摘要:

For a quarter of a century X‐ray diffraction in single crystals was unique in its ability to solve three‐dimensional structures of proteins and nucleic acids at atomic resolution. The situation changed in 1984 with the completion of a protein structure determination by nuclear magnetic resonance (NMR) spectroscopy in solution, and today NMR is a second widely used method for biomacromolecular structure determination. This review describes the method of NMR structure determination of biological macromolecules, and attempts to place NMR structure determination in perspective with X‐ray crystallography. NMR is most powerful for studies of relatively small systems with molecular weights up to about 30000, but these structures can be obtained in near‐physiological milieus. The two techniques have widely different time scales which afford different insights into internal molecular mobility as well as different views of protein or nucleic acid molecular surfaces and hydration. Generally, in addition to information on the average three‐dimensional structure, NMR provides information on a wide array of short‐lived transient conformational states. Combining information from the two methods can yield a more detailed insight into the structural basis of protein and nucleic acid functions, and thus provide a more reliable platform for rational drug design and the engineering of novel protei

ISSN:1399-0047    

DOI:10.1107/S0907444994010188

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

A protein preparation to be used for crystallization should be homogeneous and should remain so throughout the course of a prolonged crystallization experiment. General methods for preparation of pure proteins and for prevention of their covalent modification (through proteolysis, sulfhydryl oxidation,etc.) during prolonged incubation are well known. Crystallographers are less aware of general methods for stabilization of proteins against non‐covalent modifications (partial denaturation, heterogeneous aggregation) which can also introduce structural heterogeneity into a protein preparation. Related to this issue are methods to suppress protein conformational flexibility which can be a source of dynamic structural heterogeneity and which presents an entropic barrier to crystallization. However, for many years agents which stabilize protein structure have been described in the biochemical literature. Recently the most widely used of these structure‐stabilizing agents, glycerol, was used to crystallize T7 RNA polymerase. The observation that this compound has general structure‐stabilizing effects and that it was essential for crystallization of at least this one protein led to the suggestion that it might be generally useful in crystallizing flexible proteins and in inducing order in disordered segments of crystalline proteins. Subsequently, glycerol was used with good effect in the crystallization of a number of proteins. Other recent results suggest that soaking crystals in solutions containing glycerol can have `structure‐ordering' effects on the crystalline protein. These observations support the utility of glycerol in protein crystallization and suggest that the information in the biochemical literature on protein structure‐stabilizing agents will find useful application in the field of protein cryst

ISSN:1399-0047    

DOI:10.1107/S0907444994014009

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The formation of protein single crystals grown with the shape controlled by the geometry of the capillary used as a growth cell is presented. The shaped crystals show strong birefringence under crossed nicols and diffract as single crystals up to 1.74 

ISSN:1399-0047    

DOI:10.1107/S0907444994013065

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The structure of cytochromec′ from two bacterial species,Alcaligenes spandAlcaligenes denitrificans, have been determined from X‐ray diffraction data to 3.0 Å resolution using the anomalous scattering of the single Fe atom in each to identify and refine a weak molecular‐replacement solution. Molecular‐replacement studies, with the programAMORE, used two isomorphous data sets (from the two species), two independent search models (the cytochromesc′ fromRhodospirillum molischianumandRhodospirillum rubrum), both with and without side chains, and two different resolution ranges (10.0–4.0 and 15.0–3.5Å) to generate a large number of potential solutions. No single solution stood out and none appeared consistently. The Fe‐atom position in each structure was then determined from its anomalous‐scattering contribution and all molecular‐ replacement solutions were discarded which did not (i) place the Fe atom correctly and (ii) orient the molecule such that a crystallographic twofold axis generated a dimer like those of the two search models. Finally, electron‐density maps phased solely by the Fe‐atom anomalous scattering were calculated. As these were combined and subjected to solvent flattening and histogram matching (with the programSQUASH), correlation with the remaining molecular‐replacement solutions identified one as correct and enabled it to be improved and subjected to preliminary refinement. The correctness of the solution is confirmed by parallel is

ISSN:1399-0047    

DOI:10.1107/S0907444994012874

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The X‐ray unliganded structure of 6‐phosphogluconate dehydrogenase (E.C. 1.1.1.44) (6‐PGDH) from sheep liver has been determined at 2Å resolution and refined to a finalR‐factor of 19.8% for 35 031 unique reflections. The enzyme is dimeric, each subunit being comprised of an N‐terminal coenzyme‐binding domain with a Rossmann fold, a large all‐helical domain and a small C‐terminal tail. The model contains 473 residues, three sulfate ions and 346 water molecules; the two best defined sulfates are found in the active site. This structure, based on improved diffraction data, is an extension of the 2.5 Å, resolution model reported earlier. It has good geometry with 92% of the residues falling in the most favoured areas of the Ramachandran plot. Several unusual features are discussed: the incorporation of an alanine in place of the second conserved glycine of the dinucleotide‐binding fingerprint; a duplicated five‐helix motif which is unique to this enzyme; an extended water network at the dimer interface and a C‐terminal tail which is incorporated within the second subunit, forming not only a major part of the dimer interface but als

ISSN:1399-0047    

DOI:10.1107/S0907444994012229

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The crystallization of homogeneous or highly purified macromolecules depends on many variables such as precipitant, pH, choice of buffer, protein concentration, temperature, the participation of different mono‐ and divalent ions, as well as the presence of minute amounts of detergent and organic molecules. Finding the best combination among these many parameters is a multi‐variable optimization problem. This kind of problem can be treated mathematically by sampling techniques. We have used this technique for protein crystallization. The iterative procedure starts with random sampling, followed by quantitative evaluation and cycles with weighted sampling. A simple procedure, derived from this concept and called MON48, has been successfully applied to many protein crystallization probl

ISSN:1399-0047    

DOI:10.1107/S0907444994011728

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

Myotoxin II, a myotoxic calcium‐independent phospholipase‐like protein isolated from the venom ofBothrops asper, possesses no detectable phospholipase activity. The crystal structure has been determined and refined at 2.8 Å to anR‐factor of 16.5% (F>3σ) with excellent stereochemistry. Amino‐acid differences between catalytically active phospholipases and myotoxin II in the Ca2+‐binding region, specifically the substitutions Tyr28→Asn, Gly32→Leu and Asp49→Lys, result in an altered local conformation. The key difference is that the ɛ‐amino group of Lys49 fills the site normally occupied by the calcium ion in catalytically active phospholipases. In contrast to the homologous monomeric Lys49 variant fromAgkistrodon piscivorus piscivorus, myotoxin II is present as a dimer both in solution and in the crystalline state. The two molecules in the asymmetric unit are related by a nearly perfect twofold axis, yet the dimer is radically different from the dimer formed by the phospholipase fromCrotalus atrox. Whereas inC. atroxthe dimer interface occludes the active sites, in myotoxin II they

ISSN:1399-0047    

DOI:10.1107/S0907444994011455

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The oxidized recombinant flavodoxin from the cyanobacteriumAnabaena7120 has been crystallized in a trigonal form. The recombinant protein has an identical primary structure to that purified directly from Anabaena, which functions as a substitute for ferredoxin in an iron‐deficient environment for electron transfer from photosystem I to ferredoxin–NADP+reductase. X‐ray data to 1.40 Å were collected on a Siemens area detector. Of the 311 379 reflections collected, 36069 reflections were unique in space groupP3121 (a= 55.36,c= 102.59 Å) with anRmergeof 3.8%. The structure was solved by molecular replacement using coordinates from the wild‐type monoclinic structure previously solved in this laboratory [Rao, Shaffie, Yu, Satyshur, Stockman&Markley (1992).Protein Sci.1, 1413–1427]. The structure was refined withX‐PLORandSHELXL93 to a crystallographicR‐factor of 13.9% for 32963 reflections withI>2σ(I). The final structure contains 2767 atoms including 31 flavin mononucleotide (FMN) atoms, 299 water molecules, and one sulfate ion. The protein is comprised of a central five‐stranded β‐sheet surrounded by five helices and binds a single molecule of FMN at the C‐terminus of the sheet. The trigonal protein structure and the crystal packing are compared with the monoclinic wild‐type protein. Helix α3 in this structure is less distorted than in the monoclinic structure and shows additional hydrogen bonds in the N‐terminal portion of the helix. The trigonal structure is extensively hydrogen bonded in three major areas with neighboring molecules compared with five regions in the monoclinic structure, but using significantly fewer hydrogen bonds to stabilize the lattice. There are several hydrogen bonds to the amide groups from water molecules several of which stabilize an

ISSN:1399-0047    

DOI:10.1107/S0907444994011716

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

The crystal structure of a goose‐type lysozyme from the egg white of black swan has been determined at 1.9 Å resolution using a semi‐automatic procedure based on the Cα coordinates of the homologous goose

ISSN:1399-0047    

DOI:10.1107/S0907444994009893

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY

摘要:

TR1, a previously cloned gene forCrithidia fasciculatatrypanothione reductase (TR), has been overexpressed inEscherichia colistrain SG5 to produce about 20 mg enzyme 1−lof culture. Since naturalC. fasciculataTR is heterogeneous, this expression system provides an important source of homogeneousC. fasciculataTR for use in structural studies and drug design. Steady‐state kinetic constants of the purified recombinant enzyme areKm= 56 µMandkcat= 10 500 min−1. Four crystal forms of TR1 were grown using this preparation. Synchrotron radiation was crucial to discover the high level of order present in crystal form IV, which diffracts to about 1.4 Å resolution. To optimize growth and handling of form IV crystals, a novel crystallization setup called the `plug drop'

ISSN:1399-0047    

DOI:10.1107/S0907444994010772

出版商:International Union of Crystallography    

年代:1995

数据来源: WILEY