Overview of Electron Crystallography of Membrane Proteins: Crystallization and Screening Strategies Using Negative Stain Electron Microscopy

Nannenga, Brent L.; Iadanza, M.G.; Vollmar, Breanna S.; Gonen, Tamir

doi:10.1002/0471140864.ps1715s72

Cited by 29 publications

(20 citation statements)

References 30 publications

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“…6 μl of purified protein (I53-50-v0, I53-50-v1, I53-50-v2, I53-50-v3, I53-50-v4, I53-50-Btat, I53-47-v0, I53-47-v1, I53-47-Btat) at 0.04 – 0.3 mg/mL were applied to glow discharged, carbon-coated 300-mesh copper grids (Ted Pella), washed with Milli-Q water and stained with 0.75% uranyl formate as described previously 27 . Screening and sample optimization was performed on a 100 kV Morgagni M268 transmission electron microscope (FEI) equipped with an Orius charge-coupled device (CCD) camera (Gatan).…”

Section: Methodsmentioning

confidence: 99%

Evolution of a designed protein assembly encapsulating its own RNA genome

Butterfield

Lajoie

Gustafson

et al. 2017

Nature

184

237

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The challenges of evolution in a complex biochemical environment—coupling genotype to phenotype and protecting the genetic material—are solved elegantly in biological systems by nucleic acid encapsulation. In the simplest examples, viruses use capsids to surround their genomes. While these naturally occurring systems have been modified to change their tropism1 and to display proteins or peptides2–4, billions of years of evolution have favored efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a “blank slate” to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids—computationally designed icosahedral protein assemblies5, 6 with positively charged inner surfaces capable of packaging their own full-length mRNA genomes—and explore their ability to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in drastically improved genome packaging (>133-fold), stability in whole murine blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus (AAV) vectors7, 8. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at “top-down” modification of viruses to be safe and effective for drug delivery and vaccine applications1, 9, 10; the ability to computationally design synthetic nanomaterials and to optimize them through evolution now enables a complementary “bottom-up” approach with considerable advantages in programmability and control.

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Section: Methodsmentioning

confidence: 99%

Evolution of a designed protein assembly encapsulating its own RNA genome

Butterfield

Lajoie

Gustafson

et al. 2017

Nature

184

237

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“…Therefore, some optimization of blotting is necessary to strike the right balance between leaving enough solution so that the crystals are well-hydrated and preserved, but not so much that the sample is too thick for electron diffraction. Conditions for sample preparation can be screened by negative stain EM [29], however this only provides a rough starting point as conditions can deviate significantly when using stains and will need to be further optimized for cryoEM.…”

Section: Microed Sample Preparation and Data Collectionmentioning

confidence: 99%

Protein structure determination by MicroED

Nannenga

Gonen

2014

Current Opinion in Structural Biology

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In this review we discuss the current advances relating to structure determination from protein microcrystals with special emphasis on the newly developed method called MicroED. This method uses a transmission electron cryo-microscope to collect electron diffraction data from extremely small 3-dimensional (3D) crystals. MicroED has been used to solve the 3D structure of the model protein lysozyme to 2.9 Å resolution. As the method further matures, MicroED promises to offer a unique and widely applicable approach to protein crystallography using nanocrystals.

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“…An interesting alternative to conventional X-ray diffraction is an electron diffraction technique called MicroED (Brent L. Nannenga et al, 2013;B. L. Nannenga et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

“…Detergent chelators like Biobeads and cyclodextrins are most commonly used with microdialysis providing an alternative approach, albeit one that is limited by the long times required to completely remove detergents with low critical micelle concentration (Brent L. Nannenga et al, 2013).…”

Section: Lipidsmentioning

confidence: 99%

“…In general, divalent cations are believed to assist with 2D crystallisation by attracting net negatively charged surface of membranes and/or proteins, which would otherwise repel each other due to the electrostatic repulsion (Akashi et al, 1998;Bentz & Duzgunes, 1985;Ruso et al, 2003). This attraction could in turn induce the membrane fusion, which is favourable for large 2D crystal formation (Brent L. Nannenga et al, 2013). However, many experiments studying this effect used net anionic phospholipids such as phosphatidylserine and phosphatidylglycerol rather than zwitterionic phospholipids used in this experiment (phosphatidylcholine and E. coli lipid, which is largely phosphatidylethanolamine) (Akashi et al, 1998;Bentz & Duzgunes, 1985;Ruso et al, 2003).…”

Section: The Influence Of Inorganic Ions On 2d Crystallisationmentioning

confidence: 99%

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Biophysical and Structural Studies of Escherichia coli Mechanosensitive Channel of Large Conductance in Lipid Bilayers

Chi¹

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A major challenge of drug development is maximising specificity and efficacy at the lowest possible dose to minimise toxic side effects (Basile et al., 2012). Not only are the pharmaceutical windows small for both drug candidates and approved drugs alike, but they can also vary between individuals, making a dose level safe for one individual potentially harmful for another. One strategy for overcoming this problem is to develop new drug delivery methods where the drug is preferentially concentrated at the site of disease (Basile et al., 2012). In this respect, liposomal drug delivery systems have been used with success in topical applications in skin cancer treatment, and there is significant research interest in engineering similar systems for intravenous administration (Al-Jamal & Kostarelos, 2011;Fan & Zhang, 2013). Incorporating nanovalves into liposomes with controllable gating properties would further enhance the usefulness of liposomal drug delivery systems, providing a method to further regulate the release of liposome-encapsulated drugs from liposomes at the target site (Martinac et al., 2014).The mechanosensitive channel of large conductance (MscL) has been identified as a major candidate for the development of a protein-based nanovalve (Martinac et al., 2014). More generally, MscL is a prototype for the class of ion channels primarily gated by membrane tension, which includes medically significant proteins such as Piezo channels and TRP-type sodium channels (Martinac, 2011). As a well-expressing protein from Escherichia coli, MscL has been extensively studied both structurally and biophysically (Martinac, 2011). However, the channel gating mechanism of MscL, and by extension of mechanosensitive channels in general, remains poorly understood at the molecular level.This thesis aims to investigate the relationship between E. coli MscL and phospholipids both in the context of its structure and its behaviour in a lipid bilayer environment. While much has been studied on the channel gating properties of MscL in various phospholipid environments (Anishkin et al., 2005;Iscla et al., 2004;Iscla et al., 2011;Powl et al., 2008b;Tsai et al., 2005;Yoshimura et al., 2004), there remains a limited understanding of the behaviour and distribution of MscL in the bilayer. These are not only important from academic perspective as well as in the context of nanovalve development, but also for example, to identify factors which may limit the functional studies on MscL. More specifically, this thesis has focused on the identification of phospholipids closely associating with MscL, the distribution of MscL in phospholipid bilayers, and the incorporation of MscL into liposomes with heterogeneous phospholipids.The first research chapter (Chapter 2) outlines the optimisation of MscL expression and purification system for biophysical and structural studies. The original MscL construct had problems of ii heterogeneous expression and being not well-suited to reliable quantification by routine methods.New constructs were suc...

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Overview of Electron Crystallography of Membrane Proteins: Crystallization and Screening Strategies Using Negative Stain Electron Microscopy

Cited by 29 publications

References 30 publications

Evolution of a designed protein assembly encapsulating its own RNA genome

Evolution of a designed protein assembly encapsulating its own RNA genome

Protein structure determination by MicroED

Biophysical and Structural Studies of Escherichia coli Mechanosensitive Channel of Large Conductance in Lipid Bilayers

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