Experimental phasing for structure determination using membrane-protein crystals grown by the lipid cubic phase method

Li, Dianfan; Pye, V.E.; Caffrey, Martin

doi:10.1107/s1399004714010360

Cited by 19 publications

(18 citation statements)

References 78 publications

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“…They constitute a very successful host-lipid screen in the MS&FB group and beyond. With several targets, which include -barrels, -helical proteins, a GPCR-G s protein complex and an integral peptide antibiotic, crystals have been grown by the in meso method using these alternative MAGs Hö fer, Aragã o, Lyons et al, 2011;Li et al, , 2015Ring et al, 2013;Lyons et al, 2014;Malinauskaite et al, 2014;Takeda et al, 2014). In a number of cases monoolein either failed to produce crystals or the crystals that it did produce were not of diffraction quality.…”

Section: Host Lipidmentioning

confidence: 99%

“…Indeed, with careful measurements, native sulfur-SAD phasing with a single in meso crystal is possible (Weinert et al, 2014). A detailed case study of experimental phasing as applied to DgkA has recently been reported (Li et al, 2015).…”

Section: Experimental Phasingmentioning

confidence: 99%

“…We have attempted to make good this deficit by reporting full details of the screening strategies implemented in the MS&FB Group with -barrel and -helical membraneprotein targets that have led to structures ( Fig. 7; Li et al, , 2014Li et al, , 2015. Some of the lessons learned from these assorted studies are summarized below in no particular order.…”

Section: An Evolving In Meso Screening Strategymentioning

confidence: 99%

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A comprehensive review of the lipid cubic phase orin mesomethod for crystallizing membrane and soluble proteins and complexes

Caffrey

2015

Acta Crystallogr F Struct Biol Commun

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The lipid cubic phase or in meso method is a robust approach for crystallizing membrane proteins for structure determination. The uptake of the method is such that it is experiencing what can only be described as explosive growth. This timely, comprehensive and up-to-date review introduces the reader to the practice of in meso crystallogenesis, to the associated challenges and to their solutions. A model of how crystallization comes about mechanistically is presented for a more rational approach to crystallization. The possible involvement of the lamellar and inverted hexagonal phases in crystallogenesis and the application of the method to water-soluble, monotopic and lipidanchored proteins are addressed. How to set up trials manually and automatically with a robot is introduced with reference to open-access online videos that provide a practical guide to all aspects of the method. These range from protein reconstitution to crystal harvesting from the hosting mesophase, which is noted for its viscosity and stickiness. The sponge phase, as an alternative medium in which to perform crystallization, is described. The compatibility of the method with additive lipids, detergents, precipitant-screen components and materials carried along with the protein such as denaturants and reducing agents is considered. The powerful host and additive lipid-screening strategies are described along with how samples that have low protein concentration and cell-free expressed protein can be used. Assaying the protein reconstituted in the bilayer of the cubic phase for function is an important element of quality control and is detailed. Host lipid design for crystallization at low temperatures and for large proteins and complexes is outlined. Experimental phasing by heavy-atom derivatization, soaking or co-crystallization is routine and the approaches that have been implemented to date are described. An overview and a breakdown by family and function of the close to 200 published structures that have been obtained using in meso-grown crystals are given. Recommendations for conducting the screening process to give a more productive outcome are summarized. The fact that the in meso method also works with soluble proteins should not be overlooked. Recent applications of the method for in situ serial crystallography at X-ray free-electron lasers and synchrotrons are described. The review ends with a view to the future and to the bright prospects for the method, which continues to contribute to our understanding of the molecular mechanisms of some of nature's most valued proteinaceous robots.

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Section: Host Lipidmentioning

confidence: 99%

Section: Experimental Phasingmentioning

confidence: 99%

Section: An Evolving In Meso Screening Strategymentioning

confidence: 99%

See 1 more Smart Citation

A comprehensive review of the lipid cubic phase orin mesomethod for crystallizing membrane and soluble proteins and complexes

Caffrey

2015

Acta Crystallogr F Struct Biol Commun

Self Cite

234

246

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“…Both holders can be used for roomtemperature (RT, 293 K) data collection and are compatible with sample-changing robots. They provide for a wide scanning area and are suitable for post-crystallization treatments such as ligand and heavy-atom soaking (Li et al, 2015;Rucktooa et al, 2018;Huang et al, 2018). The holders can be used with a host of in situ window materials such as cyclic olefin polymer (COP) (Axford et al, 2016;Apel et al, 2019), cyclic olefin copolymer (COC) (Huang et al, 2015(Huang et al, , 2016El Ghachi et al, 2018), biaxially oriented polyethylene terephthalate (Mylar) (Broecker et al, 2016) and silicon nitride (Cherezov & Caffrey, 2007;Murray et al, 2015;Roedig et al, 2015;Owen et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

3D-printed holders forin meso in situfixed-target serial X-ray crystallography

et al. 2020

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The in meso in situ serial X-ray crystallography method was developed to ease the handling of small fragile crystals of membrane proteins and for rapid data collection on hundreds of microcrystals directly in the growth medium without the need for crystal harvesting. To facilitate mounting of these in situ samples on a goniometer at cryogenic or at room temperatures, two new 3D-printed holders have been developed. They provide for cubic and sponge phase sample stability in the X-ray beam and are compatible with sample-changing robots. The holders can accommodate a variety of window material types, as well as bespoke samples for diffraction screening and data collection at conventional macromolecular crystallography beamlines. They can be used for convenient postcrystallization treatments such as ligand and heavy-atom soaking. The design, assembly and application of the holders for in situ serial crystallography are described. Files for making the holders using a 3D printer are included as supporting information.

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“…Alternatively, denser atoms (for example mercury, gold and platinum) have successfully been used as phasing labels (Pike et al, 2016). One strategy uses cysteines for site-specific mercury labelling and has helped to solve the structures of both soluble and membrane proteins (Doyle et al, 1996;Li et al, 2015). Hg is the most successful element when it comes to forming heavyatom derivatives of proteins and their crystals, and cysteine side chains are also by far the most frequent binding or coordinating residues for Hg atoms (Sugahara et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Introducing site-specific cysteines into nanobodies for mercury labelling allowsde novophasing of their crystal structures

Hansen

Laursen

Andersen

et al. 2017

Acta Cryst Sect D Struct Biol

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-wavelength anomalous dispersion; SAD; single isomorphous replacement with anomalous signal; SIRAS.PDB references: Nb36-Nat1, 5nlu; Nb36-Nat2, 5nlw; Nb36-C85-1, 5nm0; Nb36-C85-2, 5nml The generation of high-quality protein crystals and the loss of phase information during an X-ray crystallography diffraction experiment represent the major bottlenecks in the determination of novel protein structures. A generic method for introducing Hg atoms into any crystal independent of the presence of free cysteines in the target protein could considerably facilitate the process of obtaining unbiased experimental phases. Nanobodies (single-domain antibodies) have recently been shown to promote the crystallization and structure determination of flexible proteins and complexes. To extend the usability of nanobodies for crystallographic work, variants of the Nb36 nanobody with a single free cysteine at one of four framework-residue positions were developed. These cysteines could be labelled with fluorophores or Hg. For one cysteine variant (Nb36-C85) two nanobody structures were experimentally phased using single-wavelength anomalous dispersion (SAD) and single isomorphous replacement with anomalous signal (SIRAS), taking advantage of radiationinduced changes in Cys-Hg bonding. Importantly, Hg labelling influenced neither the interaction of Nb36 with its antigen complement C5 nor its structure. The results suggest that Cys-Hg-labelled nanobodies may become efficient tools for obtaining de novo phase information during the structure determination of nanobody-protein complexes.

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Experimental phasing for structure determination using membrane-protein crystals grown by the lipid cubic phase method

Cited by 19 publications

References 78 publications

A comprehensive review of the lipid cubic phase orin mesomethod for crystallizing membrane and soluble proteins and complexes

A comprehensive review of the lipid cubic phase orin mesomethod for crystallizing membrane and soluble proteins and complexes

3D-printed holders forin meso in situfixed-target serial X-ray crystallography

Introducing site-specific cysteines into nanobodies for mercury labelling allowsde novophasing of their crystal structures

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