The 2DX robot: a membrane protein 2D crystallization Swiss Army knife.
Among the state-of-the-art techniques that provide experimental information at atomic scale for membrane proteins, electron crystallography, atomic force microscopy and solid state NMR make use of two-dimensional crystals. We present a cyclodextrin-driven method for detergent removal implemented in a fully automated robot. The kinetics of the reconstitution processes is precisely controlled, because the detergent complexation by cyclodextrin is of stoichiometric nature. The method requires smaller volumes and lower protein concentrations than established 2D crystallization methods, making it possible to explore more conditions with the same amount of protein. The method yielded highly ordered 2D crystals diffracting to high resolution from the pore-forming toxin Aeromonas hydrophila aerolysin (2.9A), the plant aquaporin SoPIP2;1 (3.1A) and the human aquaporin-8 (hAQP8; 3.3A). This new method outperforms traditional 2D crystallization approaches in terms of accuracy, flexibility, throughput, and allows the usage of detergents having low critical micelle concentration (CMC), which stabilize the structure of membrane proteins in solution.
Iacovache
Ioan
I
Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Biasini
Marco
M
Kowal
Julia
J
Kukulski
Wanda
W
Chami
Mohamed
M
van der Goot
F Gisou
FG
Engel
Andreas
A
Rémigy
Hervé-W
HW
D016980:Aeromonas hydrophila / Q000378:metabolism; D000818:Animals; D020346:Aquaporins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D001427:Bacterial Toxins / Q000737:chemistry / Q000302:isolation & purification; D020285:Cryoelectron Microscopy; D003460:Crystallization / Q000295:instrumentation / Q000379:methods*; D003505:Cyclodextrins / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure
D020346:Aquaporins; D001427:Bacterial Toxins; D003505:Cyclodextrins; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; C032676:aerolysin