Biology subjects

Takaba, K.

Publications and source records attributed to Takaba, K..

6 recordsLinked to original sources

bioRxiv2025

High throughput protein serial crystallography using a grease matrix and a large-area support film

Serial femtosecond crystallography (SFX) using ultrashort pulses from X-ray free- electron lasers (XFELs) enables the determination of crystal structures at room temperature while minimizing radiation damage to the samples. This method involves irradiating numerous crystals one by one with XFEL pulses, allowing even the capture snapshots of dynamical structures in biological macromolecules. To achieve this, an efficient sample delivery system is essential for acquiring a large number of diffraction patterns. The most common approach uses a highly viscous grease matrix containing sample crystals, injected into the XFEL path from a narrow nozzle. However, the injection often suffers from clogging issues inside the injector nozzle, resulting in additional challenges such as the need for suitably sized crystals, increased sample consumption and unstable flow rates. Alternatively, a fixed-target approach, which scans a two-dimensional substrate with dispersed samples, can circumvent these issues. However, it must ensure the integrity of biological samples and provide sufficient surface area for efficient data collection. We here present an approach that utilizes a grease matrix and a large-area support film specially designed to address these requirements. This system offers a fast and reliable solution for protein SFX, enabling high-quality structure determination while significantly reducing sample consumption.

molecular biology↗

bioRxiv2024

DrugGym: A testbed for the economics of autonomous drug discovery

Drug discovery is stochastic. The effectiveness of candidate compounds in satisfying design objectives is unknown ahead of time, and the tools used for prioritization--predictive models and assays--are inaccurate and noisy. In a typical discovery campaign, thousands of compounds may be synthesized and tested before design objectives are achieved, with many others ideated but deprioritized. These challenges are well-documented, but assessing potential remedies has been difficult. We introduce DrugGym, a frame-work for modeling the stochastic process of drug discovery. Emulating biochemical assays with realistic surrogate models, we simulate the progression from weak hits to sub-micromolar leads with viable ADME. We use this testbed to examine how different ideation, scoring, and decision-making strategies impact statistical measures of utility, such as the probability of program success within predefined budgets and the expected costs to achieve target candidate profile (TCP) goals. We also assess the influence of affinity model inaccuracy, chemical creativity, batch size, and multi-step reasoning. Our findings suggest that reducing affinity model inaccuracy from 2 to 0.5 pIC50 units improves budget-constrained success rates tenfold. DrugGym represents a realistic testbed for machine learning methods applied to the hit-to-lead phase. Source code is available at www.drug-gym.org.

biophysics↗

bioRxiv2021

Hydrogen properties and charges in a sub-1.2 A resolution cryo-EM structure revealed by a cold field emission beam

The cold field emission (CFE) beam produces the less-attenuated contrast transfer function of electron microscopy, thereby enhancing high-resolution signals and this particularly benefits higher-resolution single particle cryogenic electron microscopy. Here, we present a sub-1.2 [A] resolution structure of a standard protein sample, apoferritin. Image data were collected with the CFE beam in a high-throughput scheme while minimizing beam tilt deviations from the coma-free axis. A difference map reveals positive densities for most hydrogen atoms in the core region of the protein complex including those in water molecules, while negative densities around acidic amino-acid side chains likely represent negative charges. The position of the hydrogen densities depends on parent bonded-atom type, which is validated by an estimated level of coordinate errors.

biophysics↗

bioRxiv2021

Real-time object locator for cryo-EM data collection--- You only navigate EM once ---

In cryo-electron microscopy (cryo-EM) data collection, locating a target object is the most error-prone. Here, we present a machine learning-based approach with a real-time object locator named yoneoLocr using YOLO, a well-known object detection system. Implementation showed its effectiveness in rapidly and precisely locating carbon holes in single particle cryo-EM and for locating crystals and evaluating electron diffraction (ED) patterns in automated cryo-electron crystallography (cryo-EX) data collection.

biophysics↗

bioRxiv2020

Collecting large datasets of rotational electron diffraction with ParallEM and SerialEM

A semi-automated protocol has been developed for rotational data collection of electron diffraction patterns by combined use of SerialEM and ParallEM, where SerialEM is used for positioning of sample crystals and ParallEM for rotational data collection. ParallEM calls standard camera control software through an AutoIt script, which adapts to software operational changes and to new GUI programs guiding other cameras. Development included periodic flashing and pausing of data collection during overnight or day-long recording with a cold field-emission beam. The protocol proved to be efficient and accurate in data collection of large-scale rotational series from two JEOL electron microscopes, a general-purpose JEM-2100 and a high-end CRYO ARM 300. Efficiency resulted from simpler steps and task specialization. It is possible to collect 12-20 rotational series from [~] -68{o} to [~] 68{o} at a rotation speed of 1{o} /s in one hour without human supervision.

biophysics↗

bioRxiv2019

Left Ventricular Chamber Shape During Vena Caval Occlusion: Improved MRI-based Measurement of the End-Systolic Pressure-Volume Relationship in Normal Sheep

The left ventricular (LV) end-systolic pressure volume relationship (ES; ESPVR) is the cornerstone of systolic LV function analysis. Recently, it became possible to measure 2D LV chamber shape during vena cava occlusion (VCO) with MRI. We used an improved level-set semi-automatic segmentation method (LSSM) to determine the effect of VCO on LV geometry, ES pressure area (PA) and ESPVR. 10 healthy adult sheep were anesthetized. LV pressure transducer and inferior vena cava (IVC) balloon catheter were percutaneously inserted. Ferumoxytol (0.125 ml/kg iv; AMAG Pharmaceuticals, Waltham, MA) was given to enhance blood pool contrast. LV pressure and 2D retrospectively-gated cine MRI of LV cross sections 25 (Apex), 50 (Mid) and 75% (Base) of the distance from the apex to the base of the LV were obtained during separate IVC balloon inflations (VCO). LV pressure was digitally filtered and LV chamber segmented with the LSSM. Cross sectional area, major and minor axes, major axis orientation, ESPAR and ESPVR were calculated. The LSSM had excellent reliability. All cross sections became more elliptical during VCO. The orientation (angle) of each major axis relative to the anterior RV insertion shifted during VCO. However, the orientation remained toward the septum. There was chamber collapse (LV area < 0.25 cm2) at the apical level during VCO (7 cases). ESPAR was non-linear at all levels. ESPVR was non-linear because of apical collapse. In conclusion, MRI-based measurement of LV geometry, ESPAR and ESPVR during VCO is a valuable method that may lead to improved understanding of systolic LV function. New and NoteworthyReal-time MRI was used to continuously measure the LV PA relationship as loading conditions were transiently varied in anesthetized sheep. All three examined cross-sections became more elliptical during VCO. The ESPAR were non-linear at all three cross-sections. Chamber collapse at the apical level during VCO resulted in a non-linear ESPVR. The heart contracted in a non-concentric manner during VCO which could inform modeling studies and elucidate mechanisms underlying LV adaptations to sudden load changes.

bioengineering↗