X-ray Transparent Microfluidic Chip for Mesophase-Based Crystallization of Membrane Proteins and On-Chip Structure Determination.
X-ray Transparent Microfluidic Chip for Mesophase-Based Crystallization of Membrane Proteins and On-Chip Structure Determination.
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DOI：
10.1021/cg5011488
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发表时间：
2014-10-01
影响因子：
3.8
通讯作者：
Kenis, Paul J. A.
中科院分区：
文献类型：
--
作者：
Khvostichenko, Daria S.;Schieferstein, Jeremy M.;Pawate, Ashtamurthy S.;Laible, Philip D.;Kenis, Paul J. A.
从介晶中间相基质中结晶是获得衍射质量晶体和膜蛋白结构的一条有前途的途径。本文报道的微流体方法消除了标准的基于中间相的结晶方案的两个瓶颈：（i）手动制备粘性中间相和（ii）手动收获通常小且易碎的蛋白质晶体。在这里报道的方法中，在X射线透明的微流体芯片中配制蛋白质负载的中间相，每次结晶试验仅使用60 nL的蛋白质溶液。该芯片的X射线透明性使得能够从驻留在微流体威尔斯孔中的多个晶体收集衍射数据，消除了通常需要的手动收获和安装单个晶体。我们验证了我们的方法，通过在芯片上结晶的光合反应中心，膜蛋白从球形红细菌，然后通过解决其结构的分辨率为2.5微米，使用在环境条件下收集的X射线衍射数据芯片上。当比较芯片上的室温结构与在低温条件下获得数据的已知结构时，观察到蛋白质的亲水链中的适度构象变化。用于膜蛋白的LCP结晶的X射线透明微流体芯片每孔仅需要60 nL的蛋白质溶液，并且能够进行原位晶体X射线衍射数据收集。将球形红细菌的光合反应中心在芯片上结晶，并在室温下用芯片中的晶体将其结构解析到2.5 μ m的分辨率。
Crystallization from lipidic mesophase matrices is a promising route to diffraction-quality crystals and structures of membrane proteins. The microfluidic approach reported here eliminates two bottlenecks of the standard mesophase-based crystallization protocols: (i) manual preparation of viscous mesophases and (ii) manual harvesting of often small and fragile protein crystals. In the approach reported here, protein-loaded mesophases are formulated in an X-ray transparent microfluidic chip using only 60 nL of the protein solution per crystallization trial. The X-ray transparency of the chip enables diffraction data collection from multiple crystals residing in microfluidic wells, eliminating the normally required manual harvesting and mounting of individual crystals. We validated our approach by on-chip crystallization of photosynthetic reaction center, a membrane protein from Rhodobacter sphaeroides, followed by solving its structure to a resolution of 2.5 Å using X-ray diffraction data collected on-chip under ambient conditions. A moderate conformational change in hydrophilic chains of the protein was observed when comparing the on-chip, room temperature structure with known structures for which data were acquired under cryogenic conditions. An X-ray transparent microfluidic chip for LCP crystallization of membrane proteins requires only 60 nL of the protein solution per well and enables in situ crystal X-ray diffraction data collection. The photosynthetic reaction center from Rhodobacter sphaeroides was crystallized on-chip and its structure solved to a resolution of 2.5 Å with crystals in the chips at room temperature.
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DOI：
10.1126/science.1244142
发表时间：
2013-12-20
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Liu W;Wacker D;Gati C;Han GW;James D;Wang D;Nelson G;Weierstall U;Katritch V;Barty A;Zatsepin NA;Li D;Messerschmidt M;Boutet S;Williams GJ;Koglin JE;Seibert MM;Wang C;Shah ST;Basu S;Fromme R;Kupitz C;Rendek KN;Grotjohann I;Fromme P;Kirian RA;Beyerlein KR;White TA;Chapman HN;Caffrey M;Spence JC;Stevens RC;Cherezov V
通讯作者：
Cherezov V
DOI：
10.1107/s0907444911011589
发表时间：
2011-06
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Kisselman G;Qiu W;Romanov V;Thompson CM;Lam R;Battaile KP;Pai EF;Chirgadze NY
通讯作者：
Chirgadze NY
DOI：
10.1107/s0907444908028060
发表时间：
2008-11
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Gerdts CJ;Elliott M;Lovell S;Mixon MB;Napuli AJ;Staker BL;Nollert P;Stewart L
通讯作者：
Stewart L
影响因子：
19
作者：
Huh, Dongeun;Hamilton, Geraldine A.;Ingber, Donald E.
通讯作者：
Ingber, Donald E.
影响因子：
8.4
作者：
Cole, Matthew C.;Desai, Amit V.;Kenis, Paul J. A.
通讯作者：
Kenis, Paul J. A.