The University of Tokyo · Biochemistry, Genetics and Molecular Biology
Radostin Danev 교수의 연구실은 전자현미경 기반 단백질 구조 해석에 중점을 두며, 특히 냉동전자현미경(cryo-EM)을 활용한 단일입자 분석 기술의 혁신적 발전을 주도하고 있습니다. Volta 상한판(Volta Phase Plate, VPP)을 활용한 초고해상도 구조 해석 기법을 개발하여, 기존의 초점 조절 기반 방법에 비해 더 높은 해상도와 더 나은 대비를 실현했습니다. 주요 연구 대상은 G단백질결합수용체(GPCRs)와 대사 조절 수용체인 GLP-1R 등이며, 이들 수용체의 기능적 구조와 동역학을 원자 해상도에서 규명하고자 합니다.
Figures are computed from collected data and may differ slightly.
We describe a phase plate for transmission electron microscopy taking advantage of a hitherto-unknown phenomenon, namely a beam-induced Volta potential on the surface of a continuous thin film. The Volta potential is negative, indicating that it is not caused by beam-induced electrostatic charging. The film must be heated to ∼ 200 °C to prevent contamination and enable the Volta potential effect. The phase shift is created "on the fly" by the central diffraction beam eliminating the need for pre
With the advent of direct electron detectors, the perspectives of cryo-electron microscopy (cryo-EM) have changed in a profound way. These cameras are superior to previous detectors in coping with the intrinsically low contrast and beam-induced motion of radiation-sensitive organic materials embedded in amorphous ice, and hence they have enabled the structure determination of many macromolecular assemblies to atomic or near-atomic resolution. Nevertheless, there are still limitations and one of
We present a method for in-focus data acquisition with a phase plate that enables near-atomic resolution single particle reconstructions. Accurate focusing is the determining factor for obtaining high quality data. A double-area focusing strategy was implemented in order to achieve the required precision. With this approach we obtained a 3.2 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome. The phase plate matches or slightly exceeds the performance of the conventional
Previously, we reported an in-focus data acquisition method for cryo-EM single-particle analysis with the Volta phase plate (Danev and Baumeister, 2016). Here, we extend the technique to include a small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also removes the resolution limit inherent to the in-focus method thus allowing 3D reconstructions with resolutions be
G protein-coupled receptors (GPCRs) are key regulators of information transmission between cells and organs. Despite this, we have only a limited understanding of the behavior of GPCRs in the apo state and the conformational changes upon agonist binding that lead to G protein recruitment and activation. We expressed and purified unmodified apo and peptide-bound calcitonin gene-related peptide (CGRP) receptors from insect cells to determine their cryo-electron microscopy (cryo-EM) structures, and
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