The Structure of haemoglobin - VII. Determination of phase angles in the non-centrosymmetric [100] zone

1958 ◽  
Vol 247 (1250) ◽  
pp. 302-336 ◽  
Keyword(s):  
Standard Error ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Anomalous Scattering ◽  
Heavy Atoms ◽  
Replacement Method ◽  
Isomorphous Replacement ◽  
Arbitrary Phase ◽  
Phase Angles

In the last paper in this series a Fourier projection down the [010] axis of horse haemoglobin was given (Bragg & Perutz 1954). This projection was centrosymmetric. As a first step towards the three-dimensional analysis, the projection down [100] has now been attacked. This projection is non-centrosymmetric, and arbitrary phase angles have had to be determined. All the fundamental problems of a three-dimensional study are met, but only a small number of reflexions need be dealt with. The isomorphous replacement method has been used successfully with three mercury derivatives of haemoglobin. This provided a test of new methods for finding the vectors relating heavy atoms. Particular attention has been given to estimation of errors, and to their effect on the results. Further information about the phases has been derived from anomalous scattering by the mercury atoms, using CrKα and CuKα radiation. By combining these results, the phases of most reflexions out to a spacing of about 6 Å have been determined with a standard error of about 25°. Ambiguous results are obtained for a few reflexions. The resulting electron density projection shows peaks up to four times the estimated standard error. The prospects for three-dimensional structure analysis at 6 Å resolution are favourable. If the polypeptide chain is coiled in the α-form, the contrast should be sufficient for it to show up throughout its length.

scholarly journals Protein Crystallography: Getting in on the Ground Floor

2014 ◽  
Vol 70 (a1) ◽  
pp. C931-C931
Author(s):  
Brian Matthews
Keyword(s):  
Three Dimensional ◽  
Protein Crystal ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Egg White Lysozyme ◽  
Isomorphous Replacement ◽  
Hen Egg White ◽  
Diffraction Patterns ◽  
Dorothy Crowfoot Hodgkin

The first diffraction pattern from crystals of a protein was obtained by Desmond Bernal and Dorothy Crowfoot Hodgkin in 1934. As early as 1939, Bernal described how such diffraction patterns might be used to determine the complete three-dimensional structure of a protein. It was not until 1954, however, that Max Perutz showed how isomorphous replacement could be used to determine the phases for crystalline hemoglobin. Using this approach, Kendrew and coworkers described the three-dimensional structure of myoglobin in 1960. In 1965, David Phillips' group determined the structure of hen egg-white lysozyme. Then, in 1967, three different protein crystal structures were reported. Macromolecular crystallography had come of age. The talk will touch on some of these early events and include reminiscences of work at the MRC Lab in David Blow's group leading up to the successful determination of the alpha-chymotrypsin structure.

The structure of haemoglobin VIII. A three-dimensional Fourier synthesis at 5.5 Å resolution: determination of the phase angles

1961 ◽  
Vol 265 (1320) ◽  
pp. 15-38 ◽  
Keyword(s):  
Phase Angle ◽  
Least Squares Method ◽  
Heavy Atom ◽  
Three Dimensional ◽  
Protein Molecule ◽  
Fourier Synthesis ◽  
Shape Factors ◽  
Heavy Atoms ◽  
Phase Angles

Determination of the phase angles of a crystalline protein requires a series of isomorphous heavy-atom compounds, with heavy atoms attached to different sites on the protein molecule. The asymmetric unit of horse oxyhaem oglobin was found to combine with heavy atoms at two different sites which are now known to be sulphydryl groups. Altogether six different heavy - atom com pounds of haemoglobin were made which proved isomorphous on X -ray analysis. The positions of the heavy atoms were determined first by difference Patterson and Fourier projections on the centrosym metric plane of the monoclinic crystals, and later by three-dimensional correlation functions, ( F H 1 — F H 2 ) 2 being used as coefficients, where F H 1 and F H 2 are the structure factors of the two different heavy-atom compounds. The parameters and anisotropic shape factors of the heavy atoms were refined by a three-dimensional least-squares method. For each of the 1200 reflexions in the limiting sphere of (5.5 Å) -1 the structure amplitudes of all seven compounds were combined in an Argand diagram and the probability of the phase angle having a value a was calculated for oc = 0, 5, 10, ..., 355°. The coefficients for the final Fourier summation were then calculated in two different ways. In one method the vector from the origin to the centroid of the probability distribution, plotted around a circle of radius | F |, was chosen as the ‘best F’. The alternative set of coefficients was calculated, using the full, observed, value of F and the most probable value of the phase angle a. The most probable error in phase angle was found to be 23°, and the standard error in electron density to be expected in the final results 0.12 e/Å 3 .

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.

The crystal structure of myoglobin. VI. Seal myoglobin

1960 ◽  
Vol 258 (1293) ◽  
pp. 181-201 ◽  
Keyword(s):  
Tertiary Structure ◽  
Heavy Atom ◽  
Three Dimensional ◽  
Sperm Whale ◽  
Fourier Synthesis ◽  
Heavy Atoms ◽  
Isomorphous Replacement ◽  
Unit Cells ◽  
The Common ◽  
Sperm Whale Myoglobin

Myoglobin from the common seal ( Phoca vitulina ) when crystallized from ammonium sulphate forms monoclinic crystals with space group the unit cell, a = 57·9Å, b = 29·6Å, c = 106·4Å, β = 102°15', contains four molecules. The method of isomorphous replacement has been used in an investigation of the centrosymmetric b -axis projection in which it has been possible to determine signs for nearly all the h0l reflexions having spacings greater than 4Å. Three independent heavy-atom derivatives were employed and the signs so determined have been used to compute a map of the electron density projected on the (010) plane. This projection has been interpreted in terms of the molecule of sperm-whale myoglobin, as deduced by Bodo, Dintzis, Kendrew & Wyckoff (1959) from a three-dimensional Fourier synthesis to 6Å resolution. The results of the interpretation show that the two myoglobin molecules are very similar in form (tertiary structure) in spite of the differences in their amino-acid composition. The relative orientation of the two unit cells with respect to the myoglobin molecule is given and a comparison is made of the positions of the heavy atoms in each molecule.

scholarly journals Determination of the alpha-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis.

1994 ◽  
Vol 126 (2) ◽  
pp. 433-443 ◽  
Author(s):  
A McGough ◽  
M Way ◽  
D DeRosier
Keyword(s):  
Image Analysis ◽  
Smooth Muscle ◽  
Binding Sites ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Outer Face ◽  
Actin Binding Domain

The three-dimensional structure of actin filaments decorated with the actin-binding domain of chick smooth muscle alpha-actinin (alpha A1-2) has been determined to 21-A resolution. The shape and location of alpha A1-2 was determined by subtracting maps of F-actin from the reconstruction of decorated filaments. alpha A1-2 resembles a bell that measures approximately 38 A at its base and extends 42 A from its base to its tip. In decorated filaments, the base of alpha A1-2 is centered about the outer face of subdomain 2 of actin and contacts subdomain 1 of two neighboring monomers along the long-pitch (two-start) helical strands. Using the atomic model of F-actin (Lorenz, M., D. Popp, and K. C. Holmes. 1993. J. Mol. Biol. 234:826-836.), we have been able to test directly the likelihood that specific actin residues, which have been previously identified by others, interact with alpha A1-2. Our results indicate that residues 86-117 and 350-375 comprise distinct binding sites for alpha-actinin on adjacent actin monomers.

On the three-dimensional structure and catalytic mechanism of triose phosphate isomerase

1981 ◽  
Vol 293 (1063) ◽  
pp. 159-171 ◽  
Keyword(s):  
Catalytic Mechanism ◽  
Polypeptide Chain ◽  
Three Dimensional ◽  
Triose Phosphate Isomerase ◽  
Dimensional Structure ◽  
Dihydroxyacetone Phosphate ◽  
Triose Phosphate ◽  
Independent Determination ◽  
Chicken Muscle

Triose phosphate isomerase is a dimeric enzyme of molecular mass 56000 which catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate. The crystal structure of the enzyme from chicken muscle has been determined at a resolution of 2.5 A, and an independent determination of the structure of the yeast enzyme has just been completed at 3 A resolution. The conformation of the polypeptide chain is essentially identical in the two structures, and consists of an inner cylinder of eight strands of parallel |3-pleated sheet, with mostly helical segments connecting each strand. The active site is a pocket containing glutamic acid 165, which is believed to act as a base in the reaction. Crystallographic studies of the binding of DHAP to both the chicken and the yeast enzymes reveal a common mode of binding and suggest a mechanism for catalysis involving polarization of the substrate carbonyl group.

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