scholarly journals A new cryo-EM system for electron 3D crystallography by eEFD

2019 ◽  
Author(s):  
Koji Yonekura ◽  
Tetsuya Ishikawa ◽  
Saori Maki-Yonekura
Keyword(s):  
Electron Beam ◽  
Data Collection ◽  
Membrane Protein ◽  
Energy Loss ◽  
Protein Complexes ◽  
Efficient Operation ◽  
Membrane Protein Complex ◽  
Precise Control ◽  
First Results ◽  
Multiple Samples

AbstractA new cryo-EM system has been developed and investigated for use in protein electron 3D crystallography. The system provides parallel illumination of a coherent 300 kV electron beam to a sample, filters out energy-loss electrons through the sample with an in-column energy filter, and allows rotational data collection on a fast camera. It also possesses motorized cryo-sample loading and automated liquid-nitrogen filling for cooling of multiple samples. To facilitate its use, we developed GUI programs for efficient operation and accurate structure analysis. Here we report on the performance of the system and first results for thin 3D crystals of the protein complexes, catalase and membrane protein complex ExbBD. Data quality is remarkably improved with this approach, which we name eEFD (electron energy-filtered diffraction of 3D crystals), compared with those collected at 200 kV without energy filtration. Key advances include precise control of the microscope and recordings of lens fluctuations, which the programs process and respond to. We also discuss the merits of higher-energy electrons and filtration of energy-loss electrons in electron 3D crystallography.

scholarly journals Membrane protein megahertz crystallography at the European XFEL

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Chris Gisriel ◽  
Jesse Coe ◽  
Romain Letrun ◽  
Oleksandr M. Yefanov ◽  
Cesar Luna-Chavez ◽  
...  
Keyword(s):  
Data Collection ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Membrane Protein Complex ◽  
Unit Cell Size ◽  
Data Collection And Analysis ◽  
Rapid Pulse ◽  
Membrane Protein Complexes ◽  
Increasing Demand

Abstract The world’s first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs.

scholarly journals A Genetically Encoded Trimethylsilyl 1D 1H-NMR Probe for Conformation Change in Large Membrane Protein Complexes

2019 ◽  
Author(s):  
Qi Liu ◽  
Qing-tao He ◽  
Xiao-xuan Lyu ◽  
Fan Yang ◽  
Zhong-liang Zhu ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Complex ◽  
Protein Complexes ◽  
Signal To Noise Ratio ◽  
Conformation Change ◽  
Membrane Protein Complex ◽  
H Nmr ◽  
Highly Efficient ◽  
Membrane Protein Complexes

AbstractWhile one dimensional 1H nuclear magnetic resonance (1D 1H-NMR) spectroscopy is one of the most important and convenient method for measuring conformation change in biomacromolecules, characterization of protein dynamics in large membrane protein complexes by 1D 1H-NMR remains challenging, due to the difficulty of spectra assignment, low signal-to-noise ratio (S/N) and the need for large amount of protein. Here we report the site-specific incorporation of 4-trimethylsilyl phenylalanine (TMSiPhe) into proteins, through genetic code expansion in Escherichia coli cells, and the measurement of multiple conformational states in membrane protein complex by 1D 1H-NMR. The unique up-field 1H-NMR chemical shift of TMSiPhe, highly efficient and specific incorporation of TMSiPhe enabled facile assignment of the TMSiPhe 1H-NMR signal, and characterization of multiple conformational state in a 150 kilodalton (kD) membrane protein complex, using only 5 μM of protein and 20 min spectra accumulation time. This highly efficient and convenient methods should be broadly applicable for the investigation of dynamic conformation change of protein complexes.

Probing the Chemistry and Spectroscopy of Radiation-Sensitive Polymers by Parallel-Detection EELS

1990 ◽  
Vol 48 (2) ◽  
pp. 34-35
Author(s):  
E. G. Rightor ◽  
G. P. Young
Keyword(s):  
Electron Beam ◽  
Bisphenol A ◽  
Energy Loss ◽  
Parallel Detection ◽  
Commercial Grade ◽  
Incident Electron Beam ◽  
Ptfe Sample ◽  
Spectroscopic Information ◽  
Edge Features ◽  
Electron Energy Loss

Investigation of neat polymers by TEM is often thwarted by their sensitivity to the incident electron beam, which also limits the usefulness of chemical and spectroscopic information available by electron energy loss spectroscopy (EELS) for these materials. However, parallel-detection EELS systems allow reduced radiation damage, due to their far greater efficiency, thereby promoting their use to obtain this information for polymers. This is evident in qualitative identification of beam sensitive components in polymer blends and detailed investigations of near-edge features of homopolymers.Spectra were obtained for a poly(bisphenol-A carbonate) (BPAC) blend containing poly(tetrafluoroethylene) (PTFE) using a parallel-EELS and a serial-EELS (Gatan 666, 607) for comparison. A series of homopolymers was also examined using parallel-EELS on a JEOL 2000FX TEM employing a LaB6 filament at 100 kV. Pure homopolymers were obtained from Scientific Polymer Products. The PTFE sample was commercial grade. Polymers were microtomed on a Reichert-Jung Ultracut E and placed on holey carbon grids.

Electron beam induced current study of thin silicon layers on insulator substrate

1990 ◽  
Vol 48 (4) ◽  
pp. 618-619
Author(s):  
A. Buczkowski ◽  
Z. J. Radzimski ◽  
J. C. Russ ◽  
G. A. Rozgonyi
Keyword(s):  
Electron Beam ◽  
Energy Loss ◽  
Beam Energy ◽  
Collection Efficiency ◽  
Silicon Layer ◽  
Depletion Layer ◽  
Incident Electron Energy ◽  
Induced Current ◽  
Electron Hole ◽  
Electron Beam Induced Current

If a thickness of a semiconductor is smaller than the penetration depth of the electron beam, e.g. in silicon on insulator (SOI) structures, only a small portion of incident electrons energy , which is lost in a superficial silicon layer separated by the oxide from the substrate, contributes to the electron beam induced current (EBIC). Because the energy loss distribution of primary beam is not uniform and varies with beam energy, it is not straightforward to predict the optimum conditions for using this technique. Moreover, the energy losses in an ohmic or Schottky contact complicate this prediction. None of the existing theories, which are based on an assumption of a point-like region of electron beam generation, can be used satisfactorily on SOI structures. We have used a Monte Carlo technique which provide a simulation of the electron beam interactions with thin multilayer structures. The EBIC current was calculated using a simple one dimensional geometry, i.e. depletion layer separating electron- hole pairs spreads out to infinity in x- and y-direction. A point-type generation function with location being an actual location of an incident electron energy loss event has been assumed. A collection efficiency of electron-hole pairs was assumed to be 100% for carriers generated within the depletion layer, and inversely proportional to the exponential function of depth with the effective diffusion length as a parameter outside this layer. A series of simulations were performed for various thicknesses of superficial silicon layer. The geometries used for simulations were chosen to match the "real" samples used in the experimental part of this work. The theoretical data presented in Fig. 1 show how significandy the gain decreases with a decrease in superficial layer thickness in comparison with bulk material. Moreover, there is an optimum beam energy at which the gain reaches its maximum value for particular silicon thickness.

scholarly journals Cobamide-containing membrane protein complex inMethanobacterium

FEBS Letters ◽  
1986 ◽  
Vol 198 (2) ◽  
pp. 279-282 ◽  
Author(s):  
Harald Schulz ◽  
Georg Fuchs
Keyword(s):  
Membrane Protein ◽  
Protein Complex ◽  
Membrane Protein Complex

Conformational Flexibility in Assembly of Photosynthetic Membrane Protein Complexes in vivo

2004 ◽  
Vol 10 (S02) ◽  
pp. 1496-1497
Author(s):  
P A Bullough
Keyword(s):  
Membrane Protein ◽  
Protein Complexes ◽  
Conformational Flexibility ◽  
Photosynthetic Membrane ◽  
Membrane Protein Complexes

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

Three-dimensional crystallization of membrane proteins

1988 ◽  
Vol 21 (4) ◽  
pp. 429-477 ◽  
Author(s):  
W. Kühlbrandt
Keyword(s):  
High Resolution ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Membrane Protein Complexes ◽  
Essential Prerequisite

As recently as 10 years ago, the prospect of solving the structure of any membrane protein by X-ray crystallography seemed remote. Since then, the threedimensional (3-D) structures of two membrane protein complexes, the bacterial photosynthetic reaction centres of Rhodopseudomonas viridis (Deisenhofer et al. 1984, 1985) and of Rhodobacter sphaeroides (Allen et al. 1986, 1987 a, 6; Chang et al. 1986) have been determined at high resolution. This astonishing progress would not have been possible without the pioneering work of Michel and Garavito who first succeeded in growing 3-D crystals of the membrane proteins bacteriorhodopsin (Michel & Oesterhelt, 1980) and matrix porin (Garavito & Rosenbusch, 1980). X-ray crystallography is still the only routine method for determining the 3-D structures of biological macromolecules at high resolution and well-ordered 3-D crystals of sufficient size are the essential prerequisite.

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