Crystallographic study of calcium ion pump of sarcoplasmic reticulum

肌浆网钙离子泵的晶体学研究

• 批准号: 09480171
• 负责人: TOYOSHIMA Chikashi
• 金额: $ 8.83万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09480171/
• 关键词: ion pump; sarcoplasmic reticulum; active transport; three-dimensional structure; electron crystallography; structural analysis; membrane proteins; X-ray crystallography

Crystallographic study of the calcium ion pump (Ca^<2+>-ATPase) of sarcoplasmic reticulum(SR)has been performed to elucidate the structural basis of active transport. Two types of crystal of CA^<2+>-ATPase were known when we started this project : one was tubular crystal of native SR membrane formed in the presence of EGTA and vanadate ; the other was small three-dimensional crystals consisting of reconstituted membrane sheets formed in the presence of high concentration of calcium. Both were small and suitable for electron crystallography. Using tubular crystals, three-dimensional structures were obtained for three states at 8 A resolution : no ATP, with chromium-ATP and with thapsigargin (the last one was in collaboration with Stokes Lab at NY University). The one with bound thapsigargin was best resolved and clearly showed 10 transmembrane helices expected from secondary structure predictions (published in Nature). The one with chromium ATP indicated the position of ATP-binding site(published in Biophys. J.). We have developed an extensive set of programs for this purpose. Comparison of the projection along the b-axis of the small 3D crystals and 3D structure from tubular crystals revealed large scale conformational changes attributable to the binding of calcium (Biophys. J.).By improving the crystallisation conditions, we succeeded in preparing well ordered plate-like crystals of this ion pump. The thinnest ones could be analysed by electron and the thickest ones by X-ray crystallography. We have developed a rotation camera for collecting electron diffraction intensities and learned that even the thinnest crystals have virtually the same lattice constants with the thickest ones. We therefore proceeded to collect phase information directly from the electron micrographs and obtained a very useful set of phases for certain reflections. We are now looking for good isomorphous heavy metal derivatives for solving the structure at an atomic resolution.

本文对肌浆网（SR）钙离子泵（Ca^<2+>- atp酶）的晶体学研究阐明了主动转运的结构基础。当我们开始这个项目时，已知CA^<2+>- atp酶的两种晶体：一种是在EGTA和钒酸盐存在下形成的天然SR膜管状晶体；另一种是小的三维晶体，由在高浓度钙存在下形成的重组膜片组成。两者都很小，适合用于电子晶体学。使用管状晶体，以8 A分辨率获得了三种状态的三维结构：无ATP，有铬-ATP和有thapsigargin（最后一种是与纽约大学的Stokes实验室合作）。结合了thapsignargin的那一个得到了最好的解析，并清楚地显示出二级结构预测所期望的10个跨膜螺旋（发表在Nature上）。含铬ATP的一种表示ATP结合位点的位置（发表在《生物物理学》上）。j .)。我们为此制定了一套广泛的计划。将小三维晶体沿b轴的投影与管状晶体的三维结构进行比较，揭示了钙离子（Biophys）结合导致的大规模构象变化。j .)。通过改善结晶条件，我们成功地制备了有序的板状离子泵晶体。最薄的可以用电子分析，最厚的可以用x射线晶体学分析。我们开发了一种用于收集电子衍射强度的旋转相机，并了解到即使是最薄的晶体也与最厚的晶体具有几乎相同的晶格常数。因此，我们直接从电子显微照片中收集相位信息，并获得了一组非常有用的反射相位。我们现在正在寻找好的同构重金属衍生物，以便在原子分辨率上解决结构问题。

项目成果

P.Zhang: "Structure of the calcium pump from sarcoplasmic reticulum at 8Å resolution" Nature. 392. 835-839 (1998)

P. 张：“8Å 分辨率下肌浆网钙泵的结构”，Nature 392. 835-839 (1998)。

K.Mayanagi: "Three-dimensional electron microscopy of the photosystem II core complex." J.Struct.Biol.123. 211-224 (1998)

K.Mayanagi：“光系统 II 核心复合体的三维电子显微镜。”

K.Mayanagi: "Three-dimendional electron microscopy of the photosystem II core complex." J.Struct.Biol.123. 211-224 (1998)

K.Mayanagi：“光系统 II 核心复合体的三维电子显微镜。”

H.Ogawa: "Structure of the Ca^<2+> pump of sarcoplasmic reticulum : A view along the lipid bilayer at 9-A resolution" Biophys.J.75. 41-52 (1998)

H.Okawa：“肌浆网 Ca^2 泵的结构：9-A 分辨率下脂质双层的视图”Biophys.J.75。

K.Yonekura: "The ATP binding site of Ca^<2+> -ATP ase revealed by electron image analysis" Biophys.J.72. 997-1005 (1997)

K.Yonekura：“通过电子图像分析揭示的Ca 2+ -ATP酶的ATP结合位点”Biophys.J.72。