Electron Crystallographic Structure of the Limulus Acrosomal Bundle at 20 Å Resolution

Limulus sperm contains a dynamic macromolecular structure that rapidly extends a 50-μum process called the true discharge. The core of this structure is a bundle of ordered filaments composed of a complex of actin, scruin and calmodulin. We have shown that small segments along the bundle can be treated as single crystals with a unit cell spacing of 144 × 144 × 766 Å. A tomographic reconstruction of the bundle was done from multiple tilt series of images to ∼40 Å resolution. To extend the structural determination of the bundle at a higher resolution, we have used electron crystallographic analysis of single images of the bundles preserved in vitreous ice. Furthermore, we did not employ any helical or crystallographic symmetry (other than PI) in the reconstruction procedure.Acrosomal bundles from Limulus sperm cells were purified as described earlier. Images of frozen hydrated bundles were taken at 40,000x EM magnification in a JEOL 4000EX electron cryomicroscope using an electron dose of 6 - 10 electrons/Å2.

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Three-dimensional electron crystallography of protein microcrystals

eLife ◽

10.7554/elife.01345 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 186

Author(s):

Dan Shi ◽

Brent L Nannenga ◽

Matthew G Iadanza ◽

Tamir Gonen

Keyword(s):

Electron Diffraction ◽

Protein Structures ◽

Three Dimensional ◽

Electron Diffraction Data ◽

Electron Crystallography ◽

Electron Dose ◽

Tilt Series ◽

Diffraction Patterns ◽

A New Technique

We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1–1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name ‘MicroED’, that may have wide applicability in structural biology.

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MicroED: Three Dimensional Electron Crystallography of Protein Micro-Crystals

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314089360 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1063-C1063

Author(s):

Tamir Gonen

Keyword(s):

Electron Diffraction ◽

Protein Structures ◽

Three Dimensional ◽

Electron Diffraction Data ◽

Molecular Replacement ◽

Electron Crystallography ◽

Electron Dose ◽

Tilt Series ◽

Diffraction Patterns

We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1 - 10and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9Å resolution. In this seminar I will present our initial proof of principle study and highlight the major advances since the first publication.

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Electron-diffraction studies in synthetic polymer materials

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100075579 ◽

1983 ◽

Vol 41 ◽

pp. 362-365

Author(s):

D.T. Grubb

Keyword(s):

Electron Diffraction ◽

Synthetic Polymer ◽

Polymer Materials ◽

Crystallographic Structure ◽

High Dose ◽

Electron Dose ◽

Dose Rates ◽

First Case ◽

Diffraction Patterns ◽

The Many

Diffraction studies in polymeric and other beam sensitive materials may bring to mind the many experiments where diffracted intensity has been used as a measure of the electron dose required to destroy fine structure in the TEM. But this paper is concerned with a range of cases where the diffraction pattern itself contains the important information.In the first case, electron diffraction from paraffins, degraded polyethylene and polyethylene single crystals, all the samples are highly ordered, and their crystallographic structure is well known. The diffraction patterns fade on irradiation and may also change considerably in a-spacing, increasing the unit cell volume on irradiation. The effect is large and continuous far C94H190 paraffin and for PE, while for shorter chains to C 28H58 the change is less, levelling off at high dose, Fig.l. It is also found that the change in a-spacing increases at higher dose rates and at higher irradiation temperatures.

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Imaging of ribosomal RNA-protein interactions by dark-field STEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100153221 ◽

1989 ◽

Vol 47 ◽

pp. 250-251

Author(s):

M. Boublik ◽

V. Mandiyan ◽

S. Tumminia ◽

J.F. Hainfeld ◽

J.S. Wall

Keyword(s):

Nucleic Acid ◽

16S Rrna ◽

Dark Field ◽

Radius Of Gyration ◽

Central Domain ◽

The Core ◽

16S Rna ◽

30S Subunit ◽

Core Particles

Success in protein-free deposition of native nucleic acid molecules from solutions of selected ionic conditions prompted attempts for high resolution imaging of nucleic acid interactions with proteins, not attainable by conventional EM. Since the nucleic acid molecules can be visualized in the dark-field STEM mode without contrasting by heavy atoms, the established linearity between scattering cross-section and molecular weight can be applied to the determination of their molecular mass (M) linear density (M/L), mass distribution and radius of gyration (RG). Determination of these parameters promotes electron microscopic imaging of biological macromolecules by STEM to a quantitative analytical level. This technique is applied to study the mechanism of 16S rRNA folding during the assembly process of the 30S ribosomal subunit of E. coli. The sequential addition of protein S4 which binds to the 5'end of the 16S rRNA and S8 and S15 which bind to the central domain of the molecule leads to a corresponding increase of mass and increased coiling of the 16S rRNA in the core particles. This increased compactness is evident from the decrease in RG values from 114Å to 91Å (in “ribosomal” buffer consisting of 10 mM Hepes pH 7.6, 60 mM KCl, 2 m Mg(OAc)2, 1 mM DTT). The binding of S20, S17 and S7 which interact with the 5'domain, the central domain and the 3'domain, respectively, continues the trend of mass increase. However, the RG values of the core particles exhibit a reverse trend, an increase to 108Å. In addition, the binding of S7 leads to the formation of a globular mass cluster with a diameter of about 115Å and a mass of ∽300 kDa. The rest of the mass, about 330 kDa, remains loosely coiled giving the particle a “medusa-like” appearance. These results provide direct evidence that 16S RNA undergoes significant structural reorganization during the 30S subunit assembly and show that its interactions with the six primary binding proteins are not sufficient for 16S rRNA coiling into particles resembling the native 30S subunit, contrary to what has been reported in the literature.

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Nauwkeurige methode voor de aanwasbepaling van het grondvlak met behulp van de Pressler-boor

Silva Gandavensis ◽

10.21825/sg.v12i0.1009 ◽

1968 ◽

Vol 12 ◽

Author(s):

R. Goossens

Keyword(s):

Basal Area ◽

Precise Determination ◽

Maximum Diameter ◽

Precise Method ◽

Maximum Radius ◽

The Core ◽

Two Parameters

A precise method for the determination of the increment of the basal area using the PressIer bore. Refering to previous research showing that the basal area of the corsica pine could be characterized by an ellips, we present in this paper a precise method for the determination of the increment of the basal area. In this method we determine the direction of the maximum diameter, we measure this diameter and we take a core in one of the points of tangency of the caliper with the measured tree. The determination of the diameter perpendicular to the maximum diameter finishes the work wich is to be done in the forest. From the classical measurements effectuated on the core and from the measured diameters we can then determine the form (V) and the excentricity (e). Substituting these two parameters in the formula 2 or 2', we can also calculate the error of a radius measured on the core with respect to the representative radius, This error with them allow us to correct the measured value of the minimum or the maximum radius and we will be able to do a precise determination of the increment.

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Formation of an Iron(III) Oxo Cubane Core Fe4(μ4-O)4 from FeCl3 and the Unsymmetrical Tripodal Ligand N[(CH2CH2NH2)(CH2CH2OH)(CH2CH2CH2OH)]

Zeitschrift für Naturforschung B ◽

10.1515/znb-2004-0815 ◽

2004 ◽

Vol 59 (8) ◽

pp. 855-858 ◽

Cited By ~ 4

Author(s):

Ekkehardt Hahn ◽

Christoph Jocher ◽

Thomas Lügger

Keyword(s):

Coordination Chemistry ◽

Structure Determination ◽

Disodium Salt ◽

Ferrous Chloride ◽

Tripodal Ligand ◽

The Core ◽

Yellow Precipitate

AbstractThe coordination chemistry of the unsymmetric, aliphatic, tetradentate tripodal ligand N[(CH2CH2NH2)(CH2CH2OH)(CH2CH2CH2OH)] H4-1 with iron chlorides was investigated. The disodium salt of the deprotonated ligand Na2(H2-1) reacts with FeCl3 to yield a yellow precipitate which upon recrystallization from DMSO/CH2Cl2 gives red crystals of the octanuclear iron(III) complex [{FeIIICl(H2-1)}4FeIII4(μ4-O)4Cl4] 2 ・ 4CH2Cl2 containing a central Fe4(μ4-O)4 cubane core. Crystals of 2 ・4DMF were obtained by slow oxidation of the green iron(II) complex obtained from ferrous chloride and Na2(H2-1) after recrystallization from DMF. The structure determination of 2 ・4CH2Cl2 also revealed the presence of the iron(III) oxo cubane core. The core is surrounded by four iron atoms each coordinated by η4-(H2-1)2- and Cl- ligands.

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ChemInform Abstract: TRANSFER METAL-CATALYZED SELF-CONDENSATION OF (E)-BENZALDOXIME AND X-RAY CRYSTALLOGRAPHIC STRUCTURE DETERMINATION OF A PRODUCT

Chemischer Informationsdienst ◽

10.1002/chin.197739186 ◽

1977 ◽

Vol 8 (39) ◽

pp. no-no

Author(s):

A. GIEREN ◽

B. DEDERER ◽

I. UGI ◽

S. STUEBER

Keyword(s):

Structure Determination ◽

Crystallographic Structure ◽

X Ray ◽

Metal Catalyzed

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Crystallographic structure of the turbine C-ring from spinach chloroplast F-ATP synthase

Bioscience Reports ◽

10.1042/bsr20130114 ◽

2014 ◽

Vol 34 (2) ◽

Cited By ~ 4

Author(s):

Asha Manikkoth Balakrishna ◽

Holger Seelert ◽

Sven-Hendric Marx ◽

Norbert A. Dencher ◽

Gerhard Grüber

Keyword(s):

Atp Synthase ◽

Ring Width ◽

Atp Synthesis ◽

Crystallographic Structure ◽

Unit Cell Parameters ◽

Subunit C ◽

Cell Parameters ◽

Ring Diameter ◽

C Subunit

In eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF1FO-ATP synthase) of plants is integrated into the thylakoid membrane via its FO-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H+-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F1 sector. Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF1FO-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and β=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å.

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