scholarly journals Eukaryotic Box C/D methylation machinery has two non-symmetric protein assembly sites

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Simone Höfler ◽  
Peer Lukat ◽  
Wulf Blankenfeldt ◽  
Teresa Carlomagno
Keyword(s):  
Three Dimensional ◽  
Protein Assembly ◽  
Motif Pair ◽  
Mutant Analysis ◽  
X Ray ◽  
Guide Rna ◽  
X Ray Crystallography ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes

AbstractBox C/D ribonucleoprotein complexes are RNA-guided methyltransferases that methylate the ribose 2’-OH of RNA. The central ‘guide RNA’ has box C and D motifs at its ends, which are crucial for activity. Archaeal guide RNAs have a second box C’/D’ motif pair that is also essential for function. This second motif is poorly conserved in eukaryotes and its function is uncertain. Conflicting literature data report that eukaryotic box C’/D’ motifs do or do not bind proteins specialized to recognize box C/D-motifs and are or are not important for function. Despite this uncertainty, the architecture of eukaryotic 2’-O-methylation enzymes is thought to be similar to that of their archaeal counterpart. Here, we use biochemistry, X-ray crystallography and mutant analysis to demonstrate the absence of functional box C’/D’ motifs in more than 80% of yeast guide RNAs. We conclude that eukaryotic Box C/D RNPs have two non-symmetric protein assembly sites and that their three-dimensional architecture differs from that of archaeal 2’-O-methylation enzymes.

3-D reconstruction of molecular shape from microcrystal thin sections

1983 ◽  
Vol 41 ◽  
pp. 748-749
Author(s):  
S. Cusack ◽  
J.-C. Jésior
Keyword(s):  
Three Dimensional ◽  
Molecular Shape ◽  
Biological Molecules ◽  
Fourier Space ◽  
Single Particles ◽  
Thin Sections ◽  
Standard Methods ◽  
X Ray ◽  
X Ray Crystallography ◽  
Dimensional Reconstruction

Three-dimensional reconstruction techniques using electron microscopy have been principally developed for application to 2-D arrays (i.e. monolayers) of biological molecules and symmetrical single particles (e.g. helical viruses). However many biological molecules that crystallise form multilayered microcrystals which are unsuitable for study by either the standard methods of 3-D reconstruction or, because of their size, by X-ray crystallography. The grid sectioning technique enables a number of different projections of such microcrystals to be obtained in well defined directions (e.g. parallel to crystal axes) and poses the problem of how best these projections can be used to reconstruct the packing and shape of the molecules forming the microcrystal.Given sufficient projections there may be enough information to do a crystallographic reconstruction in Fourier space. We however have considered the situation where only a limited number of projections are available, as for example in the case of catalase platelets where three orthogonal and two diagonal projections have been obtained (Fig. 1).

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

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.

scholarly journals Low-Zpolymer sample supports for fixed-target serial femtosecond X-ray crystallography

2015 ◽  
Vol 48 (4) ◽  
pp. 1072-1079 ◽  
Author(s):  
Geoffrey K. Feld ◽  
Michael Heymann ◽  
W. Henry Benner ◽  
Tommaso Pardini ◽  
Ching-Ju Tsai ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Three Dimensional ◽  
Two Dimensional ◽  
X Ray Diffraction ◽  
Fixed Target ◽  
X Ray ◽  
X Ray Crystallography ◽  
Transmission Electron ◽  
Linac Coherent Light Source ◽  
Efficient Alternative

X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introductionviaa translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low-Zplastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. The benefits and limitations of these low-Zfixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.

scholarly journals Serial Electron Diffraction Data Processing With diffractem and CrystFEL

2021 ◽  
Vol 8 ◽  
Author(s):  
Robert Bücker ◽  
Pascal Hogan-Lamarre ◽  
R. J. Dwayne Miller
Keyword(s):  
Electron Diffraction ◽  
Data Processing ◽  
Three Dimensional ◽  
Electron Diffraction Data ◽  
New Techniques ◽  
X Ray ◽  
X Ray Crystallography ◽  
Level Of Automation ◽  
Rotation Method ◽  
High Level

Serial electron diffraction (SerialED) is an emerging technique, which applies the snapshot data-collection mode of serial X-ray crystallography to three-dimensional electron diffraction (3D Electron Diffraction), forgoing the conventional rotation method. Similarly to serial X-ray crystallography, this approach leads to almost complete absence of radiation damage effects even for the most sensitive samples, and allows for a high level of automation. However, SerialED also necessitates new techniques of data processing, which combine existing pipelines for rotation electron diffraction and serial X-ray crystallography with some more particular solutions for challenges arising in SerialED specifically. Here, we introduce our analysis pipeline for SerialED data, and its implementation using the CrystFEL and diffractem program packages. Detailed examples are provided in extensive supplementary code.

scholarly journals An Unexpected Trinuclear Cobalt(II) Complex Based on a Half-Salamo-Like Ligand: Synthesis, Crystal Structure, Hirshfeld Surface Analysis, Antimicrobial and Fluorescent Properties

Crystals ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 408 ◽  
Author(s):  
Ruo-Yan Li ◽  
Xiao-Xin An ◽  
Juan-Li Wu ◽  
You-Peng Zhang ◽  
Wen-Kui Dong
Keyword(s):  
Crystal Structure ◽  
Surface Analysis ◽  
Three Dimensional ◽  
Antimicrobial Activities ◽  
Hirshfeld Surface ◽  
Hirshfeld Surface Analysis ◽  
Fluorescent Properties ◽  
X Ray ◽  
X Ray Crystallography ◽  
Elemental Analyses

An unexpected trinuclear Co(II) complex, [Co3(L2)2(μ-OAc)2(CH3OH)2]·2CH3OH (H2L2 = 4,4′-dibromo-2,2′-[ethylenedioxybis(nitrilomethylidyne)]diphenol) constructed from a half-Salamo-based ligand (HL1 = 2-[O-(1-ethyloxyamide)]oxime-4-bromophenol) and Co(OAc)2·4H2O, has been synthesized and characterized by elemental analyses, infrared spectra (IR), UV-Vis spectra, X-ray crystallography and Hirshfeld surface analysis. The Co(II) complex contains three Co(II) atoms, two completely deprotonated (L2)2− units, two bridged acetate molecules, two coordinated methanol molecules and two crystalline methanol molecules, and finally, a three-dimensional supramolecular structure with infinite extension was formed. Interestingly, during the formation of the Co(II) complex, the ligand changed from half-Salamo-like to a symmetrical single Salamo-like ligand due to the bonding interactions of the molecules. In addition, the antimicrobial activities of HL1 and its Co(II) complex were also investigated.

scholarly journals Molecular Recognition in Proton-Transfer Compounds of Brucine with Achiral Substituted Salicylic Acid Analogues

2006 ◽  
Vol 59 (5) ◽  
pp. 320 ◽  
Author(s):  
Graham Smith ◽  
Urs D. Wermuth ◽  
Peter C. Healy ◽  
Jonathan M. White
Keyword(s):  
Proton Transfer ◽  
Three Dimensional ◽  
Water Molecules ◽  
Sulfosalicylic Acid ◽  
X Ray ◽  
X Ray Crystallography ◽  
Guest Species ◽  
Substituted Benzoic Acids ◽  
Proton Transfer Compounds ◽  
Hydrogen Bonded

The 1:1 proton-transfer brucinium compounds from the reaction of the alkaloid brucine with 5-nitrosalicylic acid, 3,5-dinitrosalicylic acid, and 5-sulfosalicylic acid, namely anhydrous brucinium 5-nitrosalicylate (1), brucinium 3,5-dinitrosalicylate monohydrate (2), and brucinium 5-sulfosalicylate trihydrate (3) have been prepared and their crystal structures determined by X-ray crystallography. All structures further demonstrate the selectivity of brucine for meta-substituted benzoic acids and comprise three-dimensional hydrogen-bonded framework polymers. Two of the compounds (1 and 3) have the previously described undulating brucine sheet host-substructures which incorporate interstitially hydrogen-bonded salicylate anion guest species and additionally in 3 the water molecules of solvation. The structure of 2 differs in having a three-centre brucinium–salicylate anion bidentate N+–H···O(carboxyl) hydrogen-bonding association linking the species through interstitial associations involving also the water molecules of solvation. A review of the crystallographic structural literature on strychnine and brucine is also given.

Teflon tape for laboratory teaching of three-dimensional x-ray crystallography

2018 ◽  
Vol 39 (5) ◽  
pp. 055502 ◽  
Author(s):  
Aitor Larrañaga ◽  
Nestor Goikoetxea ◽  
Mikel Iturrate ◽  
Erlantz Lizundia
Keyword(s):  
Three Dimensional ◽  
X Ray ◽  
X Ray Crystallography ◽  
Laboratory Teaching

Introduction

1986 ◽  
Vol 317 (1540) ◽  
pp. 293-294 ◽  
Keyword(s):  
Direct Method ◽  
Hydrolytic Enzyme ◽  
Hydrolytic Enzymes ◽  
Three Dimensional ◽  
X Ray ◽  
Enzyme Active Site ◽  
X Ray Crystallography ◽  
Protein Molecules ◽  
Biological Catalysis ◽  
The 1960S

Our understanding of the function of protein molecules was revolutionized in the 1960s by the use of X-ray crystallography to give a three-dimensional picture of their structures at atomic resolution. The structure of myoglobin was rapidly followed by the structure of several hydrolytic enzymes such as lysozyme, carboxypeptidase, ribonuclease, chymotrypsin, and subtilisin; and, not long after, by the much more complicated structure of haemoglobin, composed of four myoglobin-like molecules interacting with each other. The first hydrolytic enzyme structures showed us how enzymes perform biological catalysis by immobilizing their substrates at the enzyme active site, and gave us definite ideas about the specific functions of different parts of the protein molecules. These ideas had to be treated as hypotheses, because there was no direct method to check them. A few particular points could be proved by cunning but tedious experiments.

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