to explore the structural biology of microtubules and associated proteins, yielding new insights into their function.

biophysics↗

bioRxiv2026

Non-canonical ribosome organization enables translation stalling in neuronal RNA granules.

Neuronal outgrowth to support cognitive functions relies on a timely supply of proteins to distal sites in the neuron. To meet this demand, neurons transport mRNAs and ribosomes in large assemblies called "neuronal RNA granules". Ribosomes that integrate into these granules initiate protein synthesis in the soma, stall the process, and then become packaged into clusters that are transported to distal sites, where protein synthesis can be reactivated. The exact mechanisms and structure of these ribosome clusters remain unknown. We combined cryo-electron microscopy and cryo-electron tomography to examine these dense ribosome clusters purified from rat brains, which are included in neuronal RNA granules. We found that these clusters contain ribosomes stalled at various stages of elongation and that the mRNA sequences where the ribosomes stall facilitate stalling. The ribosome clusters contain multiple polysomal units, and ribosomes from nearby polysomes interact through rRNA expansion segments, only seen before in disomes of inactive or hibernating ribosomes in neurons. The ribosomes in the clusters form unique three-dimensional arrays that evade detection by the ribosome-associated quality-control and NO-GO decay responses, which normally lead to recycling of 40S and 60S subunits, and degradation of the mRNA and arrested nascent proteins. Overall, this study helps uncover specialized mechanisms in neurons that enable local protein translation at distal sites, supporting proper protein homeostasis, both crucial for neurodevelopment and optimal neuronal function. KEY POINTSO_LIRibosomes in neurons organize into unique clusters that are incorporated into "neuronal RNA granules". C_LIO_LIApproximately half of the ribosomes in these clusters are stalled in elongation. C_LIO_LIThe mRNA sequences read by these ribosomes facilitate stalling. C_LIO_LIRibosomes within the clusters interact through rRNA expansion segments. C_LIO_LIRibosomes in the clusters form unique 3D structures that are not detected by the ribosome-associated quality-control response. C_LI

neuroscience↗

bioRxiv2026

Structural tuning of native type V fimbriae shapes mechanical specialization in Porphyromonas gingivalis

Fimbriae are proteinaceous filaments central to bacterial adhesion, colonization, and biofilm formation. Porphyromonas gingivalis, a keystone periodontal pathogen, utilizes two type V fimbriae systems, the major Fim and the minor Mfa fimbriae, that support distinct adhesive and biofilm-associated functions. Here, we determine the high-resolution cryo-electron microscopy structures of natively assembled FimA and Mfa1 stalks purified directly from P. gingivalis. Despite a shared donor-strand exchange mechanism, the two stalks exhibit distinct helical geometries and donor-strand environments. FimA has a larger helical pitch, whereas the Mfa1 donor strand is more extensively buried and further shielded by an ordered N-terminal latch. Comparison with monomeric stalk pilin structures reveals distinct assembly-associated remodeling around the donor-strand interface. Deletion of the Mfa1 latch reduces heat-resistant oligomer accumulation and produces species consistent with incomplete processing, yet recovered filaments retain the donor-strand-exchanged stalk architecture and exhibit initial force peaks comparable to native Mfa1 during simulated axial extension. Atomic force microscopy reveals greater apparent local stiffness of FimA under indentation, whereas steered molecular dynamics simulations show multiple lower-force axial transitions in FimA but a dominant higher-force transition in Mfa1. In both systems, the exchanged donor strands remain engaged during simulated extension while distinct surrounding contact networks rearrange. Together, these findings demonstrate how a conserved polymerization linkage can support structurally and mechanically distinct adhesive filaments, providing a molecular basis for mechanical diversification of type V fimbriae.

molecular biology↗

bioRxiv2026

The native structure of the Trichonympha centriole cartwheel reveals a zigzag stacking pattern

Centrioles are essential cellular organelles with complex architectures. Their earliest assembly intermediate--the cartwheel--has a 9-fold symmetry established by Spindle assembly abnormal protein 6 (SAS-6). We used cryo-electron tomography and sub-tomogram averaging to resolve the native structure of the SAS-6 rings within the cartwheel of the exceptionally long Trichonympha proximal centriole. A 16-nm axial periodicity was formed by stacked rings of V-shaped SAS-6 tetramers, which formed the fundamental unit of the cartwheels central hub. SAS-6 head domains adopted a zigzag stacking pattern. Furthermore, the improved resolution provides new insights into the function of the previously observed central inner domain, which forms nine asymmetric densities that bridge adjacent tetramers, thereby imparting polarity and enhancing structural stability. Molecular dynamics simulations indicate increased rigidity in inter-tetramer interactions, supporting efficient ring formation and revealing how cartwheel architecture and polarity are established and stabilized.

cell biology↗

bioRxiv2025

SPEF1 mediates assembly of the central pair microtubule complexes in cilia of Tetrahymena.

SPEF1 is a conserved protein that reinforces the seam of central microtubules within the distal segment of motile cilia. We show that in Tetrahymena, loss of SPEF1 destabilized the central pair throughout the cilium length, producing gaps in the microtubule lattice, loss of proximal microtubule portions, and loss of lateral projections. While both central microtubules were affected, C1 suffered more damage than C2 and the defects were already apparent during ciliary assembly. Tetrahymena cells lacking SPEF1 had fewer cilia, which contained an excessively dense ciliary matrix and abnormal densities on the doublet microtubules. Although SPEF1 loss visibly affected only the central microtubules, it localized to all stable microtubules in the cell, both ciliary and non-ciliary. Within cilia, SPEF1 was enriched near the distal tip, but was also present throughout the middle segment, where it was more concentrated along the central than the outer doublet microtubules. In live cilia, SPEF1 particles were mostly stationary and turned over slowly, some diffused but none moved by IFT. We propose that SPEF1 acts as a stabilizer of central microtubules in both the middle and distal segment and its activity contributes to central microtubule assembly.

cell biology↗

bioRxiv2024

Structure of the ciliary tip central pair reveals the unique role of the microtubule-seam binding protein SPEF1

Motile cilia are unique organelles with the ability to autonomously move. Force generated by beating cilia propels cells and moves fluids. The ciliary skeleton is made of peripheral doublet microtubules and a central pair (CP) with a distinct structure at the tip. In this study, we present a high-resolution structure of the CP in the ciliary tip of the ciliate Tetrahymena thermophila and identify several tip proteins that bind and form unique patterns on both microtubules of the tip CP. Two of those proteins that contain tubulin polymerization-promoting protein (TPPP)-like domains, TLP1 and TLP2, bind to high curvature regions of the microtubule. TLP2, which contains two TPPP-like domains, is an unusually long protein that wraps laterally around half a microtubule and forms the bridge between the two microtubules. Moreover, we found that the conserved protein SPEF1 binds to both microtubule seams. In vitro, human SPEF1 not only binds to the microtubule seam but also crosslinks two parallel microtubules. Single-molecule microtubule dynamics assays indicate that SPEF1 stabilizes microtubules in vitro. Together, these data show that the proteins in the tip CP maintain stable microtubule structure and probably play important roles in maintaining the integrity of the axoneme.

cell biology↗

bioRxiv2023

DomainFit: Identification of Protein Domains in cryo-EM maps at Intermediate Resolution using AlphaFold2-predicted Models

Cryo-electron microscopy (cryo-EM) has revolutionized our understanding of macromolecular complexes, enabling high-resolution structure determination. With the paradigm shift to in situ structural biology recently driven by the ground-breaking development of cryo-focused ion beam milling and cryo-electron tomography, there are an increasing number of structures at sub-nanometer resolution of complexes solved directly within their cellular environment. These cellular complexes often contain unidentified proteins, related to different cellular states or processes. Identifying proteins at resolutions lower than 4 [A] remains challenging because the side chains cannot be visualized reliably. Here, we present DomainFit, a program for automated domain-level protein identification from cryo-EM maps at resolutions lower than 4 [A]. By fitting domains from artificial intelligence-predicted models such as AlphaFold2-predicted models into cryo-EM maps, the program performs statistical analyses and attempts to identify the proteins forming the density. Using DomainFit, we identified two microtubule inner proteins, one of them, a CCDC81 domain-containing protein, is exclusively localized in the proximal region of the doublet microtubule from the ciliate Tetrahymena thermophila. The flexibility and capability of DomainFit makes it a valuable tool for analyzing in situ structures.

biochemistry↗

bioRxiv2023

Effect of alpha-tubulin acetylation on the doublet microtubule structure

Acetylation of -tubulin at the lysine 40 residue (K40) by ATAT1/MEC-17 acetyltransferase modulates microtubule properties and occurs in most eukaryotic cells. Previous literatures suggest that acetylated microtubules are more stable and damage-resistant. K40 acetylation is the only known microtubule luminal post-translational modification site. The luminal location suggests that the modification tunes the lateral interaction of protofilaments inside the microtubule. In this study, we examined the effect of tubulin acetylation on the doublet microtubule in the cilia of Tetrahymena thermophila using a combination of cryo-electron microscopy, molecular dynamics, and mass spectrometry. We found that K40 acetylation exerts a small-scale effect on the doublet microtubule structure and stability by influencing the lateral rotational angle. In addition, comparative mass spectrometry revealed a link between K40 acetylation and phosphorylation in cilia.

cell biology↗

bioRxiv2023

Heterogeneity of radial spokes structural components and associated enzymes in Tetrahymena cilia

Radial spokes, RS1-RS2-RS3, are T-shaped, multiprotein complexes that transmit regulatory signals from central apparatus to outer doublet dyneins. RSs, especially RS3, differ in morphology, protein composition, and RS base-docked IDAs. Spokes defects alter cilia beating frequency, waveform, and amplitude leading, in humans, to primary ciliary dyskinesia and infertility. In contrast to RS1 and RS2, the protein composition of RS3 is partly resolved. Moreover, the role of particular spokes is unclear. Ciliate Tetrahymena thermophila has three Rsp3 paralogs and two or three paralogs of some other RSPs. Using multiple complementary approaches, we showed that Tetrahymena forms RS1 and RS2 subtypes having core composed of various Rsp3 paralogs and one type of Rsp3-less RS3. We elucidated proteomes of RS subtypes and identified novel RS-associated proteins, including enzymatic proteins involved in local regulation of the ADP/ATP levels and protein phosphorylation, whose presence further diversifies RSs properties and likely functions. In briefRadial spokes differ in their protein composition and architecture. Studies in a ciliate Tetrahymena revealed Rsp3 paralogs-dependent RS1 and RS2 subtypes, Rsp3-less RS3, and diversity of the RSP3 mutants phenotype. Known RS components and newly identified structural and enzymatic proteins were assigned to particular RSs.

cell biology↗

bioRxiv2023

Integrated modeling of the Nexin-dynein regulatory complex reveals its regulatory mechanism

Cilia are hairlike protrusions that project from the surface of eukaryotic cells and play key roles in cell signaling and motility. Ciliary motility is regulated by the conserved nexin-dynein regulatory complex (N-DRC), which links adjacent doublet microtubules and regulates and coordinates the activity of outer doublet complexes. Despite its critical role in cilia motility, the assembly and molecular basis of the regulatory mechanism are poorly understood. Here, utilizing cryo-electron microscopy in conjunction with biochemical cross-linking and integrative modeling, we localized 12 DRC subunits in the N-DRC structure of Tetrahymena thermophila. We also found that the CCDC96/113 complex is in close contact with the N-DRC. In addition, we revealed that the N-DRC is associated with a network of coiled-coil proteins that most likely mediates N-DRC regulatory activity.

cell biology↗

bioRxiv2023

Molecular architecture of the ciliary tip revealed by cryo-electron tomography

Cilia are essential organelles that protrude from the cell body. Cilia are made of a microtubule-based structure called the axoneme. In most types of cilia, the ciliary tip is distinct from the rest of the cilium. Here, we used cryo-electron tomography and subtomogram averaging to obtain the structure of the ciliary tip of the ciliate Tetrahymena thermophila. We show the microtubules in the tip are highly cross-linked with each other and stabilised by luminal proteins, plugs and cap proteins at the plus ends. In the tip region, the central pair lacks the typical projections and twists significantly. By analysing cells lacking a ciliary tip-enriched protein CEP104/FAP256 by cryo-electron tomography and proteomics, we discovered candidates for the central pair cap complex and explain potential functions of CEP104/FAP256. These data provide new insights into the function of the ciliary tip and inform about the mechanisms of ciliary assembly and length regulation.

cell biology↗

bioRxiv2022

Native doublet microtubules from Tetrahymena thermophila reveal the importance of outer junction proteins

Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory functions. The ciliary axoneme is a microtubule-based cytoskeleton consisting of two central singlets and nine outer doublet microtubules. Cryo-electron microscopy-based studies have revealed a complex network inside the lumen of both tubules composed of microtubule-inner proteins (MIPs). However, the functions of most MIPs remain unknown. Here, we present single-particle cryo-EM-based analyses of the Tetrahymena thermophila native doublet microtubule and identify 38 MIPs. These data shed light on the evolutionarily conserved and diversified roles of MIPs. In addition, we identified MIPs potentially responsible for the assembly and stability of the doublet outer junction. Knockout of the evolutionarily conserved outer junction component CFAP77 moderately diminishes Tetrahymena swimming speed and beat frequency, indicating the important role of CFAP77 and outer junction stability in cilia beating generation and/or regulation.

biophysics↗

bioRxiv2022

Cryo-EM structure of the Agrobacterium tumefaciens T-pilus reveals the importance of positive charges in the lumen

Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants and this is the most frequently applied process for the generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system localized in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2[A] resolution and that of the related plasmid pKM101-determined N-pilus at 3[A] resolution. Both pili contain a main pilus protein (VirB2 in A. tumefaciens and TraM in pKM101) and phospholipids arranged in a 5-start helical assembly. They contain positively charged amino acids in the pilus lumen and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge to the lumen. Mutagenesis of the lumen-exposed Arg91 residue in VirB2 resulted in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion system pili and that the charge of the lumen is of functional importance.

biochemistry↗

bioRxiv2022

Regulatory mechanisms of the dynein-2 motility by post-translational modification revealed by molecular dynamic simulation

Intraflagellar transport for ciliary assembly and maintenance is driven by dynein and kinesins specific for the cilia. It has been shown that anterograde and retrograde transports run on different regions of the doublet microtubule, i.e., separate train tracks. However, little is known about the regulatory mechanism of this selective process. Since the doublet microtubule is known to display specific post-translational modifications of tubulins, i.e. "tubulin code", for molecular motor regulations, we investigated the motility of ciliary specific dynein-2 under different post-translational modification by coarse-grained molecular dynamics. Our setup allows us to simulate the stochastic stepping behaviors of dynein-2 on un-modified, detyrosinated, poly-glutamylated and poly-glycylated microtubules in silico. Our study revealed that poly-glutamylation can play an inhibitory effect on dynein-2 motility. Our result indicates that poly-glutamylation of the B-tubule of the doublet microtubule can be used as an efficient means to target retrograde intraflagellar transport onto the A-tubule.

biophysics↗

bioRxiv2021

Chloroplast-localized translation for protein targeting in Chlamydomonas reinhardtii

Translation is localized within cells to target proteins to their proper locations. We asked whether translation occurs on the chloroplast surface in Chlamydomonas and, if so, whether it is involved in co-translational protein targeting, aligned spatially with localized translation by the bacterial-type ribosomes within this organelle, or both. Our results reveal a domain of the chloroplast envelope which is bound by translating ribosomes. Purified chloroplasts retained ribosomes and mRNAs encoding two chloroplast proteins specifically on this "translation domain", but not a mRNA encoding a cytoplasmic protein. Ribosomes clusters were seen on this domain by electron tomography. Activity of the chloroplast-bound ribosomes is supported by results of the ribopuromycylation and puromycin-release assays. Co-translational chloroplast protein import is supported by nascent polypeptide dependency of the ribosome-chloroplast associations. This cytoplasmic translation domain aligns localized translation by organellar bacterial-type ribosomes in the chloroplast. This juxtaposition the dual translation systems facilitates the targeting and assembly of the polypeptide products. One-Sentence SummaryTranslation is localized to a domain of the chloroplast envelope for co-translational protein targeting in Chlamydomonas.

cell biology↗

bioRxiv2020

Remodeling and activation mechanisms of outer arm dynein revealed by cryo-electron microscopy

Cilia are thin microtubule-based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs conformational changes caused by ATP hydrolysis. Here, we report the native ODA complex structure attaching to the doublet microtubule by cryoelectron microscopy. The structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with molecular dynamics simulations, we present a model of how the attachment of the ODA to the doublet microtubule induces remodeling and activation of the ODA complex.

cell biology↗

bioRxiv2020

Local computational methods to improve the interpretability and analysis of cryo-EM maps

Cryo-electron microscopy (cryo-EM) maps usually show heterogeneous distributions of B-factors and electron density occupancies and are typically B-factor sharpened to improve their contrast and interpretability at high-resolutions. However, over-sharpening due to the application of a single global B-factor can distort processed maps causing connected densities to appear broken and disconnected. This issue limits the interpretability of cryo-EM maps, i.e. ab initio modelling. In this work, we propose 1) approaches to enhance high-resolution features of cryo-EM maps, while preventing map distortions and 2) methods to obtain local B-factors and electron density occupancy maps. These algorithms have as common link the use of the spiral phase transformation and are called LocSpiral, LocBSharpen, LocBFactor and LocOccupancy. Our results, which include improved maps of recent SARS-CoV-2 structures, show that our methods can improve the interpretability and analysis of obtained reconstructions.

biophysics↗