相关实验视频

Updated: Jun 10, 2025

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Published on: January 9, 2015

通过冷电子显微镜和X射线晶体学对L-氨酸氧化酶的开放和封闭结构进行分析

Hiroki Yamaguchi^1,2, Kazutoshi Takahashi¹, Nobutaka Numoto³

¹Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co. Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan.

Journal of biochemistry

|October 18, 2024

概括

研究人员确定了来自Pseudomonas sp.的L-氨酸氧化酶 (AROD) 的结构. TPU 7192使用冷电子显微镜. 这种结构洞察力揭示了酶的关键特征.

科学领域:

生物化学生物化学
结构生物学结构生物学
酶学是一种酶学.

背景情况:

L-氨酸氧化酶 (AROD) 是一种FAD依赖的氧化还原酶,可催化L-氨酸去胺.
来自Pseudomonas sp.的PT-AROD是一种 TPU 7192对L-氨酸具有很高的选择性,因此对生物测试有价值.
对于PT-AROD的功能和选择性的结构基础仍然没有被阐明.

研究的目的:

为了确定PT-AROD的三维结构.
阐明有助于PT-AROD高基底选择性的结构特征.
为了解FAD依赖的L-氨酸氧化机制提供见解.

主要方法:

使用冷电子显微镜 (cryo-EM) 在2.3 Å分辨率下确定PT-AROD的结构.
使用质量摄影法估计了酶在溶液中的分子量.
进行了与X射线结晶学数据和局部定向突变发生的比较结构分析.

主要成果:

发现PT-AROD采用了具有D4对称性的八米结构.
低温电磁探测揭示了不同开放和闭合的形状,类似盖子的循环控制了基板的访问.
在基质结合部位内Glu493的突变显著降低了基质选择性.

结论:

确定的结构提供了FAD依赖的AROD的第一个高分辨率视图.
盖状循环和Glu493残留物对于PT-AROD的基质识别和选择性至关重要.
这些发现为理解和潜在地为特定应用设计AROD酶提供了结构基础.

关键词:

对于L-氨酸氧化酶来说,这是一个很好的方法.在X射线晶体学.氨基酸氧化酶是一种氨基酸氧化酶.低温电磁波冷却器 (Cryo-EM) 是一个非常好的方法.单个粒子分析

更多相关视频

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies

Published on: November 20, 2021

Author Spotlight: Exploring Cellular Processes by Modeling Ligands in Cryo-EM Maps

Author Spotlight: Exploring Cellular Processes by Modeling Ligands in Cryo-EM Maps

Published on: July 19, 2024

相关概念视频

Protein and Protein Structure

Protein and Protein Structure

Proteins are one of the most abundant organic molecules in living systems and have the most diverse range of functions of all macromolecules. Proteins may be structural, regulatory, contractile, or protective. They may serve in transport, storage, or membranes; or they may be toxins or enzymes. Their structures, like their functions, vary greatly. They are all, however, amino acid polymers arranged in a linear sequence.
A protein's shape is critical to its function. For example, an enzyme...

Cryo-electron Microscopy

Cryo-electron Microscopy

Conventional electron microscopy (EM) involves dehydration, fixation, and staining of biological samples, which distorts the native state of biological molecules and results in several artifacts. Also, the high-energy electron beam damages the sample and makes it difficult to obtain high-resolution images. These issues can be addressed using cryo-EM, which uses frozen samples and gentler electron beams. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, for...

Gene Families

Gene Families

Gene families consist of groups of genes proposed to have originated from a common ancestor. Typically these arise through events in which a gene or genes are mistakenly duplicated during cell division. Unlike their parent genes (which are subject to selection pressure to maintain function), these gene copies do not need to preserve their sequences and may evolve at a relatively faster rate.
Occasionally these regions can be adapted to take on new roles within the organism, becoming novel genes...