Looking for Hydrogen Atoms: Neutron Crystallography Provides Novel Insights Into Protein Structure and Function

2014 ◽  
Vol 67 (12) ◽  
pp. 1751 ◽  
Author(s):  
Emily A. Golden ◽  
Alice Vrielink
Keyword(s):  
Protein Structures ◽  
Biological Macromolecules ◽  
Binding Affinities ◽  
Hydrogen Atoms ◽  
Hydronium Ions ◽  
Direct Localization ◽  
Neutron Protein Crystallography ◽  
And Function ◽  
Neutron Crystallography

Neutron crystallography allows direct localization of hydrogen positions in biological macromolecules. Within enzymes, hydrogen atoms play a pivotal role in catalysis. Recent advances in instrumentation and sample preparation have helped to overcome the difficulties of performing neutron diffraction experiments on protein crystals. The application of neutron macromolecular crystallography to a growing number of proteins has yielded novel structural insights. The ability to accurately position water molecules, hydronium ions, and hydrogen atoms within protein structures has helped in the study of low-barrier hydrogen bonds and hydrogen-bonding networks. The determination of protonation states of protein side chains, substrates, and inhibitors in the context of the macromolecule has provided important insights into enzyme chemistry and ligand binding affinities, which can assist in the design of potent therapeutic agents. In this review, we give an overview of the method and highlight advances in knowledge attained through the application of neutron protein crystallography.

A high-performance neutron diffractometer for biological crystallography (BIX-3)

2002 ◽  
Vol 35 (1) ◽  
pp. 34-40 ◽  
Author(s):  
I. Tanaka ◽  
K. Kurihara ◽  
T. Chatake ◽  
N. Niimura
Keyword(s):  
High Performance ◽  
Signal To Noise Ratio ◽  
Neutron Imaging ◽  
Bragg Diffraction ◽  
Imaging Plate ◽  
Biological Macromolecules ◽  
Hydrogen Atoms ◽  
Neutron Diffractometer ◽  
Technical Innovations ◽  
Neutron Protein Crystallography

A high-performance neutron diffractometer for biological crystallography (BIX-3) has been constructed at JRR-3M in the Japan Atomic Energy Research Institute (JAERI) in order to determine the hydrogen-atom positions in biological macromolecules. It uses several recent technical innovations, such as a neutron imaging plate and an elastically bent silicon monochromator developed by the authors. These have made it possible to realise a compact vertical arrangement of the diffractometer. Diffraction data have been collected from the proteins rubredoxin and myoglobin in about one month, to a resolution of 1.5 Å. The data were good enough to identify the hydrogen atoms with high accuracy. By adopting a crystal-step scan method for measuring Bragg diffraction intensities, the signal-to-noise ratio was much better than that of the Laue method. This shows that BIX-3 is one of the best-performing machines for neutron protein crystallography in the world today.

High resolution neutron protein crystallography. Hydrogen and hydration in proteins

2003 ◽  
Vol 218 (2) ◽  
Author(s):  
N. Niimura ◽  
T. Chatake ◽  
A. Ostermann ◽  
K. Kurihara ◽  
I. Tanaka
Keyword(s):  
High Resolution ◽  
Dynamical Behavior ◽  
Protein Crystallography ◽  
Neutron Imaging ◽  
Imaging Plate ◽  
Biological Macromolecules ◽  
Hydrogen Atoms ◽  
Neutron Diffractometer ◽  
Neutron Protein Crystallography ◽  
Acidic Hydrogen

AbstractNeutron diffraction provides an experimental method of directly locating hydrogen atoms in proteins, and the development of the neutron imaging plate (NIP) became a breakthrough event in neutron protein crystallography. The general features of the NIP are reviewed. A high resolution neutron diffractometer dedicated to biological macromolecules (BIX-3) with the NIP has been constructed at Japan Atomic Energy Research Institute and this has enabled 1.5 Å resolution structural analyses of several proteins to be carried out. The specifications of BIX-3 and LADI (a quasi-Laue type diffractometer installed in the Institut Laue-Langevin) are compared. The crystal structures of myoglobin, wild type rubredoxin and a mutant of rubredoxin have been carried out using BIX-3. From these studies, several topics, such as the location of hydrogen bonds and certain acidic hydrogen atoms, the identification of methyl hydrogen atoms, details of H/D exchange and dynamical behavior of hydration structures have been investigated, and important information has been extracted from the structural results. Finally, a systematic procedure to grow large single crystals of proteins or nucleic acids is described.

scholarly journals Programming conventional electron microscopes for solving ultrahigh-resolution structures of small and macro-molecules

2019 ◽  
Author(s):  
Heng zhou ◽  
Feng Luo ◽  
Zhipu Luo ◽  
Dan Li ◽  
Cong Liu ◽  
...  
Keyword(s):  
Organic Compounds ◽  
Structure Determination ◽  
Low Cost ◽  
Ccd Camera ◽  
Biological Macromolecules ◽  
Hydrogen Atoms ◽  
Ultrahigh Resolution ◽  
Electron Microscopes ◽  
Camera Synchronization

AbstractMicrocrystal electron diffraction (MicroED) is becoming a powerful tool in determining the crystal structures of biological macromolecules and small organic compounds. However, wide applications of this technique are still limited by the special requirement for radiation-tolerated movie-mode camera and the lacking of automated data collection method. Herein, we develop a stage-camera synchronization scheme to minimize the hardware requirements and enable the use of the conventional electron cryo-microscope with single-frame CCD camera, which ensures not only the acquisition of ultrahigh-resolution diffraction data but also low cost in practice. This method renders the structure determination of both peptide and small organic compounds at ultrahigh resolution up to ~0.60 Å with unambiguous assignment of nearly all hydrogen atoms. The present work provides a widely applicable solution for routine structure determination of MicroED, and demonstrates the capability of the low-end 120kV microscope with a CCD camera in solving ultra-high resolution structures of both organic compound and biological macromolecules.

scholarly journals Perdeuteration: Vital to visualising solvent in neutron crystallography?

2014 ◽  
Vol 70 (a1) ◽  
pp. C1208-C1208
Author(s):  
Stuart Fisher ◽  
Matthew Blakeley ◽  
Eduardo Howard ◽  
Isabelle Petit-Haertlein ◽  
Michael Haertlein ◽  
...  
Keyword(s):  
Water Molecules ◽  
Nuclear Density ◽  
Labile Hydrogen ◽  
Hydrogen Atoms ◽  
High Quality ◽  
Density Maps ◽  
Unit Cells ◽  
Solvent Molecules ◽  
Neutron Protein Crystallography ◽  
Neutron Crystallography

Early neutron crystallography studies replaced hydrogen with deuterium by soaking the crystal in heavy water prior to data collection, which exchanged labile hydrogen atoms (OH, NH, and SH) and solvent molecules only. Carbon bonded hydrogen atoms were not replaced, and their negative scattering density resulted in cancellation in nuclear density maps with resolution worse than 1.8 Å. Furthermore complications arise due to partial exchange, where deuterium is present in some unit cells and hydrogen in others. More recently it has become possible to completely replace hydrogen with deuterium through expression in a deuterated medium, using facilities such as the Deuteration Laboratory (DLAB) in Grenoble. As this is a complex and expensive task, the question arises as to the importance of its use. As well as allowing the study of radically smaller crystals (<0.05mm3), it also has the possibility to avoid the cancellation problems discussed above. We have obtained data from high quality crystals of partially hydrogenated type III antifreeze protein, where methyl protonated valine and leucine residues were incorporated into the perdeuterated protein. This provides an excellent opportunity to assess the effects of negative scattering from hydrogen atoms not only on the visibility of neighbouring carbon atoms but also on water molecules in close vicinity. The observation of these cancellation effects gives a further reason to use full deuteration in neutron protein crystallography.

Membrane protein crystallography in the era of modern structural biology

2020 ◽  
Vol 48 (6) ◽  
pp. 2505-2524
Author(s):  
Tristan O. C. Kwan ◽  
Danny Axford ◽  
Isabel Moraes
Keyword(s):  
Structural Biology ◽  
Molecular Level ◽  
Protein Structures ◽  
Three Dimensional ◽  
Biological Macromolecules ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cellular Processes ◽  
Biochemical Level

The aim of structural biology has been always the study of biological macromolecules structures and their mechanistic behaviour at molecular level. To achieve its goal, multiple biophysical methods and approaches have become part of the structural biology toolbox. Considered as one of the pillars of structural biology, X-ray crystallography has been the most successful method for solving three-dimensional protein structures at atomic level to date. It is however limited by the success in obtaining well-ordered protein crystals that diffract at high resolution. This is especially true for challenging targets such as membrane proteins (MPs). Understanding structure-function relationships of MPs at the biochemical level is vital for medicine and drug discovery as they play critical roles in many cellular processes. Though difficult, structure determination of MPs by X-ray crystallography has significantly improved in the last two decades, mainly due to many relevant technological and methodological developments. Today, numerous MP crystal structures have been solved, revealing many of their mechanisms of action. Yet the field of structural biology has also been through significant technological breakthroughs in recent years, particularly in the fields of single particle electron microscopy (cryo-EM) and X-ray free electron lasers (XFELs). Here we summarise the most important advancements in the field of MP crystallography and the significance of these developments in the present era of modern structural biology.

scholarly journals A beginner’s guide to neutron macromolecular crystallography

2020 ◽  
Vol 42 (6) ◽  
pp. 16-20
Author(s):  
Flora Meilleur
Keyword(s):  
Three Dimensional ◽  
Structure And Function ◽  
Macromolecular Crystallography ◽  
Biological Structure ◽  
Hydrogen Atoms ◽  
Neutron Data ◽  
Biochemical Processes ◽  
And Function ◽  
Neutron Crystallography

Hydrogen atoms drive biological structure and function, but the lightest element is often unseen in three-dimensional macromolecule structures, hampering our understanding of biochemical processes. This guide will i) present how neutron crystallography uniquely reveals the experimental positions of hydrogen atoms and resolves mechanical controversies, ii) briefly introduce beamlines at neutron facilities, iii) discuss sample requirements and preparation and iv) familiarize the reader with neutron data and refinement statistics.

Conformation of Ribosomes from Escherichia Coli

1974 ◽  
Vol 32 ◽  
pp. 210-211
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Fluorescent Dyes ◽  
Protein Biosynthesis ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Complete Understanding ◽  
And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

Concentration determination of chromatin in unstained resin-embedded sections by means of Z-contrast in STEM

1990 ◽  
Vol 48 (2) ◽  
pp. 186-187
Author(s):  
M. Haider ◽  
B. Bohrmann
Keyword(s):  
Energy Loss ◽  
Freeze Substitution ◽  
Biological Macromolecules ◽  
Local Concentration ◽  
E Coli ◽  
Concentration Determination ◽  
Phosphorous Content ◽  
Z Contrast ◽  
Electron Energy Loss

The technique of Z-contrast in STEM offers the possibility to determine the local concentration of macromolecules like lipids, proteins or DNA. Contrast formation depends on the atomic composition of the particular structure. In the case of DNA, its phosphorous content discriminates it from other biological macromolecules. In our studies, sections of E. coli, the dinoflagellate Amphidinium carterae and Euglena spec. cells were used which were obtained by cryofixation followed by freeze-substitution into acetone with 3% glutaraldehyde. The samples were then embedded either in Lowicryl HM20 at low temperature or in Epon at high temperature. Sections were coated on both sides with 30Å carbon.The DF- and the inelastic image have been recorded simultaneously with a Cryo-STEM. This Cryo-STEM is equipped with a highly dispersive Electron Energy Loss Spectrometer. With this instrument pure Z-contrast can be achieved either with a Filtered DF-image divided by the inelastic image or, as is used in this paper, by dividing the conventional DF-image by an inelastic image which has been recorded with an inelastic detector whose response is dependent on the total energy loss of the inelastically scattered electrons.

Depletion and Reconstitution of Active E. Coli Ribosomes

1975 ◽  
Vol 33 ◽  
pp. 640-641
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Protein Biosynthesis ◽  
Large Subunit ◽  
High Resolution Electron Microscopy ◽  
Small Subunit ◽  
Structure And Function ◽  
Biologically Active ◽  
Biological Macromolecules ◽  
E Coli ◽  
Resolution Electron ◽  
And Function

Correlations between structure and function of biological macromolecules have been studied intensively for many years, mostly by indirect methods. High resolution electron microscopy is a unique tool which can provide such information directly by comparing the conformation of biopolymers in their biologically active and inactive state. We have correlated the structure and function of ribosomes, ribonucleoprotein particles which are the site of protein biosynthesis. 70S E. coli ribosomes, used in this experiment, are composed of two subunits - large (50S) and small (30S). The large subunit consists of 34 proteins and two different ribonucleic acid molecules. The small subunit contains 21 proteins and one RNA molecule. All proteins (with the exception of L7 and L12) are present in one copy per ribosome.This study deals with the changes in the fine structure of E. coli ribosomes depleted of proteins L7 and L12. These proteins are unique in many aspects.

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