电子科技大学：《生物物理 Biophysics》课程教学资源（课件讲稿）蛋白质三级结构解析（冷冻电镜三维重构）

上节课主要知识点回顾 D 3000 2500 X-RAY 2000 0154nm X-RAY 1500 1000 0.l-0.5nm PROTEIN CRYSTAL X-RAY DIFFRACTION 500 PATTERN ATOMS PROTEIN X光的波長興原子問的距雅料 ELECTRON DENSITY 300 320 34036 plate Wavelength(nm) 教信号：scien1ci相 检测蛋白质三级结 X-射线晶体衍射 构变化？

上节课主要知识点回顾 检测蛋白质三级结 构变化？ X-射线晶体衍射 严禁复制

解释1X-射线产生 阴极（发射电子） P. Crystallization and X-Ray Data C 高重频X射线自由电子激光O production was solubilized in 150 mM NaCl],and the prote 0 exclusion chromatography tc 低重频X射线自由电子激光● 20 第四代 corresponding to the monom 第三代光源 光源 protein was concentrated tc 4 (pH 7.5)and 150 mM NaCl. tering showed the presence of For preparation of the HS 第二代光源可 阴极（接受电子，发射X射线） acid at a concentration of 2.5 KPO4 (pH 7.5)and warmed 第一代光源● cooled to ~30C,and 0.2 mM 棒球 细胞蛋白质原子 夸克 was incubated for 20 min,co 10 ò 。为 12,000×gfor4 min at4C 103103101103103107101010B10510t The supernatant was concent X射线管 波长/m exchanged in 20 mM KPO4 (I 增强器 可见光 同步辐射源 acic 1900 1960198020002020 in 直线加速器 年份 pnate ouner (pH 7.3),and 150 mM KCI.Ine crystais or usrHsA ○电子枪 10107105103101101021010210光子量/e were flash-frozen in liquid nitrogen and the x-ray diffraction 光束线 无线微被管电灯 同步辐射 辐射性源 data were collected at the synchrotron beamline 2.05 A res- @ ￥ olution with the crystal mal at thre-temperlure of 100 K. 带电粒子、弧形磁 (b)同步辐射波夏 场、光速运动、切 线发射辐射

解释-1 X-射线产生 阴极（接受电子，发射X-射线） 同步辐射源 阴极（发射电子） 带电粒子、弧形磁 场、光速运动、切 线发射辐射 严禁复制

同步辐射源的重要性 1997年 2003年 2006年 ATP合酶 水通道、离子通道 真核转录 2009年 202件甲 核糖体研究 G蛋白偶联受体 Venkatraman Ramakrishnan Thomas A.Steitz Ada E.Yonath Robert J.Lefkowitz Brian Kobilka

同步辐射源的重要性 核糖体研究 G-蛋白偶联受体 ATP合酶 水通道、离子通道 真核转录 严禁复制

解释-2蛋白质如何结晶 (混乱→有序熵不利)？ Temperature HANGING DROP Concentration PH value Protein Conc.x l/2 Precipitant Conc.x 1/2 饱和蒸气 足够 Vol.x 2 Purity Bvffer 压变化 Additives Precipitants 500uL REAGENT I-2uL PROTEIN Crystals of HSA-myristic acid complex were obtained with the hanging diffusion method in a 28%PEG 3350,50 mM phos phate buffer(pH 7.5),and 150 mM KCI.The crystals of OsrHSA were flash-frozen in liguid pitrogen and the Xrav diffraction data were collected at the synchrotron beamline to 2.05 A res- olution with the crystal maintained at the temperature of 100 K. https://www.bilibili.com/video/BV10E411d7Vx/?spm_id_from=autoN 高度有序排列（含水） ext&t=7.3

解释-2 蛋白质如何结晶（混乱→有序熵不利）？ 纯度高 足够浓 https://www.bilibili.com/video/BV1oE411d7Vx/?spm_id_from=autoN ext&t=7.3 饱和蒸气 压变化 高度有序排列（含水） 严禁复制

解释-3如何采集衍射信号和分析？ beam Inverse Fourier Transform:Fourier Summation p(xyz)= F(hkl).e-2ilhx+ky+Iz-(hk)] are labelled according to how ma https://www.bilibili.com/video/av838677109/?t=1.9

解释-3 如何采集衍射信号和分析？ https://www.bilibili.com/video/av838677109/?t=1.9 严禁复制

数据分析小故事 焦急等待的日子 最困难也没放弃…

数据分析小故事 最困难也没放弃...... 焦急等待的日子...... 严禁复制

讨论 思考-1是否可以检测到水分子？ 思考-2是否可以检测到17对二硫键？

讨论 思考-1 是否可以检测到水分子？ 严禁复制 思考-2 是否可以检测到17对二硫键？

提问--根据文中信息进行判断？ Crystallization and X-Ray Data Collection.OsrHSA from large-scale production was solubilized in buffer [50 mM KPO4 (pH 7.5), 150 mM NaCl],and the protein was further purified using size- exclusion chromatography to remove any dimers.The peak corresponding to the monomeric species was collected and the protein was concentrated to 100 mg/mL in 50 mM KPO4 (pH 7.5)and 150 mM NaCl.Results from dynamic light scat- tering showed the presence of only the monomeric species. For preparation of the HSA-myristic acid complex,myristic acid at a concentration of 2.5 mM was resuspended in 20 mM KPO4 (pH 7.5)and warmed to 50 C.The sample was then cooled to ~30 C,and 0.2 mM of rHSA was added.The sample was incubated for 20 min,cooled to 4 C,and centrifuged at 12,000 xg for 4 min at 4 C to pellet any excess myristic acid. The supernatant was concentrated to ~120 mg/mL and buffer exchanged in 20 mM KPO4(pH 7.5)and 0.1 mM myristic acid. Crystals of HSA-myristic acid complex were obtained with the hanging diffusion method in a 28%PEG 3350,50 mM phos- phate buffer(pH 7.5),and 150 mM KCl.The crystals of OsrHSA were flash-frozen in liquid nitrogen,and the X-ray diffraction data were collected at the synchrotron beamline to 2.05 A res- olution with the crystal maintained at the temperature f 100 K. 分辨率

提问 --- 根据文中信息进行判断？ 分辨率 严禁复制

分辨率查询结果 3.0A 5A 大部分主链 Resolution 3.5A 35A 3.5A 主链+大侧链 201 27A 2.5A 主链+小侧链 图1 olution 2.0 A 1.2A 主链+侧链+某些氢原子

分辨率查询结果 5A 大部分主链 3.5A 主链+大侧链 2.5A 主链+小侧链 1.2A 主链+侧链+某些氢原子 严禁复制

为什么？分辨率--最小可观察到的距离 C-C键 C-0键 0-H键 1.5A 1.4-1.5A 0.9A S-C键 S-H键 0-H键 1.7A 1.7A 0.9A

为什么？分辨率 --- 最小可观察到的距离 1.5A 1.4-1.5A 0.9A C-C键 C-O键 O-H键 1.7A 1.7A 0.9A S-C键 严禁复制 S-H键 O-H键