Biology subjects

Wu, K.-P.

Publications and source records attributed to Wu, K.-P..

11 recordsLinked to original sources

bioRxiv2026

AI agent supervision of structural model building and refinement

Structural model building and refinement need many decisions between different programs. These decisions often depend on an experienced researcher who knows how the programs work together and how to read experimental data. We used Claude Code with Claude Opus 5.5 to supervise this work for 24 cryo-EM and X-ray cases. The work finished in about 36 hours on one workstation. The agent prepared the inputs, ran established programs, read the validation results, compared candidate models and chose the next step. For 15 deposited cryo-EM models with weak starting geometry, the median MolProbity score improved from 2.62 to 1.76. For three crystal structures, the agent models came close to the published Rfree values. Chemical information such as ligands, ions and modifications must be provided by the researcher, in the same way as the sequence. With this information, the agent can place and check these components. These results show that agent supervision is acceptable when every decision can be inspected. The researcher makes the final scientific judgment. We suggest practical requirements for this kind of use. Future AI agents may help researchers obtain high quality and reliable structures of biological macromolecules.

biochemistry↗

bioRxiv2026

Specimen-Dependent Sampling and Signal Limitations Govern the Effectiveness of Low-Magnification Super-Resolution in Cryo-EM

Single-particle cryo-EM routinely delivers near-atomic structures; however, optimizing data collection requires a delicate balance among magnification, sampling bandwidth, and particle throughput. Although low-magnification super-resolution imaging can recover information beyond the physical Nyquist limit, its benefit varies substantially among specimens. This variability suggests that the effectiveness of super-resolution may depend on whether reconstruction is limited by detector sampling bandwidth or by the recoverable particle signal. However, the conditions that distinguish these two regimes remain poorly defined. Here, we systematically compared super-resolution and physical-pixel workflows at two magnifications using apoferritin (APO) and malate synthase G (MSG) as representative specimens with contrasting molecular size, symmetry, and image contrast. At low magnification, APO exhibited sampling-limited behavior, with super-resolution processing achieving 1.72 Angstrom compared with 2.74 Angstrom for physical-pixel processing. In contrast, MSG exhibited predominantly signal-limited behavior under the same conditions, yielding comparable resolutions of 3.05 Angstrom and 2.98 Angstrom for super-resolution and physical-pixel processing, respectively, despite the increased sampling bandwidth. These contrasting responses were further supported by particle-number saturation, per-particle motion correction, and Q-score analyses, which provided complementary evidence for the underlying sampling-limited and signal-limited regimes. Together, these results provide a practical framework for assessing whether reconstruction quality is predominantly constrained by sampling bandwidth or recoverable particle signal and offer a rational basis for balancing achievable resolution and particle throughput when selecting acquisition strategies.

biophysics↗

bioRxiv2026

Calcium modulates intramolecular long-range contacts to form a polymorphic α-synuclein A53T fibril

Human -synuclein (aSyn) is an intrinsically disordered protein, and aggregations of its amyloid fibrils are associated with Parkinsons disease (PD). Apart from familial aSyn mutations, accumulated environmental calcium exacerbates aSyn aggregation and accelerates symptoms in aging PD patients. Here, we explored the effects of Ca2+ ions on aSyn A53T, an aggregation-prone mutant variant, from disordered states to fibrillar structures. Paramagnetic nuclear magnetic resonance (NMR) revealed that binding of Ca2+ ions to the aSyn C-terminal (residues 110-140) relaxed the aSyn conformation, resulting in more aggressive fibrillogenesis. Cryo-electron microscopy structures of aSyn A53T with or without Ca2+ ion revealed substantial differences in amyloid folds and fibril assemblies. We characterized N1 (residues 61-66), N2 (residues 69-79), and N3 (residues 89-95) segments in the central non-amyloid {beta} component (NAC) crucial for forming localized structural contacts during early-step aggregation. Our work establishes the contacts governing aSyn misfolding from disordered monomer to aggregated fibril and provides insights into the structural changes elicited by Ca2+ ions.

biophysics↗

bioRxiv2025

Structural basis of K11/K48-branched ubiquitin chain recognition by the human 26S proteasome

Beyond the canonical K48-linked homotypic polyubiquitination for proteasome-targeted proteolysis, K11/K48-branched ubiquitin (Ub) chains are involved in fast-tracking protein turnover during cell cycle progression and proteotoxic stress. Here, we report cryo-EM structures of human 26S proteasome in a complex with a K11/K48-branched Ub chain. The structures revealed a multivalent substrate recognition mechanism involving a hitherto unknown K11-linked Ub binding site at the groove formed by RPN2 and RPN10 in addition to the canonical K48-linkage binding site formed by RPN10 and RPT4/5 coiled-coil. Additionally, RPN2 recognizes an alternating K11-K48 linkage through a conserved motif similar to the K48-specific T1 binding site of RPN1. The insights gleaned from these structures explain the molecular mechanism underlying the recognition of the K11/K48 branched Ub as a priority signal in the ubiquitin-mediated proteasomal degradation.

biochemistry↗

bioRxiv2024

Elucidating the Mechanism Underlying UBA7-UBE2L6 Disulfide Complex Formation

We elucidate cryo-EM structure and formation of the ubiquitin-associated bovine UBA7*UBE2L6 disulfide complex, shedding light on a highly specific and evolutionarily conserved mechanism governing ISG15 conjugation, a pivotal process in the immune response. UBA7 displays a unique capacity to recognize UBE2L6, distinct from this latters homolog UBE2L3, highlighting the intricacies of cellular regulation. Inter-species interactions of the resulting complex further underscore its significance. We characterize three crucial factors that influence UBA7*UBE2L6 disulfide complex formation: (1) strong binding affinity and specificity; (2) conformational differences in the catalytic cysteine capping loop (CCL); and (3) increased thiolate/thiol ratios at catalytic cysteines. Modification of any of these factors profoundly impacts complex activation and the ISG15 transfer cascade. This redox-sensitive complex implies a link between oxidative stress and regulation of the immune response, highlighting a potential therapeutic target for modulating immune reactions arising from infections and inflammatory conditions.

biophysics↗

bioRxiv2023

Engineering Nanomolar Potent Protein-based Inhibitors for Papain-like Protease Guided by Residue Correlation Network

Identifying critical residues in protein-protein binding and efficiently designing stable and specific protein binders is challenging. In addition to direct contacts in a protein-protein binding interface, our study employs computation modeling to reveal the essential network of residue interaction and dihedral angle correlation critical in protein-protein recognition. We propose that mutating residues regions exhibited highly correlated motions within the interaction network can efficiently optimize protein-protein interactions to create tight and selective protein binders. We validated our strategy using ubiquitin (Ub) and MERS coronaviral papain-like protease (PLpro) complexes, where Ub is one central player in many cellular functions and PLpro is an antiviral drug target. Our designed UbV with 3 mutated residues resulted in a [~]3,500-fold increase in functional inhibition, compared with the wild-type Ub. Further optimization by incorporating 2 more residues within the network, the 5-point mutant achieved a KD of 1.5 nM and IC50 of 9.7 nM. The modification led to a 27,500-fold and 5,500-fold enhancements in affinity and potency, respectively, as well as improved selectivity, without destabilizing the UbV structure. Our study highlights residue correlation and interaction networks in protein-protein interaction, introduces an effective approach to design high affinity protein binders for cell biology and future therapeutics solutions.

biophysics↗

bioRxiv2022

RE2DC: a robust and efficient 2D classifier with visualization for processing massive and heterogeneous cryo-EM data

Single-particle cryo-electron microscopy (cryo-EM) increasingly generates millions of particle images, yet two-dimensional (2D) classification remains a major bottleneck because existing approaches balance computational efficiency against robustness to noise, outliers and structural heterogeneity. We introduce RE2DC (Robust and Efficient 2D Classifier), an algorithmic framework that resolves this trade-off through dynamic linear-time clustering, dimension-reduction multi-reference alignment, and offers real-time interactive t-SNE visualization. Rather than relying primarily on hardware acceleration, RE2DC reduces the computational cost of robust clustering and employs de-noised images for alignment, enabling efficient execution on standard multi-core CPUs. Across diverse benchmark datasets, RE2DC achieves class homogeneity comparable to ISAC while processing datasets three- to ten-fold faster per classification round than RELION. Notably, RE2DC resolves rare, structurally coherent particle populations, enabling detection of transient conformational intermediates and supporting near real-time cryo-EM analysis. By addressing algorithmic complexity, RE2DC establishes a general framework for robust, scalable analysis of massive and heterogeneous image datasets.

bioinformatics↗

bioRxiv2022

Cryo-EM reveals the structure and dynamics of a 723-residue malate synthase G

Determination of sub-100 kilodalton (kDa) structures by cryo-electron microscopy (EM) is a longstanding but not straightforward goal. Here, we present a 2.9-[A] cryo-EM structure of a 723-amino acid apo-form malate synthase G (MSG) from Escherichia coli. The cryo-EM structure of the 82-kDa MSG exhibits the same global folding as structures resolved by crystallography and nuclear magnetic resonance (NMR) spectroscopy, and the crystal and cryo-EM structures are indistinguishable. Analyses of MSG dynamics reveal consistent conformational flexibilities among the three structural approaches, most notably that the /{beta} domain exhibits heterogeneity. We observed that sidechains of F453, L454, M629, and E630 residues involved in hosting the cofactor acetyl-CoA and substrate rotate differently between the cryo-EM apo-form and complex crystal structures. Our work demonstrates that the cryo-EM technique can be used to determine structures and conformational heterogeneity of sub-100 kDa biomolecules to a quality as high as that obtained from crystallography and NMR spectroscopy.

biophysics↗

bioRxiv2022

Structural basis of transcriptional activation by the OmpR/PhoB-family response regulator PmrA

PmrA, an OmpR/PhoB-family response regulator, activates gene transcription responsible for polymyxin resistance in bacteria by recognizing promoters in which the canonical -35 element is replaced by the pmra-box, representing the PmrA recognition sequence. Here, we report a cryo-electron microscopy-derived structure of a bacterial PmrA-dependent transcription activation complex (TAC) containing a PmrA dimer, an RNA polymerase {sigma}70-holoenzyme (RNAPH), and the pbgP promoter DNA. Our structure reveals that the RNAPH mainly contacts the PmrA C-terminal DNA binding domain (DBD) via electrostatic interactions and reorients the DBD three base pairs upstream of the pmra-box, resulting in a dynamic TAC conformation. In vivo assays show that substitution of PmrA DNA-recognition residues eliminated its transcriptional activity, but variants with altered RNAPH-interacting residues exhibited elevated transcriptional activity. Our study indicates that both PmrA recognition-induced DNA distortion and PmrA promoter escape play important roles in its transcriptional activation.

biophysics↗

bioRxiv2020

Simeprevir suppresses SARS-CoV-2 replication and synergizes with remdesivir

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp). Our results thus reveal the viral protein targets of simeprevir, and provide preclinical rationale for the combination of simeprevir and remdesivir for the pharmacological management of COVID-19 patients. One Sentence SummaryDiscovery of simeprevir as a potent suppressor of SARS-CoV-2 viral replication that synergizes with remdesivir.

microbiology↗

bioRxiv2018

Stout camphor tree genome fills gaps in understanding of flowering plant genome and gene family evolution

We present reference-quality genome assembly and annotation for the stout camphor tree (SCT; Cinnamomum kanehirae [Laurales, Lauraceae]), the first sequenced member of the Magnoliidae comprising four orders (Laurales, Magnoliales, Canellales, and Piperales) and over 9,000 species. Phylogenomic analysis of 13 representative seed plant genomes indicates that magnoliid and eudicot lineages share more recent common ancestry relative to monocots. Two whole genome duplication events were inferred within the magnoliid lineage, one before divergence of Laurales and Magnoliales and the other within the Lauraceae. Small scale segmental duplications and tandem duplications also contributed to innovation in the evolutionary history of Cinnamomum. For example, expansion of terpenoid synthase subfamilies within the Laurales spawned the diversity of Cinnamomum monoterpenes and sesquiterpenes.

genomics↗