scholarly journals Termini restraining of small membrane proteins enables structure determination at near-atomic resolution

2020 ◽  
Vol 6 (51) ◽  
pp. eabe3717
Author(s):  
Shixuan Liu ◽  
Shuang Li ◽  
Yihu Yang ◽  
Weikai Li
Keyword(s):  
Membrane Proteins ◽  
Structure Determination ◽  
Protein Crystallization ◽  
Cysteine Proteases ◽  
Catalytic Triad ◽  
Atomic Resolution ◽  
High Resolution Structure ◽  
Improved Stability ◽  
Functional Families ◽  
Thiol Oxidoreductase

Small membrane proteins are difficult targets for structural characterization. Here, we stabilize their folding by restraining their amino and carboxyl termini with associable protein entities, exemplified by the two halves of a superfolder GFP. The termini-restrained proteins are functional and show improved stability during overexpression and purification. The reassembled GFP provides a versatile scaffold for membrane protein crystallization, enables diffraction to atomic resolution, and facilitates crystal identification, phase determination, and density modification. This strategy gives rise to 14 new structures of five vertebrate proteins from distinct functional families, bringing a substantial expansion to the structural database of small membrane proteins. Moreover, a high-resolution structure of bacterial DsbB reveals that this thiol oxidoreductase is activated through a catalytic triad, similar to cysteine proteases. Overall, termini restraining proves exceptionally effective for stabilization and structure determination of small membrane proteins.

scholarly journals Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins

FEBS Letters ◽  
2010 ◽  
Vol 584 (12) ◽  
pp. 2539-2547 ◽  
Author(s):  
Yo Sonoda ◽  
Alex Cameron ◽  
Simon Newstead ◽  
Hiroshi Omote ◽  
Yoshinori Moriyama ◽  
...  
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Structure Determination ◽  
High Resolution Structure ◽  
Resolution Structure

scholarly journals Femtosecond crystallography of membrane proteins in the lipidic cubic phase

2014 ◽  
Vol 369 (1647) ◽  
pp. 20130314 ◽  
Author(s):  
Wei Liu ◽  
Daniel Wacker ◽  
Chong Wang ◽  
Enrique Abola ◽  
Vadim Cherezov
Keyword(s):  
Membrane Proteins ◽  
Structure Determination ◽  
Cubic Phase ◽  
Technological Advances ◽  
Lipidic Cubic Phase ◽  
High Resolution Structure ◽  
Effects Of Radiation ◽  
G Protein Coupled ◽  
Serial Femtosecond Crystallography

Despite recent technological advances in heterologous expression, stabilization and crystallization of membrane proteins (MPs), their structural studies remain difficult and require new transformative approaches. During the past two years, crystallization in lipidic cubic phase (LCP) has started gaining a widespread acceptance, owing to the spectacular success in high-resolution structure determination of G protein-coupled receptors (GPCRs) and to the introduction of commercial instrumentation, tools and protocols. The recent appearance of X-ray free-electron lasers (XFELs) has enabled structure determination from substantially smaller crystals than previously possible with minimal effects of radiation damage, offering new exciting opportunities in structural biology. The unique properties of LCP material have been exploited to develop special protocols and devices that have established a new method of serial femtosecond crystallography of MPs in LCP (LCP-SFX). In this method, microcrystals are generated in LCP and streamed continuously inside the same media across the intersection with a pulsed XFEL beam at a flow rate that can be adjusted to minimize sample consumption. Pioneering studies that yielded the first room temperature GPCR structures, using a few hundred micrograms of purified protein, validate the LCP-SFX approach and make it attractive for structure determination of difficult-to-crystallize MPs and their complexes with interacting partners. Together with the potential of femtosecond data acquisition to interrogate unstable intermediate functional states of MPs, LCP-SFX holds promise to advance our understanding of this biomedically important class of proteins.

scholarly journals Be Cautious with Crystal Structures of Membrane Proteins or Complexes Prepared in Detergents

Crystals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 86 ◽  
Author(s):  
Youzhong Guo
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Crystal Structures ◽  
Structure Determination ◽  
Protein Crystallization ◽  
Energy Coupling ◽  
Careful Examination ◽  
Cubic Phase ◽  
Native Cell ◽  
Membrane Protein Crystallization

Membrane proteins are an important class of macromolecules found in all living organisms and many of them serve as important drug targets. In order to understand their biological and biochemical functions and to exploit them for structure-based drug design, high-resolution and accurate structures of membrane proteins are needed, but are still rarely available, e.g., predominantly from X-ray crystallography, and more recently from single particle cryo-EM — an increasingly powerful tool for membrane protein structure determination. However, while protein-lipid interactions play crucial roles for the structural and functional integrity of membrane proteins, for historical reasons and due to technological limitations, until recently, the primary method for membrane protein crystallization has relied on detergents. Bicelle and lipid cubic phase (LCP) methods have also been used for membrane protein crystallization, but the first step requires detergent extraction of the protein from its native cell membrane. The resulting, crystal structures have been occasionally questioned, but such concerns were generally dismissed as accidents or ignored. However, even a hint of controversy indicates that methodological drawbacks in such structural research may exist. In the absence of caution, structures determined using these methods are often assumed to be correct, which has led to surprising hypotheses for their mechanisms of action. In this communication, several examples of structural studies on membrane proteins or complexes will be discussed: Resistance-Nodulation-Division (RND) family transporters, microbial rhodopsins, Tryptophan-rich Sensory Proteins (TSPO), and Energy-Coupling Factor (ECF) type ABC transporters. These analyses should focus the attention of membrane protein structural biologists on the potential problems in structure determination relying on detergent-based methods. Furthermore, careful examination of membrane proteins in their native cell environments by biochemical and biophysical techniques is warranted, and completely detergent-free systems for membrane protein research are crucially needed.

scholarly journals High-resolution structure determination by continuous-rotation data collection in MicroED

2014 ◽  
Vol 11 (9) ◽  
pp. 927-930 ◽  
Author(s):  
Brent L Nannenga ◽  
Dan Shi ◽  
Andrew G W Leslie ◽  
Tamir Gonen
Keyword(s):  
High Resolution ◽  
Data Collection ◽  
Structure Determination ◽  
Continuous Rotation ◽  
High Resolution Structure ◽  
Resolution Structure

Use of a crystallization robot to set up sitting-drop vapor-diffusion crystallization andin situcrystallization screens

2000 ◽  
Vol 33 (2) ◽  
pp. 344-349 ◽  
Author(s):  
Christopher F. Snook ◽  
Michael D. Purdy ◽  
Michael C. Wiener
Keyword(s):  
Membrane Proteins ◽  
Protein Crystallization ◽  
Integral Membrane Proteins ◽  
Vapor Diffusion ◽  
Liquid Handling ◽  
General Utility ◽  
Crystallization Experiments ◽  
Automated Screening ◽  
Set Up

A commercial crystallization robot has been modified for use in setting up sitting-drop vapor-diffusion crystallization experiments, and for setting up protein crystallization screensin situ. The primary aim of this effort is the automated screening of crystallization of integral membrane proteins in detergent-containing solutions. However, the results of this work are of general utility to robotic liquid-handling systems. Sources of error that can prevent the accurate dispensing and mixing of solutions have been identified, and include local environmental, machine-specific and solution conditions. Solutions to each of these problems have been developed and implemented.

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