scholarly journals What macromolecular crystallogenesis tells us – what is needed in the future

IUCrJ ◽  
2017 ◽  
Vol 4 (4) ◽  
pp. 340-349 ◽  
Author(s):  
Richard Giegé
Keyword(s):  
Self Assembly ◽  
Inorganic Materials ◽  
Biological Processes ◽  
X Ray ◽  
Biologically Relevant ◽  
Cellular Processes ◽  
The Future

Crystallogenesis is a longstanding topic that has transformed into a discipline that is mainly focused on the preparation of crystals for practising crystallographers. Although the idiosyncratic features of proteins have to be taken into account, the crystallization of proteins is governed by the same physics as the crystallization of inorganic materials. At present, a diversified panel of crystallization methods adapted to proteins has been validated, and although only a few methods are in current practice, the success rate of crystallization has increased constantly, leading to the determination of ∼105X-ray structures. These structures reveal a huge repertoire of protein folds, but they only cover a restricted part of macromolecular diversity across the tree of life. In the future, crystals representative of missing structures or that will better document the structural dynamics and functional steps underlying biological processes need to be grown. For the pertinent choice of biologically relevant targets, computer-guided analysis of structural databases is needed. From another perspective, crystallization is a self-assembly process that can occur in the bulk of crowded fluids, with crystals being supramolecular assemblies. Life also uses self-assembly and supramolecular processes leading to transient, or less often stable, complexes. An integrated view of supramolecularity implies that proteins crystallizing eitherin vitroorin vivoor participating in cellular processes share common attributes, notably determinants and antideterminants that favour or disfavour their correct or incorrect associations. As a result, underin vivoconditions proteins show a balance between features that favour or disfavour association. If this balance is broken, disorders/diseases occur. Understanding crystallization underin vivoconditions is a challenge for the future. In this quest, the analysis of packing contacts and contacts within oligomers will be crucial in order to decipher the rules governing protein self-assembly and will guide the engineering of novel biomaterials. In a wider perspective, understanding such contacts will open the route towards supramolecular biology and generalized crystallogenesis.

scholarly journals Structure of the SAS-6 cartwheel hub from Leishmania major

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Mark van Breugel ◽  
Rainer Wilcken ◽  
Stephen H McLaughlin ◽  
Trevor J Rutherford ◽  
Christopher M Johnson
Keyword(s):  
Small Molecules ◽  
Self Assembly ◽  
Leishmania Major ◽  
Cell Organelles ◽  
Cylindrical Cell ◽  
X Ray ◽  
Cilia And Flagella ◽  
Proof Of Principle

Centrioles are cylindrical cell organelles with a ninefold symmetric peripheral microtubule array that is essential to template cilia and flagella. They are built around a central cartwheel assembly that is organized through homo-oligomerization of the centriolar protein SAS-6, but whether SAS-6 self-assembly can dictate cartwheel and thereby centriole symmetry is unclear. Here we show that Leishmania major SAS-6 crystallizes as a 9-fold symmetric cartwheel and provide the X-ray structure of this assembly at a resolution of 3.5 Å. We furthermore demonstrate that oligomerization of Leishmania SAS-6 can be inhibited by a small molecule in vitro and provide indications for its binding site. Our results firmly establish that SAS-6 can impose cartwheel symmetry on its own and indicate how this process might occur mechanistically in vivo. Importantly, our data also provide a proof-of-principle that inhibition of SAS-6 oligomerization by small molecules is feasible.

scholarly journals A microtubule RELION-based pipeline for cryo-EM image processing

2019 ◽  
Author(s):  
Alexander D. Cook ◽  
Szymon W. Manka ◽  
Su Wang ◽  
Carolyn A. Moores ◽  
Joseph Atherton
Keyword(s):  
Electron Microscopy ◽  
Image Processing ◽  
Structural Similarity ◽  
Geometric Lattice ◽  
Contrast Transfer Function ◽  
Biologically Relevant ◽  
Cryo Electron Microscopy

AbstractMicrotubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The determination of the αβ-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.AbbreviationsMiRP, Microtubule RELION-based Pipeline; cryo-EM, cryo-electron microscopy; MT, microtubule; CTF, contrast transfer function; PF, protofilament.

Determination of ruthenium and chromium by X-ray fluorescence spectrometry and the use of inert ruthenium (II) phenanthroline as a solid phase marker in sheep digestion studies

1977 ◽  
Vol 89 (1) ◽  
pp. 17-22 ◽  
Author(s):  
C. C. Evans ◽  
J. C. Macrae ◽  
S. Wilson
Keyword(s):  
Solid Phase ◽  
Quantitative Estimation ◽  
Fluorescence Spectrometry ◽  
Fatty Acid Production ◽  
X Ray ◽  
Coefficients Of Variation ◽  
Lower Limits

SummaryThe quantitative estimation of ruthenium and chromium in faeces and digesta from sheep by X-ray fluorescence spectrometry is described. Estimates of calibration parameters, established by regression analysis, were applicable to a range of matrices. Lower limits of detection of 2·4 mg Ru/kg (solid), 1·8 mg Ru/1 (liquid), 1·6 mg Cr/kg (solid) and 1·1 mg Cr/1 (liquid) and precisions, expressed as coefficients of variation, of 5·4% at 17·7 mg Ru/kg (solid), 5·1% at 182·7 mg Cr/kg (solid) and 1·1% at 152 mg Cr/1 (liquid) were acceptable for the routine analysis of Ru and Cr.The use of inert ruthenium phenanthroline (Ru-P) and inert chromium ethylenediamine tetra-acetic acid (Cr-EDTA) as solid:liquid dual phase markers in digestive physiological studies was evaluated. The specificity of Ru-P for the particulate phase of digesta, established by both in vitro and in vivo experiments, was found to be 92–98%, and in vivo recovery of intraruminally administered Ru-P in faeces was 97–101%. In vitro incubation of rumen liquor showed that concentrations of Ru-P up to 10−4 M had little effect on volatile fatty acid production rates but at 10−3 M there was virtually complete inhibition of acetate and propionate production.Advantages of using inert Ru-P as a solid phase marker are discussed.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

Storage of Human Blood Platelets

1974 ◽  
Vol 32 (02/03) ◽  
pp. 405-416 ◽  
Author(s):  
M. R Hardeman ◽  
Carina J L. Heynens
Keyword(s):  
Storage Temperature ◽  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Storage Period ◽  
Serotonin Uptake ◽  
Hypotonic Shock ◽  
Glycolytic Intermediates

SummaryStorage experiments were performed at 4°, 25° and 37° C with platelet-rich plasma under sterile conditions. In some experiments also the effect of storing platelets at 4° C in whole blood was investigated.Before, during and after three days of storage, the platelets were tested at 37° C for their serotonin uptake and response to hypotonic shock. In addition some glycolytic intermediates were determined.A fair correlation was noticed between the serotonin uptake and hypotonic shock experiments. Both parameters were best maintained at 25° C. Also platelet counting, performed after the storage period, indicated 25° C as the best storage temperature. Determination of glycolytic intermediates did not justify any conclusion regarding the optimal storage temperature. Of the various anticoagulants studied, ACD and heparin gave the best results as to the serotonin uptake and hypotonic shock response, either with fresh or stored platelets. The use of EDTA resulted in the lowest activity, especially after storage.The results of these storage experiments in vitro, correspond well with those in vivo reported in the literature.

SiRNA technology, the gene therapy of the future?

2008 ◽  
Vol 149 (4) ◽  
pp. 153-159 ◽  
Author(s):  
Zsuzsanna Rácz ◽  
Péter Hamar
Keyword(s):  
Gene Therapy ◽  
Short Interfering Rna ◽  
The Future ◽  
Interfering Rna

A genetikában új korszak kezdődött 17 éve, amikor a petúniában felfedezték a koszuppressziót. Később a koszuppressziót azonosították a növényekben és alacsonyabb rendű eukariótákban megfigyelt RNS-interferenciával (RNSi). Bár a növényekben ez ősi vírusellenes gazdaszervezeti védekezőmechanizmus, emlősökben az RNSi élettani szerepe még nincs teljesen tisztázva. Az RNSi-t rövid kettős szálú interferáló RNS-ek (short interfering RNA, siRNS) irányítják. A jelen cikkben összefoglaljuk az RNSi történetét és mechanizmusát, az siRNS-ek szerkezete és hatékonysága közötti összefüggéseket, a célsejtbe való bejuttatás virális és nem virális módjait. Az siRNS-ek klinikai alkalmazásának legfontosabb akadálya az in vivo alkalmazás. Bár a hidrodinamikus kezelés állatokban hatékony, embereknél nem alkalmazható. Lehetőséget jelent viszont a szervspecifikus katéterezés. A szintetizált siRNS-ek ismert mellékhatásait szintén tárgyaljuk. Bár a génterápia ezen új területén számos problémával kell szembenézni, a sikeres in vitro és in vivo kísérletek reményt jelentenek emberi betegségek siRNS-sel történő kezelésére.

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