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N Grigorieff

Publications and source records attributed to N Grigorieff.

10 recordsLinked to original sources
PubMed2001

Three-dimensional structure of a voltage-gated potassium channel at 2.5 nm resolution.

BACKGROUND: The voltage-gated potassium channel Shaker from Drosophila consists of a tetramer of identical subunits, each containing six transmembrane segments. The atomic structure of a bacterial homolog, the potassium channel KcsA, is much smaller than Shaker. It does not have a voltage sensor and other important domains like the N-terminal tetramerization (T1) domain. The structure of these additional elements has to be studied in the more complex voltage-gated channels. RESULTS: We determined the three-dimensional structure of the entire Shaker channel at 2.5 nm resolution using electron microscopy. The four-fold symmetric structure shows a large and a small domain linked by thin 2 nm long connectors. To interpret the structure, we used the crystal structures of the isolated T1 domain and the KcsA channel. A unique density assignment was made based on the symmetry and dimensions of the crystal structures and domains, identifying the smaller domain as the cytoplasmic mass of Shaker containing T1 and the larger domain as embedded in the membrane. CONCLUSIONS: The two-domain architecture of the Shaker channel is consistent with the recently proposed "hanging gondola" model for the T1 domain, putting the T1 domain at a distance from the membrane domain but attached to it by thin connectors. The space between the two domains is sufficient to permit cytoplasmic access of ions and the N-terminal inactivation domain to the pore region. A hanging gondola architecture has also been observed in the nicotinic acetylcholine receptor and the KcsA structure, suggesting that it is a common element of ion channels.

Animals↗
PubMed2001

Projection structure of a ClC-type chloride channel at 6.5 A resolution.

Virtually all cells in all eukaryotic organisms express ion channels of the ClC type, the only known molecular family of chloride-ion-selective channels. The diversity of ClC channels highlights the multitude and range of functions served by gated chloride-ion conduction in biological membranes, such as controlling electrical excitability in skeletal muscle, maintaining systemic blood pressure, acidifying endosomal compartments, and regulating electrical responses of GABA (gamma-aminobutyric acid)-containing interneurons in the central nervous system. Previously, we expressed and purified a prokaryotic ClC channel homologue. Here we report the formation of two-dimensional crystals of this ClC channel protein reconstituted into phospholipid bilayer membranes. Cryo-electron microscopic analysis of these crystals yields a projection structure at 6.5 A resolution, which shows off-axis water-filled pores within the dimeric channel complex.

Chloride Channels↗
PubMed2000

Resolution measurement in structures derived from single particles.

Analytical expressions are derived and computer simulations are presented to assess the accuracy of procedures commonly used to estimate the resolution of three-dimensional (3D) structures derived from images of single protein molecules or complexes. It is shown that in the case of a low signal-to-noise ratio in the images, the Fourier ring correlation between two structures, each calculated using one half of the data, significantly overestimates the resolution when the two half data sets were aligned against the same reference structure. The overestimate arises because of a correlation between the noise components present in the images. The correlation is introduced by the alignment and becomes more serious as the signal-to-noise ratio is reduced. A reliable resolution measure is only obtained when the two half data sets are aligned against two independent reference structures. It is further shown that the noise correlation also significantly affects the spectral signal-to-noise ratio and the Q factor, making them unreliable measures of signal present in a 3D structure and in the original images, respectively. It is concluded that the alignment of images is always accompanied by a correlation of the noise and that this correlation is indistinguishable from a correlation arising from a signal.

Crystallography, X-Ray↗
PubMed1999

Structure of the respiratory NADH:ubiquinone oxidoreductase (complex I)

Three-dimensional structures of NADH:ubiquinone oxidoreductase (or complex I) from the respiratory chain of mitochondria and bacteria have been recently studied by electron microscopy. The low-resolution structures all reveal a characteristic L shape for complex I; however, some of the differences among these structures may have important implications for the location of the functional elements of complex I, for example, the ubiquinone-binding site.

Animals↗
PubMed1998

Clathrin coats at 21 A resolution: a cellular assembly designed to recycle multiple membrane receptors.

We present a map at 21 A resolution of clathrin assembled into cages with the endocytic adaptor complex, AP-2. The map was obtained by cryo-electron microscopy and single-particle reconstruction. It reveals details of the packing of entire clathrin molecules as they interact to form a cage with two nested polyhedral layers. The proximal domains of each triskelion leg depart from a cage vertex in a skewed orientation, forming a slightly twisted bundle with three other leg domains. Thus, each triskelion contributes to two connecting edges of the polyhedral cage. The clathrin heavy chains continue inwards under the vertices with local 3-fold symmetry, the terminal domains contributing to 'hook-like' features which form an intermediate network making possible contacts with the surface presented by the inner adaptor shell. A node of density projecting inwards from the vertex may correspond to the C-termini of clathrin heavy chains which form a protrusion on free triskelions at the vertex. The inter-subunit interactions visible in this map provide a structural basis for considering the assembly of clathrin coats on a membrane and show the contacts which will need to be disrupted during disassembly.

Adaptor Protein Complex alpha Subunits↗
PubMed1998

Three-dimensional structure of bovine NADH:ubiquinone oxidoreductase (complex I) at 22 A in ice.

NADH:ubiquinone oxidoreductase (complex I) is the first and largest complex in the electron transport chain of mitochondria. The bovine complex purified from cardiac muscle consists of at least 42 different subunits with a combined molecular mass of about 890 kDa. The three-dimensional structure of the complex was determined at 22 A from single particles embedded in vitrified ice using electron cryo-microscopy. The structure was calculated using a new program to align particles, to correct for the contrast transfer function of the microscope, and to carry out the three-dimensional reconstruction of the complex. The bovine complex has the overall L-shaped appearance found in earlier studies of the closely related complex I from Neurospora crassa, but it differs by having a thin stalk region linking the membrane-bound globular arm with the intrinsic membrane domain. Thus, the stalk which measures about 30 A in diameter is likely to contain part of the electron transfer pathway linking the NADH binding site in the globular arm with the ubiquinone binding site in the membrane domain. The globular domain of bovine complex I is significantly bigger than that of the N. crassa enzyme, suggesting that the apparent additional subunit complexity of the bovine enzyme is associated with the globular part.

Animals↗
PubMed1998

Electron Crystallography of Two-Dimensional Crystals of Membrane Proteins.

Electron microscopy has become a powerful technique, along with X-ray crystallography and nuclear magnetic resonance spectroscopy, to study the three-dimensional structure of biological molecules. It has evolved into a number of methods dealing with a wide range of biological samples, with electron crystallography of two-dimensional crystals being so far the only method allowing data collection at near-atomic resolution. In this paper, we review the methodology of electron crystallography and its application to membrane proteins, starting with the pioneering work on bacteriorhodopsin, which led to the first visualization of the secondary structure of a membrane protein in 1975. Since then, improvements in instrumentation, sample preparation, and data analysis have led to atomic models for bacteriorhodopsin and light-harvesting complex II from higher plants. The structures of many more membrane proteins have been studied by electron crystallography and in this review examples are included where a resolution of better than 10 Å has been achieved. Indeed, in some of the given examples an atomic model can be expected in the near future. Finally, a brief outlook is given on current and future developments of electron crystallographic methods. Copyright 1998 Academic Press.

Journal Article↗
PubMed1996

Electron-crystallographic refinement of the structure of bacteriorhodopsin.

Using electron diffraction data corrected for diffuse scattering together with additional phase information from 30 new images of tilted specimens, an improved experimental density map has been calculated for bacteriorhodopsin. The atomic model has then been rebuilt into this new map with particular attention to the surface loops. All the residues from 7 to 227 as well as ten lipid molecules are now included, although a few amino acid residues in three of the six surface loops, about half of the lipid hydrophobic chains and all of the lipid head groups are disordered. The model has then been refined against the experimental diffraction amplitudes to an R-factor of 28% at 3.5 angstrom resolution with strict geometry (0.005 angstrom) bond length deviation) using the improvement of the "free" phase residual between calculated and experimental phases from images as an objective criterion of accuracy. For the refinement some new programs were developed to restrain the number of parameters, to be compatible with the limited resolution of our data. In the final refined model of the protein (2BRD), compared with earlier co-ordinates (1BRD), helix D has been moved towards the cytoplasm by almost 4 angstrom, and the overall accuracy of the co-ordinates of residues in the other six helices has been improved. As a result the positions of nearly all the important residues in bacteriorhodopsin are now well determined. In particular, the buried, protonated Asp115 is 7 angstrom from, and so not in contact with, the retinal and Met118 forms a cap on the pocket occupied by the beta-ionone ring. No clear density exists for the side-chain of Arg82, which forms a central part of the extracellular half-channel. The only arginine side-chain built into good density is that of Arg134 at the extracellular end of helix E, the others being disordered near one of the two surfaces. The interpretation of the end of helix F on the extracellular surface is now clearer; an extra loose helical turn has been built bringing the side-chain of Glu194 close to Arg134 to form a probable salt bridge. The model provides an improved framework for understanding the mechanism of the light-driven proton pumping. A number of cavities that could contain water molecules were found by searching the refined model, most of them above or below the Schiff base in the half-channels leading to the two surfaces. The ordered and disordered regions of the structure are described by the temperature factor distribution.

Amino Acid Sequence↗
PubMed1995

Lipid location in deoxycholate-treated purple membrane at 2.6 A.

A high resolution projection at 2.6 A of deoxycholate-treated purple membrane using only images has been obtained with a 200 keV FEG microscope operated at liquid helium temperature. Examination of this high quality map has allowed the following conclusions to be made: Comparison between the internal structure of the trimers of the native and the deoxycholate-treated crystal forms shows that almost every detail of the structure at high resolution is identical. The cell dimension change from 62.4 A to 57.9 A is accompanied by a loss of about half the normal lipids and a 2 degrees anticlockwise rotation of the trimer as a rigid body. Three of the lipids per bacteriorhodopsin molecule remain in identical positions relative to the trimer. In addition, from the projection map together with a packing analysis using the atomic model for bacteriorhodopsin, space for three further lipids has been identified making a total of six lipids per bacteriorhodopsin molecule in this crystal form. Finally, the surprisingly small rotation of the trimer between the two crystal forms with completely different Van der Waals contacts suggest that the crystals are held together by strong, long-range electrostatic interactions.

Bacteriorhodopsins↗