Harnessing SAXS and X-ray crystallography for high-resolution structural studies of macromolecules

Proteolytic digestion of the immunoglobulin IgG with papain cleaves the molecule into an antigen binding fragment, Fab, and a compliment binding fragment, Fc. Structures of intact immunoglobulin, Fab and Fc from various sources have been solved by X-ray crystallography. Rabbit Fc can be crystallized as thin platelets suitable for high resolution electron microscopy. The structure of rabbit Fc can be expected to be similar to the known structure of human Fc, making it an ideal specimen for comparing the X-ray and electron crystallographic techniques and for the application of the molecular replacement technique to electron crystallography. Thin protein crystals embedded in ice diffract to high resolution. A low resolution image of a frozen, hydrated crystal can be expected to have a better contrast than a glucose embedded crystal due to the larger density difference between protein and ice compared to protein and glucose. For these reasons we are using an ice embedding technique to prepare the rabbit Fc crystals for molecular structure analysis by electron microscopy.

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Synthesis, Characterisation and Structural Studies of [Zn(phen)3][Fe(CN)5(NO)]·2H2O·0.25MeOH and [(bipy)2(H2O)Zn(μ-NC)Fe(CN)4(NO)]·0.5H2O

Zeitschrift für Naturforschung B ◽

10.1515/znb-2003-0913 ◽

2003 ◽

Vol 58 (9) ◽

pp. 916-921 ◽

Cited By ~ 3

Author(s):

Amitabha Datta ◽

Samiran Mitra ◽

Georgina Rosair

Keyword(s):

Infrared Spectroscopy ◽

Thermogravimetric Analysis ◽

Octahedral Geometry ◽

Distorted Octahedral Geometry ◽

Structural Studies ◽

Crystal Lattices ◽

X Ray ◽

X Ray Crystallography ◽

Distorted Octahedral ◽

Solvent Molecules

Two new bimetallic complexes [Zn(phen)3][Fe(CN)5(NO)] · 2 H2O · 0.25 MeOH, (1) and [(bipy)2(H2O)Zn(μ-NC)Fe(CN)4(NO)] · 0.5 H2O, (2), have been isolated (where phen = 1,10-phenanthroline and bipy = bipyridyl) and characterised by X-ray crystallography [as the 2 H2O · 0.25 CH3OH solvate for (1) and hemihydrate for (2)] infrared spectroscopy and thermogravimetric analysis. Substitution of phenanthroline for bipyridyl resulted in a cyano-bridged bimetallic species rather than two discrete mononuclear metal complexes. The bond angles of Fe-N-O were shown to be practically linear for both 1 [179.2(7)°] and 2 [178.3(3)°], and the Zn atoms have distorted octahedral geometry. The solvent molecules in both crystal lattices take part in forming hydrogen-bonded networks.

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Complementary Use of Electron Cryomicroscopy and X-Ray Crystallography: Structural Studies of Actin and Actomyosin Filaments

Advances in Experimental Medicine and Biology - Integrative Structural Biology with Hybrid Methods ◽

10.1007/978-981-13-2200-6_4 ◽

2018 ◽

pp. 25-42

Author(s):

Takashi Fujii ◽

Keiichi Namba

Keyword(s):

Structural Studies ◽

Electron Cryomicroscopy ◽

X Ray ◽

X Ray Crystallography

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Three-dimensional crystallization of membrane proteins

Quarterly Reviews of Biophysics ◽

10.1017/s0033583500004625 ◽

1988 ◽

Vol 21 (4) ◽

pp. 429-477 ◽

Cited By ~ 156

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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The fluoride permeation pathway and anion recognition in Fluc family fluoride channels

eLife ◽

10.7554/elife.69482 ◽

2021 ◽

Vol 10 ◽

Author(s):

Benjamin C McIlwain ◽

Roja Gundepudi ◽

B Ben Koff ◽

Randy B Stockbridge

Keyword(s):

Binding Sites ◽

Anion Recognition ◽

Anion Binding ◽

Structural Studies ◽

Planar Bilayer ◽

X Ray ◽

X Ray Crystallography ◽

Molecular Determinants ◽

Cytoplasmic Accumulation ◽

Oriented System

Fluc family fluoride channels protect microbes against ambient environmental fluoride by undermining the cytoplasmic accumulation of this toxic halide. These proteins are structurally idiosyncratic, and thus the permeation pathway and mechanism have no analogy in other known ion channels. Although fluoride binding sites were identified in previous structural studies, it was not evident how these ions access aqueous solution, and the molecular determinants of anion recognition and selectivity have not been elucidated. Using x-ray crystallography, planar bilayer electrophysiology and liposome-based assays, we identify additional binding sites along the permeation pathway. We use this information to develop an oriented system for planar lipid bilayer electrophysiology and observe anion block at one of these sites, revealing insights into the mechanism of anion recognition. We propose a permeation mechanism involving alternating occupancy of anion binding sites that are fully assembled only as the substrate approaches.

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Mechanism of IPPase shown by high resolution Neutron and X-ray crystallography

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314087889 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1211-C1211

Author(s):

Joseph Ng ◽

Ronny Hughes ◽

Michelle Morris ◽

Leighton Coates ◽

Matthew Blakeley ◽

...

Keyword(s):

High Resolution ◽

Active Site ◽

Inorganic Pyrophosphatase ◽

Inorganic Pyrophosphate ◽

X Ray ◽

X Ray Crystallography ◽

Cellular Processes ◽

Crystallographic Structures ◽

Hydrolysis Of ◽

Low Energy Conformations

Soluble inorganic pyrophosphatase (IPPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to form orthophosphate (Pi). The action of this enzyme shifts the overall equilibrium in favor of synthesis during a number of ATP-dependent cellular processes such as in the polymerization of nucleic acids, production of coenzymes and proteins and sulfate assimilation pathways. Two Neutron crystallographic (2.10-2.50Å) and five high-resolution X-ray (0.99Å-1.92Å) structures of the archaeal IPPase from Thermococcus thioreducens have been determined under both cryo and room temperatures. The structures determined include the recombinant IPPase bound to Mg+2, Ca+2, Br-, SO2-2 or PO4-2 involving those with non-hydrolyzed and hydrolyzed pyrophosphate complexes. All the crystallographic structures provide snapshots of the active site corresponding to different stages of the hydrolysis of inorganic pyrophosphate. As a result, a structure-based model of IPPase catalysis is devised showing the enzyme's low-energy conformations, hydration states, movements and nucleophile generation within the active site.

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Синтез, структура и магнитные свойства многослойных наногетероструктур Fe|MgO|Cr|MgO|Fe

Физика твердого тела ◽

10.21883/ftt.2021.09.51256.21h ◽

2021 ◽

Vol 63 (9) ◽

pp. 1306

Author(s):

Е.М. Якунина ◽

Е.А. Кравцов ◽

Yu.N. Khaydukov ◽

Н.О. Антропов ◽

В.В. Проглядо

Keyword(s):

Magnetic Properties ◽

High Resolution ◽

Exchange Interaction ◽

Magnetic Moments ◽

Ferromagnetic Material ◽

Structural Studies ◽

X Ray ◽

Structural And Magnetic Properties ◽

Metal Layers

Layered nanoheterostructures Fe | MgO | Cr | MgO | Fe is an artificial ferromagnetic material in which the exchange interaction of the magnetic moments of Fe layers through intermediate dielectric and metal layers can lead to magnetic configurations that are not realized in the well-studied Fe | MgO | Fe and Fe | Cr | Fe. In this work, we investigated the correlations of the structural and magnetic properties of layered nanoheterostructures Fe (10 nm) | MgO (1.5 nm) | Cr (t nm) | MgO (1.5 nm) | Fe (7 nm) (thickness t = 0.9 and 1.8 nm). Structural studies performed using X-ray diffractometry and high-resolution reflectometry confirmed the formation of a textured structure and revealed its well-defined layered character with sharp interlayer boundaries.

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Homogeneously N-glycosylated proteins derived from the GlycoDelete HEK293 cell line enable diffraction-quality crystallogenesis

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798320013753 ◽

2020 ◽

Vol 76 (12) ◽

pp. 1244-1255

Author(s):

Sandra Kozak ◽

Yehudi Bloch ◽

Steven De Munck ◽

Aleksandra Mikula ◽

Isabel Bento ◽

...

Keyword(s):

Cell Line ◽

Protein Interactions ◽

Successful Implementation ◽

Structural Studies ◽

Protein Fragments ◽

X Ray ◽

X Ray Crystallography ◽

Cryo Electron Microscopy ◽

Acid Protein ◽

Stimulating Factor

Structural studies of glycoproteins and their complexes provide critical insights into their roles in normal physiology and disease. Most glycoproteins contain N-linked glycosylation, a key post-translation modification that critically affects protein folding and stability and the binding kinetics underlying protein interactions. However, N-linked glycosylation is often an impediment to yielding homogeneous protein preparations for structure determination by X-ray crystallography or other methods. In particular, obtaining diffraction-quality crystals of such proteins and their complexes often requires modification of both the type of glycosylation patterns and their extent. Here, we demonstrate the benefits of producing target glycoproteins in the GlycoDelete human embryonic kidney 293 cell line that has been engineered to produce N-glycans as short glycan stumps comprising N-acetylglucosamine, galactose and sialic acid. Protein fragments of human Down syndrome cell-adhesion molecule and colony-stimulating factor 1 receptor were obtained from the GlycoDelete cell line for crystallization. The ensuing reduction in the extent and complexity of N-glycosylation in both protein molecules compared with alternative glycoengineering approaches enabled their productive deployment in structural studies by X-ray crystallography. Furthermore, a third successful implementation of the GlycoDelete technology focusing on murine IL-12B is shown to lead to N-glycosylation featuring an immature glycan in diffraction-quality crystals. It is proposed that the GlycoDelete cell line could serve as a valuable go-to option for the production of homogeneous glycoproteins and their complexes for structural studies by X-ray crystallography and cryo-electron microscopy.

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Synthesis and characterization of new tetra-substituted porphyrins with exo-donor carboxylic groups as building blocks for supramolecular architectures: Catalytic and structural studies of their metalated derivatives

Journal of Porphyrins and Phthalocyanines ◽

10.1142/s1088424610002641 ◽

2010 ◽

Vol 14 (09) ◽

pp. 804-814 ◽

Cited By ~ 4

Author(s):

Lucia Carlucci ◽

Gianfranco Ciani ◽

Simona Maggini ◽

Davide M. Proserpio ◽

Fabio Ragaini ◽

...

Keyword(s):

Building Blocks ◽

Supramolecular Organization ◽

Silver Complexes ◽

Structural Studies ◽

Polymeric Compound ◽

X Ray ◽

X Ray Crystallography ◽

Supramolecular Architectures ◽

Carboxylic Groups

We report herein the synthesis of the porphyrins 5,10,15,20-tetrakis(4-carboxybiphenyl)-porphyrin (H2TCBP) and 5,10,15,20-tetrakis(4-carboxy-2,6-dimethylbiphenyl)porphyrin (H2TCDMBP) bearing diphenyl units on meso-positions, and of their cobalt and silver derivatives. The silver complexes of H2TCDMBP and of H2TCPP ( H2TCPP = 5 ,10,15,20-tetrakis(4-carboxyphenyl)porphyrin) were investigated by X-ray crystallography and their supramolecular organization elucidated. Co(TCBP) was reacted with copper formate, yielding a polymeric compound that showed a catalytic activity in the benzylic amination of hydrocarbons using arylazide as aminating agent.

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