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1.
The membrane protein bacteriorhodopsin was imaged in buffer solution at room temperature with the atomic force microscope. Three different substrates were used: mica, silanized glass and lipid bilayers. Single bacteriorhodopsin molecules could be imaged in purple membranes adsorbed to mica. A depression was observed between the bacteriorhodopsin molecules. The two dimensional Fourier transform showed the hexagonal lattice with a lattice constant of 6.21 +/- 0.20 nm which is in agreement with results of electron diffraction experiments. Spots at a resolution of approximately 1.1 nm could be resolved. A protein, cationic ferritin, could be imaged bound to the purple membranes on glass which was silanized with aminopropyltriethoxysilane. This opens the possibility of studying receptor/ligand binding under native conditions. In addition, purple membranes bound to a lipid bilayer were imaged. These images may help in interpreting results of functional studies done with purple membranes adsorbed to black lipid membranes.  相似文献   

2.
Recent advances in ab initio direct methods have enabled the solution of crystal structures of small proteins from native X-ray data alone, that is, without the use of fragments of known structure or the need to prepare heavy-atom or selenomethionine derivatives, provided that the data are available to atomic resolution. These methods are also proving to be useful for locating the selenium atoms or other anomalous scatterers in the multiple wavelength anomalous diffraction phasing of larger proteins at lower resolution.  相似文献   

3.
The projected structures of two unstained periodic biological specimens, the purple membrane and catalase, have been determined by electron microscopy to resolutions of 7 Å and 9 Å, respectively. Glucose was used to facilitate their in vacuo preservation and extremely low electron doses were applied to avoid their destruction.The information on which the projections are based was extracted from defocussed bright-field micrographs and electron diffraction patterns. Fourier analysis of the micrograph data provided the phases of the Fourier components of the structures; measurement of the electron diffraction patterns provided the amplitudes.Large regions of the micrographs (3000 to 10,000 unit cells) were required for each analysis because of the inherently low image contrast (<1%) and the statistical noise due to the low electron dose.Our methods appear to be limited in resolution only by the performance of the microscope at the unusually low magnifications which were necessary. Resolutions close to 3 Å should ultimately be possible.  相似文献   

4.
Three isomorphous heavy-atom derivatives have been used to calculate a 2.5 Å resolution electron density map of tosyl-elastase at pH 5.0, from which an accurate atomic model has been constructed. Atomic co-ordinates measured from this model have been refined using model building, real-space refinement and energy minimization programs. The three-dimensional conformation of the polypeptide chain is described in terms of conformational angles, hydrogen-bonding networks and the environment of different types of amino acid side-chain.Difference Fourier calculation of the high resolution structure of native elastase at pH 5.0 shows it to be virtually identical to that of the tosyl derivative, except near the tosyl group. The conformation of the catalytically important residues in native elastase is very similar to that of native α-chymotrypsin, except for the orientation of the active centre serine oxygen. The significance of important structural similarities and differences between these two enzymes is discussed.Elastase contains 25 internal water molecules which play an important role in stabilizing the active conformation of the enzyme. Many of these water molecules are in identical positions to those found in the interior of α-chymotrypsin  相似文献   

5.
A method is described for the selective introduction of heavy atoms into structured membrane proteins by a two step modification. The procedure is applied for the purple membrane protein bacteriorhodopsin. Selective heavy-atom modification of this protein is achieved by placing a mercury reagent of intermediate polarity into phenylthiocarbamoylated bacteriorhodopsin. Incorporation of mercury requires the selective phenylthiocarbamoylation of a lysine residue. Optical investigations including circular dichroism document unchanged chromophore-protein and protein-protein interactions in mercury labeled purple membranes.  相似文献   

6.
The three-dimensional structure of the deoxycholate-treated form of purple membrane has been determined to a resolution of about 6 Å. Using low temperature electron diffraction data, room temperature electron microscope images and improved methods of data analysis, higher resolution has been reached than was obtained using native membranes of the same size. Statistical analysis of the data shows that the new map is considerably better than earlier maps. The map indicates the probable sites for the lipid molecules that remain in the deoxycholate-treated membranes; some of these sites differ from those suggested by the projection map of Glaeser et al. (1985). Comparison of the bacteriorhodopsin structures now determined independently from three crystal forms shows that the monomer structure is independent of the detailed contacts with lipid molecules. The average of the three structures gives a picture with very little noise showing seven similar rod-like features which are clearly best interpreted as -helices; there is no indication that part of the structure is -sheet as suggested by Jap et al. (1983). Phases from the averaged structure at 6 Å resolution will enable better refinement of the parameters that will be required in the analysis of higher resolution images from tilted specimens needed to extend the projection map at 3.5 Å resolution (Henderson et al. 1986) to produce a three-dimensional atomic resolution map.  相似文献   

7.
The X-ray structure of the nucleosome core particle was solved at 7 Å resolution using the method of multiple isomorphous replacement based on two isomorphous derivatives, each containing a different multiple heavy-atom compound. The preparation of these heavy-atom compounds and their application to this macromolecular structure determination are described. The first of these reagents, TAMM (tetrakis(acetoxymercuri)methane), was solubilized by the addition of an excess of glycylglycine and, when added to crystals of the nucleosome core particle, produced a derivative with a single major site. Despite the large mass of 206,000 daltons per asymmetric unit, the position of the TAMM molecule was found in these crystals using the difference Patterson technique. This compound was sufficiently electron-dense to produce a unique solution, whereas the mono-mercurial, methylmercury nitrate had been inadequate. The second reagent, PIP (di-μ-iodobis(ethylenediamine)diplatinum(II) nitrate), is freely soluble in aqueous solution and, on addition to the crystals, labelled the histone proteins at several sites. The locations of the PIP groups were determined from difference Fourier and Patterson maps. The X-ray structure and solution characterization of this compound are reported. These multiple heavy-atom compounds appear to be generally applicable to X-ray structure determination, and are particularly useful in conjunction with crystals having asymmetric units of large volume but lacking non-crystallographic symmetry elements.  相似文献   

8.
A neutral protease, i.e., a zinc-containing metalloendoprotease from Streptomyces caespitosus, has been crystallized using acetone as a precipitating agent. The crystals diffract to better than 1.5 A resolution when a rotating anode X-ray generator is used as an X-ray source. Protein phase angles were calculated by the multiple isomorphous replacement method using two heavy-atom derivatives (HgCl2 and CH3HgCl). A 6 A resolution electron density map clearly showed molecular boundaries. Although its amino acid sequence is not known, the folding pattern of the polypeptide chain could be traced on a 2.5 A resolution electron density map. A large cleft, which is located on the molecular surface, was proved to be the active site of the enzyme by structure analyses of inhibitor-complex crystals. The highest electron density peak, which corresponds to the cleft, was assigned to a catalytically essential zinc atom on difference Fourier synthesis between native and EDTA-soaked crystals.  相似文献   

9.
In electron crystallography, membrane protein structure is determined from two-dimensional crystals where the protein is embedded in a membrane. Once large and well-ordered 2D crystals are grown, one of the bottlenecks in electron crystallography is the collection of image data to directly provide experimental phases to high resolution. Here, we describe an approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We use the strengths of electron crystallography in rapidly obtaining accurate experimental phase information from low-resolution images and accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α helix fragments and extended to high resolution using phases from the fragments. Phases were further improved by density modifications followed by fragment expansion and structure refinement against the high-resolution diffraction data. Using this approach, structures of three membrane proteins were determined rapidly and accurately to atomic resolution without high-resolution image data.  相似文献   

10.
Gap junctions are clusters of closely packed intercellular membrane channels embedded in the plasma membranes of two adjoining cells. The central pore of the membrane channels serves as a conduit between cell cytoplasms for molecules less than 1000 Da in size. Advances in the purification of gap junctions and electron cryocrystallography and computer reconstruction techniques have produced new insights into the intercellular channel structure. Methods are described here for the purification of gap junction membranes, biochemical treatments to produce hemichannel layers ("split junctions"), assessment of the purity of gap junction preparations, electron cryomicroscopy, image processing and reconstruction, three-dimensional visualization, and interpretation. The critical step in electron crystallographic structure determination remains the isolation of crystalline material in sufficient and pure quantities for recording of electron microscope images. Along with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, the quality of gap junction purification is assessed using electron microscopy of negatively stained preparations. Electron microscopy is also used to assess the crystallinity of the purified gap junctions and split junctions. Electron cryocrystallography is a powerful technique for high-resolution structural characterization. Image processing is used to combine and enhance two-dimensional images. Electron crystallographic analysis is used to generate a three-dimensional structure from a set of electron micrographs. This three-dimensional information is extracted from a set of images recorded after tilting the specimen in the electron microscope stage and recombined using Fourier analysis techniques analogous to those used in X-ray crystallography. Computer modeling of the three-dimensional gap junction structures is a useful tool for analyzing hemichannel docking.  相似文献   

11.
The 43 kDa N-terminal ATPase domain of the Escherichia coli DNA gyrase B protein has been purified from an over-expressing strain. This protein has been crystallized in two crystal forms, both in the presence of the non-hydrolysable ATP analogue 5'-adenylyl-beta,gamma-imidodiphosphate. The first crystal form is monoclinic P2(1), with cell dimensions a = 76 A, b = 88 A, c = 82 A, beta = 105.5 degrees, and diffracts to at least 2.7 A resolution using synchrotron radiation. Crystal density measurements suggest that there are two molecules in the asymmetric unit (Vm = 3.08 A3/Da). The second crystal form is orthorhombic C222(1), with cell dimensions a = 89.2 A, b = 143.1 A and c = 79.8 A. The crystals diffract to beyond 3 A and are stable for at least 100 hours when exposed to X-rays from a rotating anode source. The asymmetric unit of this crystal form appears to contain one molecule (Vm = 2.96 A3/Da). Data have already been collected to 5 A resolution from native crystals of this second form, and to 6 A resolution from three heavy-atom derivatives. Electron density maps calculated using phases obtained from these derivatives show features consistent with secondary structural elements, and have allowed the molecular boundary to be determined. Higher resolution native and derivative data are being collected.  相似文献   

12.
A method is described for merging in reciprocal space of electron microscopic data from three-dimensional crystals of the acrosomal bundle. Permutation of indices was required to find the proper alignment of data from different bundles. The method utilizes a statistical evaluation of the significance of the cross-correlation results to indicate the proper order for merging. The three-dimensional (3D) merging is a reference-free operation that does not depend on choosing a "zero-tilt" user-defined starting point. Results from the merging are given and the merged 3D data to 9.5A resolution is evaluated for coherence. General issues such as statistical significance of cross-correlation function peaks, symmetry evaluation, and phase coherence as a function of amplitude are discussed.  相似文献   

13.
Colicins are antibiotic proteins that kill sensitive Escherichia coli cells. The structure of the pore-forming fragment of colicin A has been solved to 2.5 A resolution using the techniques of X-ray crystallography and genetic engineering. Site-directed mutagenesis was used to construct a number of cysteine-containing mutant proteins, one of which yielded an excellent mercurial derivative. Our experiences suggest strategies for obtaining useful heavy-atom derivatives for protein crystallography using genetic engineering techniques.  相似文献   

14.
The atomic force microscope has developed into a powerful tool in structural biology allowing information to be acquired at submolecular resolution on the protruding structures of membrane proteins. It is now a complementary technique to X-ray crystallography and electron microscopy for structure determination of individual membrane proteins after extraction, purification and reconstitution into lipid bilayers. Moving on from the structures of individual components of biological membranes, atomic force microscopy has recently been demonstrated to be a unique tool to identify in situ the individual components of multi-protein assemblies and to study the supramolecular architecture of these components allowing the efficient performance of a complex biological function. Here, recent atomic force microscopy studies of native membranes of different photosynthetic bacteria with different polypeptide contents are reviewed. Technology, advantages, feasibilities, restrictions and limits of atomic force microscopy for the acquisition of highly resolved images of up to 10 A lateral resolution under native conditions are discussed. From a biological point of view, the new insights contributed by the images are analysed and discussed in the context of the strongly debated organisation of the interconnected network of membrane-associated chlorophyll-protein complexes composing the photosynthetic apparatus in different species of purple bacteria.  相似文献   

15.
We have used site-specific heavy-atom labelling and X-ray diffraction to localize single amino acid residues in the cytoplasmic domain of the integral membrane protein rhodopsin, the dim-light photoreceptor of retinal vertebrate rod cells. Two-dimensional orthorhombic crystals of the space group p22(1)2(1) (a=59.5(+/-1) A and b=82.7(+/-1.5) A) were produced from detergent-solubilized, partially delipidated rhodopsin. To obtain milligram amounts of two-dimensional crystals, which are required for X-ray diffraction, the yield of the crystalline material was significantly increased by reconstitution of rhodopsin in the presence of cholesterol (1:2 to 1:10 mol/mol) and by adding polar organic solvents to the dialysis buffer. The native cysteine residues C140 and C316 were then selectively labelled with mercury using the sulphydryl-specific reagent p-chloromercuribenzoate (1.6-2.1 mol Hg per mol rhodopsin). The labelling did not affect the unit cell dimensions. Optical absorption spectra of labelled and native two-dimensional rhodopsin crystals showed the characteristic 11-cis-retinal peak at 498 nm, which corresponds to the dark state of rhodopsin. The in-plane position of the mercury label was calculated at 9.5 A resolution from the intensity differences in the X-ray diffraction patterns of labelled and native crystals using Fourier difference methods and the phase information from electron crystallography. The label positions were in excellent agreement with the positions of C140 at the cytoplasmic end of helix 3 and of C316 in the cytoplasmic helix 8 recently obtained from three-dimensional rhodopsin crystals. Whereas these high-resolution diffraction studies were performed under cryogenic conditions (100 K), our results were obtained at room temperature with fully hydrated membranes and in the absence of loop-loop crystal contacts. To study the structural changes of the cytoplasmic loops involved in activation and signal transduction, our more physiological conditions offer important advantages. Furthermore, the localization of C316 is the first direct proof that the electron density on top of helix 1 observed by cryo-electron microscopy is a part of the C-terminal loop. Our approach is of particular interest for investigations of other membrane proteins, for which 3D crystals are not available. Structural constraints from heavy-atom labels at strategic sites enable the assignment of a position in the amino acid sequence to features visible in a low-resolution density map and the study of conformational changes associated with different functional states of the membrane protein.  相似文献   

16.
Electron density levels of sarcoplasmic reticulum membranes   总被引:1,自引:0,他引:1  
Low-angle X-ray diffraction has been recorded from oriented preparations of sacroplasmic reticulum membranes in fluid media containing glycerol solutions in different concentrations. Discrete diffraction orders of a lamellar repeat distance ranging from 200 Å to 250 Å have been recorded. Fourier synthesis at a resolution of 17 Å for 0, 10, 20, and 30% glycerol-treated sarcoplasmic reticulum membranes are described. An electron density scale in electrons/A?3 for these Fourier syntheses has been determined. The question of the correctness of our asymmetric electron density profile for the sarcoplasmic reticulum membrane is critically examined. A study is made on the choice of phases and on the method used to process the X-ray intensities.  相似文献   

17.
Crystal structure of recombinant human interleukin-1 beta at 2.0 A resolution   总被引:11,自引:0,他引:11  
The crystal structure of recombinant human interleukin-1 beta (IL-1 beta) has been determined at 2.0 A resolution and refined to a crystallographic R-factor of 0.19. Three heavy-atom derivatives were identified and used for multiple isomorphous replacement phasing. Interpretation of the resulting electron density map revealed a structure in which there are 12 antiparallel beta-strands and no alpha-helix. The single 153-residue polypeptide chain is folded into a six-stranded beta-barrel similar in architecture to the Kunitz-type trypsin inhibitor found in soybeans. The molecule displays approximate 3-fold symmetry about the axis of the beta-barrel. Each successive pair of component strands of the barrel brackets an extensive sequence outside the barrel that includes an additional pair of beta-strands and a prominent loop. Together, these three external segments conceal much of the perimeter and one end of the barrel, leaving only the end supporting the chain termini fully exposed. The structure can be used to identify portions of the polypeptide chain that are exposed on the surface of the molecule, some of which must be epitopes recognized by interleukin-1 beta receptors.  相似文献   

18.
Electron diffraction patterns of two-dimensional crystals of light-harvesting chlorophyll a/b-protein complex (LHC-II) from photosynthetic membranes of pea chloroplasts, tilted at different angles up to 60°, were collected to 3.2 Å resolution at -125°C. The reflection intensities were merged into a three-dimensional data set. The Friedel R-factor and the merging R-factor were 21.8 and 27.6%, respectively. Specimen flatness and crystal size were critical for recording electron diffraction patterns from crystals at high tilts. The principal sources of experimental error were attributed to limitations of the number of unit cells contributing to an electron diffraction pattern, and to the critical electron dose. The distribution of strong diffraction spots indicated that the three-dimensional structure of LHC-II is less regular than that of other known membrane proteins and is not dominated by a particular feature of secondary structure.  相似文献   

19.
A sensitive high-resolution electron diffraction assay for change in structure is described and harnessed to analyze the binding of divalent cations to the purple membrane (PM) of Halobacterium halobium. Low-dose electron diffraction patterns are subject to a matched filter algorithm (Spencer, S. A., and A. A. Kossiakoff. 1980. J. Appl. Crystallogr. 13:563-571). to extract accurate values of reflection intensities. This, coupled with a scheme to account for twinning and specimen tilt in the microscope, yields results that are sensitive enough to rapidly quantitate any structure change in PM brought about by site-directed mutagenesis to the level of less than two carbon atoms. Removal of tightly bound divalent cations (mainly Ca2+ and Mg2+) from PM causes a color change to blue and is accompanied by a severely altered photocycle of the protein bacteriohodopsin (bR), a light-driven proton pump. We characterize the structural changes that occur upon association of 3:1 divalent cation to PM, versus membranes rendered purple by addition of excess Na+. High resolution, low dose electron diffraction data obtained from glucose-embedded samples of Pb2+ and Na+ reconstituted PM preparations at room temperature identify several sites with total occupancy of 2.01 +/- 0.05 Pb2+ equivalents. The color transition as a function of ion concentration for Ca2+ or Mg2+ and Pb2+ are strictly comparable. A (Pb2(+)-Na+) PM Fourier difference map in projection was synthesized at 5 A using the averaged data from several nominally untilted patches corrected for twinning and specimen tilt. We find six major sites located on helices 7, 5, 4, 3, 2 (nomenclature of Engelman et al. 1980. Proc. Natl. Acad. Sci. USA. 77:2023-2027) in close association with bR. These partially occupied sites (0.55-0.24 Pb2+ equivalents) represent preferential sites of binding for divalent cations and complements our earlier result by x-ray diffraction (Katre et al. 1986. Biophys. J. 50:277-284).  相似文献   

20.
The projected structure of the purple membrane has been determined to 3·7 Å resolution by low dose electron imaging and diffraction. The specimens were maintained at low temperature in order to minimize the effects of radiation damage.To obtain phases at high resolution, a treatment of errors in electron imaging has been developed that makes possible the systematic combination of data from several image areas. This treatment is analogous to that used in protein crystallography for phase determination by multiple isomorphous replacement. Phase probability distributions are calculated for each reflection, and distributions from many sets of image data are combined. The use of probability distributions allows such factors as image motion, defocus, defocus uncertainty and image degradation to be taken into account in the weighting of data from individual image areas. Centroid phases and figures of merit are computed, allowing the map with the least-squares error in electron scattering potential to be synthesized from the combined data.  相似文献   

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