Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography.
Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural information, typically obtained by crystallography; however MPs are difficult to crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization methods, which typically yield well-diffracting, but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. The development of new-generation X-ray free-electron laser (XFEL) sources that produce extremely bright femtosecond pulses has enabled room temperature data collection from microcrystals with no or negligible radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, which represent a notoriously difficult target for structure determination. In the LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of MP microcrystals to the intersection of the injector stream with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can substantially improve the diffraction resolution when only sub-10 µm crystals are available, or when the use of smaller crystals at room temperature can overcome various problems associated with larger cryocooled crystals, such as accumulation of defects, high mosaicity and cryocooling artifacts. Future advancements in X-ray sources and detector technologies should make serial crystallography highly attractive and practicable for implementation not only at XFELs, but also at more accessible synchrotron beamlines. Here we present detailed visual protocols for the preparation, characterization and delivery of microcrystals in LCP for serial crystallography experiments. These protocols include methods for conducting crystallization experiments in syringes, detecting and characterizing the crystal samples, optimizing crystal density, loading microcrystal laden LCP into the injector device and delivering the sample to the beam for data collection.
The Bridge Institute, University of Southern California; Department of Chemistry, University of Southern California.
Andrii
A
Ishchenko
The Bridge Institute, University of Southern California; Department of Chemistry, University of Southern California; cherezov@usc.edu.
Vadim
V
Cherezov
School of Molecular Sciences, Center for Applied Structural Discovery at the Biodesign Institute, Arizona State University; w.liu@asu.edu.
Wei
W
Liu
D003460:Crystallization; D003461:Crystallography; D018360:Crystallography, X-Ray; D006801:Humans; D008055:Lipids; D008565:Membrane Proteins; D017356:Synchrotrons; D013594:Syringes; D013696:Temperature
D008055:Lipids; D008565:Membrane Proteins
Nature. 2000 Aug 17;406(6797):752-7
10963603
Nature. 2011 Jul 19;477(7366):549-55
21772288
Nat Struct Mol Biol. 2015 Mar;22(3):265-8
25686086
Cell. 2015 May 7;161(4):833-44
25913193
Nature. 2015 Jul 30;523(7562):561-7
26200343
Science. 2007 Nov 23;318(5854):1258-65
17962520
IUCrJ. 2015 Jan 27;2(Pt 2):168-76
25866654
Curr Opin Struct Biol. 2011 Aug;21(4):559-66
21775127
IUCrJ. 2015 Aug 04;2(Pt 5):545-51
26306196
Nat Methods. 2015 Jan;12(1):61-3
25384243
Science. 2013 Dec 20;342(6165):1521-4
24357322
Nat Protoc. 2009;4(5):706-31
19390528
Science. 2012 Jul 13;337(6091):232-6
22798613
Nat Protoc. 2014 Sep;9(9):2123-34
25122522
Acta Crystallogr D Biol Crystallogr. 2015 Feb;71(Pt 2):387-97
25664750
J Vis Exp. 2011 Mar 28;(49):null
21490572
IUCrJ. 2015 Jun 30;2(Pt 4):421-30
26177184
Nat Commun. 2014;5:3309
24525480
Anal Chem. 2010 Jan 15;82(2):491-7
20025250
Nature. 2011 Feb 3;470(7332):73-7
21293373
Nature. 2014 Sep 11;513(7517):261-5
25043005
Cryst Growth Des. 2013 Jul 3;13(7):2846-2857
23956688
Science. 2014 Dec 5;346(6214):1242-6
25477465
Phys Chem Chem Phys. 2011 Feb 28;13(8):3004-21
21183976