An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography.
An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography.
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DOI：
10.1038/s41467-021-24757-7
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发表时间：
2021-07-22
影响因子：
16.6
通讯作者：
Orville AM
中科院分区：
文献类型：
--
作者：
Butryn A;Simon PS;Aller P;Hinchliffe P;Massad RN;Leen G;Tooke CL;Bogacz I;Kim IS;Bhowmick A;Brewster AS;Devenish NE;Brem J;Kamps JJAG;Lang PA;Rabe P;Axford D;Beale JH;Davy B;Ebrahim A;Orlans J;Storm SLS;Zhou T;Owada S;Tanaka R;Tono K;Evans G;Owen RL;Houle FA;Sauter NK;Schofield CJ;Spencer J;Yachandra VK;Yano J;Kern JF;Orville AM
连续飞秒晶体学在结构生物学中开辟了许多新的机会。近年来，出现了几种采用光诱导系统的方法，以实现时间分辨实验，从而在高原子和时间分辨率下揭示蛋白质动力学。然而，很少有酶是光依赖性的，而需要配体扩散到活性位点的大分子是普遍存在的。在这项工作中，我们提出了一种滴滴样品输送系统，可以研究微晶浆料中的酶催化反应。该系统提供了一个更大的含晶体的液滴诱导湍流混合的顶部上的多个皮升大小的液滴的突发配体溶液和运输的混合物的X-射线相互作用区域的时间分辨率。我们演示了混合使用荧光染料，数值模拟和时间分辨串行飞秒晶体学，这表明通过微滴快速配体扩散。滴上滴法有可能被广泛应用于系列晶体学研究，特别是与小分子底物的酶反应。目前，许多时间分辨连续飞秒（SFX）晶体学实验是用光驱动的蛋白质系统进行的，而非光触发酶的反应引发仍然是一个主要的瓶颈。在这里，作者提出了一种扩展的滴剂胶带系统，其中底物或抑制剂的皮升大小的液滴与微晶浆料的纳升大小的液滴混合，并且他们使用它来进行抑制剂与溶菌酶结合的时间分辨SFX测量，其次，β-内酰胺抗生素与细菌丝氨酸β-内酰胺酶的结合。
Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates. Currently many of the time resolved serial femtosecond (SFX) crystallography experiments are done with light driven protein systems, whereas the reaction initiation for non-light triggered enzymes remains a major bottle neck. Here, the authors present an expanded Drop-on-Tape system, where picoliter-sized droplets of a substrate or inhibitor are turbulently mixed with nanoliter sized droplets of microcrystal slurries, and they use it for time-resolved SFX measurements of inhibitor binding to lysozyme and secondly, binding of a β-lactam antibiotic to a bacterial serine β-lactamase.
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影响因子：
4.4
作者：
BUNKER, DL;GARRETT, B;LONG, GS
通讯作者：
LONG, GS
DOI：
10.1107/s0907444909029436
发表时间：
2009-10-01
影响因子：
2.2
作者：
Moriarty, Nigel W.;Grosse-Kunstleve, Ralf W.;Adams, Paul D.
通讯作者：
Adams, Paul D.
DOI：
10.1063/1.4972069
发表时间：
2017-07
期刊：
Structural dynamics (Melville, N.Y.)
影响因子：
--
作者：
Kupitz C;Olmos JL Jr;Holl M;Tremblay L;Pande K;Pandey S;Oberthür D;Hunter M;Liang M;Aquila A;Tenboer J;Calvey G;Katz A;Chen Y;Wiedorn MO;Knoska J;Meents A;Majriani V;Norwood T;Poudyal I;Grant T;Miller MD;Xu W;Tolstikova A;Morgan A;Metz M;Martin-Garcia JM;Zook JD;Roy-Chowdhury S;Coe J;Nagaratnam N;Meza D;Fromme R;Basu S;Frank M;White T;Barty A;Bajt S;Yefanov O;Chapman HN;Zatsepin N;Nelson G;Weierstall U;Spence J;Schwander P;Pollack L;Fromme P;Ourmazd A;Phillips GN Jr;Schmidt M
通讯作者：
Schmidt M
影响因子：
48
作者：
Fuller FD;Gul S;Chatterjee R;Burgie ES;Young ID;Lebrette H;Srinivas V;Brewster AS;Michels-Clark T;Clinger JA;Andi B;Ibrahim M;Pastor E;de Lichtenberg C;Hussein R;Pollock CJ;Zhang M;Stan CA;Kroll T;Fransson T;Weninger C;Kubin M;Aller P;Lassalle L;Bräuer P;Miller MD;Amin M;Koroidov S;Roessler CG;Allaire M;Sierra RG;Docker PT;Glownia JM;Nelson S;Koglin JE;Zhu D;Chollet M;Song S;Lemke H;Liang M;Sokaras D;Alonso-Mori R;Zouni A;Messinger J;Bergmann U;Boal AK;Bollinger JM Jr;Krebs C;Högbom M;Phillips GN Jr;Vierstra RD;Sauter NK;Orville AM;Kern J;Yachandra VK;Yano J
通讯作者：
Yano J
DOI：
10.1126/science.1217737
发表时间：
2012-07-20
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Boutet S;Lomb L;Williams GJ;Barends TR;Aquila A;Doak RB;Weierstall U;DePonte DP;Steinbrener J;Shoeman RL;Messerschmidt M;Barty A;White TA;Kassemeyer S;Kirian RA;Seibert MM;Montanez PA;Kenney C;Herbst R;Hart P;Pines J;Haller G;Gruner SM;Philipp HT;Tate MW;Hromalik M;Koerner LJ;van Bakel N;Morse J;Ghonsalves W;Arnlund D;Bogan MJ;Caleman C;Fromme R;Hampton CY;Hunter MS;Johansson LC;Katona G;Kupitz C;Liang M;Martin AV;Nass K;Redecke L;Stellato F;Timneanu N;Wang D;Zatsepin NA;Schafer D;Defever J;Neutze R;Fromme P;Spence JC;Chapman HN;Schlichting I
通讯作者：
Schlichting I