scholarly journals Structure determination of a membrane protein with data collected from micro-crystals in lipidic cubic phase at room temperature in low-background CrystalDirect crystallization plates

2016 ◽  
Vol 72 (a1) ◽  
pp. s15-s16
Author(s):  
Thomas R. Schneider ◽  
Gleb Bourenkov ◽  
Maria Martinez-Molledo ◽  
Ivars Karpics ◽  
Esben M. Quistgaard ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Structure Determination ◽  
Room Temperature ◽  
Cubic Phase ◽  
Low Background ◽  
Lipidic Cubic Phase

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 Structure determination of an integral membrane protein at room temperature from crystalsin situ

2015 ◽  
Vol 71 (6) ◽  
pp. 1228-1237 ◽  
Author(s):  
Danny Axford ◽  
James Foadi ◽  
Nien-Jen Hu ◽  
Hassanul Ghani Choudhury ◽  
So Iwata ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Structure Determination ◽  
Room Temperature ◽  
Protein Structures ◽  
Integral Membrane Protein ◽  
Data Sets ◽  
X Ray Diffraction ◽  
Haemophilus Influenza

The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystalsin situeliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination ofHaemophilus influenzaTehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines.

Synthesis and Structure of the Binuclear 1 : 1 Silver(I) 2-Hydroxy-3,5-dinitrobenzoate/Triphenylphosphine Complex

2003 ◽  
Vol 56 (7) ◽  
pp. 718
Author(s):  
A. Hamid bin Othman ◽  
Brian W. Skelton ◽  
Allan H. White
Keyword(s):  
Structure Determination ◽  
Room Temperature ◽  
The Other ◽  
Triphenyl Phosphine ◽  
X Ray ◽  
Carboxylate Oxygen ◽  
Phenolic Oxygen ◽  
Triphenylphosphine Complex ◽  
Silver Atoms

A room-temperature single-crystal X-ray structure determination of the 1 : 1 adduct of silver(I) 2-hydroxy-3,5-dinitrobenzoate/triphenyl-phosphine (AgL/PPh3) was recorded, showing it to be a binuclear centrosymmetric system with the silver atoms bridged by one of the carboxy oxygen atoms of each ligand, [(PPh3)Ag(μ-O)2Ag(PPh3)] as in the parent acetate; the phenolic oxygen, retaining its protonation, is hydrogen bonded within the ligand to the other feebly chelating carboxylate oxygen.

scholarly journals Ab initio structure determination of nanocrystals of organic pharmaceutical compounds by electron diffraction at room temperature using a Timepix quantum area direct electron detector. Corrigendum

2018 ◽  
Vol 74 (6) ◽  
pp. 709-709 ◽  
Author(s):  
E. van Genderen ◽  
M. T. B. Clabbers ◽  
P. P. Das ◽  
A. Stewart ◽  
I. Nederlof ◽  
...  
Keyword(s):  
Electron Diffraction ◽  
Ab Initio ◽  
Structure Determination ◽  
Room Temperature ◽  
Pharmaceutical Compounds ◽  
Acta Cryst ◽  
Electron Detector ◽  
Ab Initio Structure

Corrections are made to Table 1 in the article by van Genderen et al. [Acta Cryst. (2016), A72, 236–242].

scholarly journals A Comparison of Structure Determination of Small Organic Molecules by 3D Electron Diffraction at Cryogenic and Room Temperature

Symmetry ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2131
Author(s):  
Taimin Yang ◽  
Steve Waitschat ◽  
Andrew Kentaro Inge ◽  
Norbert Stock ◽  
Xiaodong Zou ◽  
...  
Keyword(s):  
Electron Diffraction ◽  
Data Collection ◽  
Data Quality ◽  
Structure Determination ◽  
Room Temperature ◽  
Organic Molecules ◽  
Small Organic Molecules ◽  
3D Electron ◽  
Cryogenic Conditions

3D electron diffraction (3D ED), also known as micro-crystal electron diffraction (MicroED), is a rapid, accurate, and robust method for structure determination of submicron-sized crystals. 3D ED has mainly been applied in material science until 2013, when MicroED was developed for studying macromolecular crystals. MicroED was considered as a cryo-electron microscopy method, as MicroED data collection is usually carried out in cryogenic conditions. As a result, some researchers may consider that 3D ED/MicroED data collection on crystals of small organic molecules can only be performed in cryogenic conditions. In this work, we determined the structure for sucrose and azobenzene tetracarboxylic acid (H4ABTC). The structure of H4ABTC is the first crystal structure ever reported for this molecule. We compared data quality and structure accuracy among datasets collected under cryogenic conditions and room temperature. With the improvement in data quality by data merging, it is possible to reveal hydrogen atom positions in small organic molecule structures under both temperature conditions. The experimental results showed that, if the sample is stable in the vacuum environment of a transmission electron microscope (TEM), the data quality of datasets collected under room temperature is at least as good as data collected under cryogenic conditions according to various indicators (resolution, I/σ(I), CC1/2 (%), R1, Rint, ADRA).

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.

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