Two-dimensional crystals of proteins on lipid layers

doi:10.1016/S0959-440X(05)80090-9

Current Opinion in Structural Biology

Volume 1, Issue 4, August 1991, Pages 642-646

https://doi.org/10.1016/S0959-440X(05)80090-9 Get rights and content

Proteins bound to lipid layers form single-layer (two-dimensional) crystals amenable to structure determination by electron microscopy and image processing. Recent studies have extended the range of the lipid-layer crystallization approach. In some cases, a simple electrostatic interaction between a protein and a charged lipid will suffice to direct binding, obviating the requirement for a specific lipid-binding ligand for two-dimensional crystal growth. Alternatively, streptavidin can be used to couple a biotinylated protein to a biotinylated lipid layer. Streptavidin itself forms large two-dimensional crystals that diffract to 2.8 Å resolution, showing that ordering on lipid layers can be comparable to that in three dimensions.

References and recommended reading (28)

DarstSA et al.
Two-Dimensional Crystals of Escherichia coli RNA Polymerase Holoenzyme on Positively Charged Lipid Layers
J Mol Biol
(1988)
DarstSA et al.
Two-Dimensional and Epitaxial Crystallization of a Mutant Form of Yeast RNA Polymerase II
J Mol Biol
(1991)
BlankenburgR et al.
Interaction Between Biotin Lipids and Streptavidin in Monolayers: Formation of Oriented Two-Dimensional Protein Domains Induced By Surface Recognition
Biochemistry
(1989)
MosserG et al.
Sub-Domain Structure of Lipid-Bound Annexin-V Resolved By Electron Image Analysis
J Mol Biol
(1991)
LebeauL et al.
Systematic Study of Phospholipids Linked to a Steroid Derivative, Spread Into a Monolayer at the Air/Water Interface
Biochim Biophys Acta
(1988)
DarstSA et al.
Three-Dimensional Structure of Escherichia coli RNA Polymerase Holoenzyme Determined by Electron Crystallography
Nature
(1989)
KühlbrandtW
Three-Dimensional Crystallization of Membrane Proteins
Q Rev Biophys
(1988)
UzgirisEE et al.
Two-Dimensional Crystallization Technique for Imaging Macromolecules, With Application to Antigen—Antibody—Complement Complexes
Nature
(1983)
RibiHO et al.
Two-Dimensional Crystals of Enzyme—Effector Complexes: Ribonucleotide Reductase at 18Å Resolution
Biochemistry
(1987)
EdwardsAM et al.
Purification and Lipid Layer Crystallization of Yeast RNA Polymerase II

SchultzP et al.

Structural Study of the Yeast RNA Polymerase A: Electron Microscopy of Lipid-Bound Molecules and Two-Dimensional Crystals

J Mol Biol

(1990)

LebeauL et al.

Two-Dimensional Crystallization of DNA Gyrase B Subunit on Specifically Designed Lipid Monolayers

FEBS Lett

(1990)

LudwigDS et al.

Two-Dimensional Crystals of Cholera Toxin B Subunit—Receptor Complexes: Projected Structure at 17 Å Resolution

RobinsonJP et al.

Three-Dimensional Structural Analysis of Tetanus Toxin by Electron Crystallography

J Mol Biol

(1988)

Cited by (109)

Double-headed binding of myosin II to F-actin shows the effect of strain on head structure
2023, Journal of Structural Biology
Force production in muscle is achieved through the interaction of myosin and actin. Strong binding states in active muscle are associated with Mg·ADP bound to the active site; release of Mg·ADP allows rebinding of ATP and dissociation from actin. Thus, Mg·ADP binding is positioned for adaptation as a force sensor. Mechanical loads on the lever arm can affect the ability of myosin to release Mg·ADP but exactly how this is done is poorly defined. Here we use F-actin decorated with double-headed smooth muscle myosin fragments in the presence of Mg·ADP to visualize the effect of internally supplied tension on the paired lever arms using cryoEM. The interaction of the paired heads with two adjacent actin subunits is predicted to place one lever arm under positive and the other under negative strain. The converter domain is believed to be the most flexible domain within myosin head. Our results, instead, point to the segment of heavy chain between the essential and regulatory light chains as the location of the largest structural change. Moreover, our results suggest no large changes in the myosin coiled coil tail as the locus of strain relief when both heads bind F-actin. The method would be adaptable to double-headed members of the myosin family. We anticipate that the study of actin-myosin interaction using double-headed fragments enables visualization of domains that are typically noisy in decoration with single-headed fragments.
Structural studies of the endogenous spliceosome – The supraspliceosome
2017, Methods
Citation Excerpt :
We therefore tried a different approach of concentrating the particles on charged lipid monolayer films. This methodology has been successfully used in electron crystallography studies of biologically active complexes [58,59]. We adapted this method to reproducibly concentrate and generate, on a positively charged monolayer, a layer with a high density of supraspliceosomes that can be picked up on holey carbon grids, blotted and plunged into liquid ethane, and visualized in the frozen-hydrated state [36].
Pre-mRNA splicing is executed in mammalian cell nuclei within a huge (21 MDa) and highly dynamic molecular machine – the supraspliceosome – that individually package pre-mRNA transcripts of different sizes and number of introns into complexes of a unique structure, indicating their universal nature. Detailed structural analysis of this huge and complex structure requires a stepwise approach using hybrid methods. Structural studies of the supraspliceosome by room temperature electron tomography, cryo-electron tomography, and scanning transmission electron microscope mass measurements revealed that it is composed of four native spliceosomes, each resembling an in vitro assembled spliceosome, which are connected by the pre-mRNA. It also elucidated the arrangement of the native spliceosomes within the intact supraspliceosome. Native spliceosomes and supraspliceosomes contain all five spliceosomal U snRNPs together with other splicing factors, and are active in splicing. The structure of the native spliceosome, at a resolution of 20 Å, was determined by cryo-electron microscopy, and a unique spatial arrangement of the spliceosomal U snRNPs within the native spliceosome emerged from in silico studies. The supraspliceosome also harbor components for all pre-mRNA processing activities. Thus the supraspliceosome – the endogenous spliceosome – is a stand-alone complete macromolecular machine capable of performing splicing, alternative splicing, and encompass all nuclear pre-mRNA processing activities that the pre-mRNA has to undergo before it can exit from the nucleus to the cytoplasm to encode for protein. Further high-resolution cryo-electron microscopy studies of the endogenous spliceosome are required to decipher the regulation of alternative splicing, and elucidate the network of processing activities within it.
Assemblies of pore-forming toxins visualized by atomic force microscopy
2016, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
There are two approaches for preparing 2D assemblies of PFTs. The first is very similar to the techniques employed for the EM visualization of 2D protein crystals [81,82]. In this approach, a lipid monolayer is first transferred onto mica using the LB method; then, the mica-supported lipid monolayer is horizontally lowered onto a separately prepared lipid monolayer at the air/water interface, and the protein is injected into the subphase.
A number of pore-forming toxins (PFTs) can assemble on lipid membranes through their specific interactions with lipids. The oligomeric assemblies of some PFTs have been successfully revealed either by electron microscopy (EM) and/or atomic force microscopy (AFM). Unlike EM, AFM imaging can be performed under physiological conditions, enabling the real-time visualization of PFT assembly and the transition from the prepore state, in which the toxin does not span the membrane, to the pore state. In addition to characterizing PFT oligomers, AFM has also been used to examine toxin-induced alterations in membrane organization. In this review, we summarize the contributions of AFM to the understanding of both PFT assembly and PFT-induced membrane reorganization. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.
Electron crystallography - The waking beauty of structural biology
2012, Current Opinion in Structural Biology
Since its debut in the mid 1970s, electron crystallography has been a valuable alternative in the structure determination of biological macromolecules. Its reliance on single-layered or double-layered two-dimensionally ordered arrays and the ability to obtain structural information from small and disordered crystals make this approach particularly useful for the study of membrane proteins in a lipid bilayer environment. Despite its unique advantages, technological hurdles have kept electron crystallography from reaching its full potential. Addressing the issues, recent initiatives developed high-throughput pipelines for crystallization and screening. Adding progress in automating data collection, image analysis and phase extension methods, electron crystallography is poised to raise its profile and may lead the way in exploring the structural biology of macromolecular complexes.
Effect of surface chemistry of novel templates on crystallization of proteins
2012, Chemical Engineering Science
Citation Excerpt :
This means the crystallization of macromolecules will depend more on surface properties than small salt compounds. Attempts to find correlations between proteins and surfaces that can improve or just change their crystallization parameters are also recorded in the literature and different substrates have been used for this purpose—mineral surfaces (McPherson and Schilichta, 1988; Kimble et al., 1998), lipid films (Edwards et al., 1994; Kornberg and Darst, 1991; Kubo et al., 2007), polymers (Grzesiak and Matzger, 2008), protein thin film (Pechkova and Nicolini, 2001,2002), molecular imprinted polymer (Saridakis et al., 2011), as well as applying chemical modification of mica (Falini et al., 2002; Simone et al., 2006; Tang et al., 2005; Tosi et al., 2008) or glass surface (Liu et al., 2007; Nanev and Tsekova, 2000; Rong et al., 2002; Sun et al., 2010, Tsekova et al., 2002). Here we report the preparation of new surface chemical treated crystallization templates, the determination of their dispersive and polar surface energies components and crystallization performance of Lysozyme, Thaumatin, Catalase and Ferritin on them.
The heterogeneous crystallization of four model proteins (Lysozyme, Thaumatine, Catalase and Ferritin) on novel organic templates is reported. The chemical surface modification of glass substrates is described. The organic layers formed are characterized by contact angles' measurement with their dispersive and polar surface energy components determinated. Crystallization studies were completed using the hanging drop technique. The substrate surface chemistry was found to influence several aspects of protein crystallization: the number of crystals formed, the sizes of crystals and the crystal morphology. Data obtained confirm the theoretical suggestions that templates with specific surface chemistry can be successfully used for the control of nucleation and morphology of protein crystals.
Membrane Crystallization Technology
2010, Comprehensive Membrane Science and Engineering
The interest to combine membrane operations with crystallization, in order to increase the overall process efficiency, has been growing for several years. This approach has been put in practice in the form of the membrane crystallization (MCr) technology. The main features of MCr are: (1) the use of membranes as precision devices for mass flow, in vapor phase, by opposing a well-defined and tunable resistance and (2) the action of the porous surface of the membrane as a suitable tool to activate heterogeneous nucleation. Thanks to these two fundamental advantages, combined together in the same apparatus, an improved control of the crystallization processes, the possibility to be initiated at low supersaturation, and the enhancement of the crystallization kinetics can be achieved even for large molecules such as proteins.
Furthermore, MCr technology is expected to represent an attractive option for realizing integrated and highly efficient industrial productive processes, well satisfying the requirements of the process-intensification strategy. A significant example in this context is seawater desalination, where a rational integration of MCr technology with traditional pressure-driven membrane operations might determine substantial improvements in water quality, total product recovery factor, overall cost, environmental impact, and brine disposal management.

View all citing articles on Scopus

View full text

Article preview

Current Opinion in Structural Biology

References and recommended reading (28)

Two-Dimensional Crystals of Escherichia coli RNA Polymerase Holoenzyme on Positively Charged Lipid Layers

J Mol Biol

Two-Dimensional and Epitaxial Crystallization of a Mutant Form of Yeast RNA Polymerase II

J Mol Biol

Interaction Between Biotin Lipids and Streptavidin in Monolayers: Formation of Oriented Two-Dimensional Protein Domains Induced By Surface Recognition

Biochemistry

Sub-Domain Structure of Lipid-Bound Annexin-V Resolved By Electron Image Analysis

J Mol Biol

Systematic Study of Phospholipids Linked to a Steroid Derivative, Spread Into a Monolayer at the Air/Water Interface

Biochim Biophys Acta

Three-Dimensional Structure of Escherichia coli RNA Polymerase Holoenzyme Determined by Electron Crystallography

Nature

Three-Dimensional Crystallization of Membrane Proteins

Q Rev Biophys

Two-Dimensional Crystallization Technique for Imaging Macromolecules, With Application to Antigen—Antibody—Complement Complexes

Nature

Two-Dimensional Crystals of Enzyme—Effector Complexes: Ribonucleotide Reductase at 18Å Resolution

Biochemistry

Purification and Lipid Layer Crystallization of Yeast RNA Polymerase II

Structural Study of the Yeast RNA Polymerase A: Electron Microscopy of Lipid-Bound Molecules and Two-Dimensional Crystals

J Mol Biol

Two-Dimensional Crystallization of DNA Gyrase B Subunit on Specifically Designed Lipid Monolayers

FEBS Lett

Two-Dimensional Crystals of Cholera Toxin B Subunit—Receptor Complexes: Projected Structure at 17 Å Resolution

Three-Dimensional Structural Analysis of Tetanus Toxin by Electron Crystallography

J Mol Biol

Cited by (109)

Double-headed binding of myosin II to F-actin shows the effect of strain on head structure

Structural studies of the endogenous spliceosome – The supraspliceosome

Assemblies of pore-forming toxins visualized by atomic force microscopy

Electron crystallography - The waking beauty of structural biology

Effect of surface chemistry of novel templates on crystallization of proteins

Membrane Crystallization Technology