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Snake venom thrombin-like enzymes (SVTLEs) constitute the major portion (10-24%) of snake venom and these are the second most abundant enzymes present in the crude venom. During envenomation, these enzymes had shown prominently the various pathological effects, such as disturbance in hemostatic system, fibrinogenolysis, fibrinolysis, platelet aggregation, thrombosis, neurologic disorders, activation of coagulation factors, coagulant, procoagulant etc. These enzymes also been used as a therapeutic agent for the treatment of various diseases such as congestive heart failure, ischemic stroke, thrombotic disorders etc. Although the crystal structures of five SVTLEs are available in the Protein Data Bank (PDB), there is no single article present in the literature that has described all of them. The current work describes the structural aspects, structure-based mechanism of action, processing and inhibition of these enzymes. The sequence analysis indicates that these enzymes show a high sequence identity (57-85%) with each other and low sequence identity with trypsin (36-43%), human alpha-thrombin (29-36%) and other snake venom serine proteinases (57-85%). Three-dimensional structural analysis indicates that the loops surrounding the active site are variable both in amino acids composition and length that may convey variable substrate specificity to these enzymes. The surface charge distributions also vary in these enzymes. Docking analysis with suramin shows that this inhibitor preferably binds to the C-terminal region of these enzymes and causes the destabilization of their three-dimensional structure., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Proteins isolated from marine invertebrates are frequently characterized by exceptional structural and functional properties. ShPI-1, a BPTI Kunitz-type inhibitor from the Caribbean Sea anemone Stichodactyla helianthus, displays activity not only against serine-, but also against cysteine-, and aspartate proteases. As an initial step to evaluate the molecular basis of its activities, we describe the crystallographic structure of ShPI-1 in complex with the serine protease bovine pancreatic trypsin at 1.7Å resolution. The overall structure and the important enzyme-inhibitor interactions of this first invertebrate BPTI-like Kunitz-type inhibitor:trypsin complex remained largely conserved compared to mammalian BPTI-Kunitz inhibitor complexes. However, a prominent stabilizing role within the interface was attributed to arginine at position P3. Binding free-energy calculations indicated a 10-fold decrease for the inhibitor affinity against trypsin, if the P3 residue of ShPI-1 is mutated to alanine. Together with the increased role of Arg(11) at P3 position, slightly reduced interactions at the prime side (Pn') of the primary binding loop and at the secondary binding loop of ShPI-1 were detected. In addition, the structure provides important information for site directed mutagenesis to further optimize the activity of rShPI-1A for biotechnological applications., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Esperase is a highly alkalophilic bacterial proteinase produced by Bacillus lentus. The enzyme hydrolyzes peptide bonds comprising the carboxylic groups of hydrophobic as well as hydrophilic residues in the oxidized insulin B chain. Some of these bonds are not attacked by other alkaline microbial proteinases. P1-P4 specificity was determined by a series of peptide nitroanilides. The S1 recognition loop exhibits a preference for Phe. The "cleft" of the smallest subsite S2 prefers Ala and exhibits low affinity for the larger chain of Leu. S3 is more open than the other subsites and can accept a variety of residues. Hydrophobic interactions predominate in the S4-P4 interactions because S4 can accommodate Phe very well. The results characterize Esperase as an endopeptidase with a broader specificity in comparison with other microbial serine proteinases. This is probably owing to a more flexible substrate binding site.

Savinase and Esperase are closely related highly alkalophilic proteinases produced by Bacillus lentus. They are suitable couple for investigating the structural basis of proteinase specificity due to the identity of the catalytic and the differences in the substrate binding sites. Two of the substitutions in these sites are very important: T129P and G131P. The two prolines provide an extra rigidity of the Savinase-binding site. The substitutions S166N and Q191T in the S1 recognition loop change the binding geometry of the substrate P1 residue. The geometry of S1 in Esperase is more favorable for binding and catalysis in comparison to that in Savinase. Differences in P3 specificity are probably created by the substitution V104L, which influences the conformation of S3. Leu in position 104 is more favorable for the binding of Phe to S4 than Val. The lower affinity and catalytic efficiency as well as more narrow proteolytic specificity of Savinase in comparison to those of Esperase are explained with the extra rigidity and unfavorable changes in geometry of the substrate binding site of the first enzyme.

Fluorescence emission properties of the alkaline protease Esperase have been investigated using steady-state and time-resolved fluorescence spectroscopy. The local polarity and solvent accessibility of the tryptophyl chromophores is characterized. Quenching studies demonstrated that Trp 6 and Trp 113 are 'buried' to acrylamide, iodide ions and caesium ions. An abnormally low tryptophan quantum yield was calculated showing that the emission of the two indole rings is significantly quenched by nearby side chains or peptide bonds. The fluorescence decay of PMS-Esperase was well fitted by two exponentials with lifetimes of 2.7 and 0.35 ns. X-ray data for Esperase (S. Klupsch, Ph.D. Thesis, University of Hamburg, Hamburg, Germany) in the region of the two tryptophans were used to explain the observed emission properties. Gln 182 and Asn 204 as well as Asn 117 and Met 119 are the most likely quenchers, respectively, of the Trp 6 and Trp 113 fluorescence. The two tryptophans in Esperase are 'buried' in hydrophobic regions and are excellent intrinsic probes to study folding-unfolding reactions. Experiments in the presence and absence of added calcium ions demonstrated the stabilizing role of the Ca(2+)-binding sites.

Savinase and Esperase (EC. 3.4.21.14) are secreted by the alkalophilic bacterium Bacillus lentus and are representatives of that subgroup of subtilisin enzymes with maximum stability in the range of pH 7 to 10 and high activity in the range of pH 8 to 12. The crystal structures of native Savinase and diisopropyl fluorophosphate (DFP) inhibited Esperase have been refined using X-ray data to 1.4 angstroms and 1.8 angstroms resolution respectively collected with synchrotron radiation. The structures were refined to R-factors (=(Sigma//Fo/-/Fc//)/(Sigma/Fo/)) of 16.4% for Esperase and 17.3% for Savinase. The sequence identity between the two enzymes is 66%. The structures are very similar to those of other Bacillus subtilisins. There are two calcium ions in each, equivalent to the strong and the weak sites in subtilisins Carlsberg and BPN'. The structures show novel features which can to some extent be related to their stability and activity. The large number of salt bridges in Esperase and Savinase is likely to contribute to the high thermal stability. Non-conservative substitutions and deletions in the hydrophobic binding pocket S1 as well as the more hydrophobic character of the substrate binding region probably contribute to the alkaline activity profile of the enzymes. Towards the end of the binding site there is an extra proline, Pro131, in Savinase near proline 129, forming a cluster that provides extra active-site rigidity compared with other subtilisins. On the other side of the active site of Esperase and Savinase, the tyrosine found in most other subtilisins is replaced by leucine and valine respectively. The tyrosine potentially interacts with substrate residue P6. At high pH, the negatively charged deprotonated tyrosine could interact unfavorably with the substrate, a possibility that is overcome by substitution with a neutral residue. This is probably one explanation for the shift of the activity profile of Esperase and Savinase to more alkaline pH.

The three dimensional crystal structures of thermitase-bacitracin (TMTBAC), Savinase- bacitracin (SAVBAC) and Savinase-zinc/bacitracin (SAVBAC/ZN) have been determined by X-ray diffraction to 2.2 angstroms, 2.2 angstroms and 1.95 angstroms resolution, respectively. The multifunctional dodecapeptide bacitracin A secreted by Bacillus licheniformis is well known as an antibiotic against gram-positive bacteria but also as an inhibitor for different proteases. The bacteriocidal activity requires the presence of divalent metal cations such as zinc or nickel. It also could be shown that bacitracin A is bound to subtilisin in the Bacillus licheniformis. This complex is stable throughout the purification by chromatography. Therefore the subtilisin proteases thermitase and Savinase were used for cocrystallization with bacitracin A and zinc/bacitracin A. The complexes are formed from two enzyme molecules and two bacitracin A molecules. All three complexes show the same novel mode of enzyme inhibition. Each bacitracin A chain binds non-covalently to two protease molecules: to the catalytic side of one and to the substrate recognition side of the second protease molecule. In that way the two bacitracin A molecules link two subtilisin molecules together to form a dimer. Despite this common feature we found some important differences in the conformations of bacitracin A in the three complex structures which were analysed and described in detail in this paper. An examination of the solvent structure of the complexes shows water molecules in the region around the bacitracin A molecules are not conserved and play a different role in the stabilization of the bacitracin A conformation.

Fluorescence decay kinetics of indole groups in five proteinases from microorganisms are reported. The data show differences between the excited state lifetimes of the tryptophans located in identical positions in the polypeptide chains of the closely related proteinases mesentericopeptidase and subtilisin Novo. The lifetime of the single Trp 113 in subtilisins DY and Carlsberg are identical. The microenvironments of this residue in the four subtilisins are identical and probably its fluorescence is quenched in these proteins. The crystallographic models of the enzymes investigated were analysed in the region of the tryptophyl residues and provide an explanation for the observed emission properties.

The stability towards thermal and chemical (guanidine hydrochloride, GnHCl) denaturation of six inhibited subtilases (mesentericopeptidase, subtilisins BPN', Carlsberg and DY, proteinase K and thermitase) has been investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroic and fluorescence spectroscopy. Experiments in the absence and presence of extraneous calcium in the concentration range 2 x 10(-3)-10(-1) M were performed. The presence of calcium in the weak calcium binding site changes the denaturation drastically. The heat- (or GnHCl-) induced unfolding curves obtained using CD spectroscopy show two independent transitions which seem not to have been resolved before. The presence of Ca2+ in the second (third in the case of thermitase) binding site increases the Tm values by 11-21 degrees C and the delta GD(H2O) values obtained from denaturation experiments in GnHCl by 6.7-7.2 kcal/mol when an extraneous Ca2+ concentration of 2 x 10(-2) M was used. One interpretation is that the initial step of denaturation in the presence of added calcium is the formation of a partially unfolded intermediate form, retaining a highly ordered structure with 60-85% of the alpha-helix structure of the native enzyme. This intermediate then unfolds at a temperature considerably higher than that of the same proteinases in the absence of added Ca2+. The free energy of stabilization of the intermediates is increased by 1.8-2.8 times in comparison with that for the unfolding reactions of the subtilases with empty Ca2/Ca3 binding sites. A second interpretation is that the two steps in the unfolding curves correspond to enzyme without and with calcium in the weak binding site. Fluorescence experiments confirm the mechanism involving the formation of intermediate states. The results are discussed in relation to the X-ray models of the six subtilases.

The subtilisin molecule possesses several internal water molecules, which may be characterised as an integral part of the protein structure. We have introduced specific mutations (T71I, T71S, T71V, T71A and T71G) at position 71 in the subtilisin variant Savinase from Bacillus lentus. This position is involved in a hydrogen bonded network with several internal water molecules, forming a water channel. The water channel and most of the other internal water molecules are positioned in the interface between two half-domains of the subtilisin molecule. The data presented here indicate that the internal water molecules are structural, and may be the result of trapping during the folding process.

The effect of high pH (pH 10.5) on Savinase, a subtilisin-like serine proteinase, has been investigated using X-ray crystallography. The structures of two Savinase mutants were determined at two different pH values, namely pH 6.0, where the enzyme is inactive (this is the pH at which most of the structural work has been carried out on other serine proteinases), and pH 10.5, where Savinase is active. Comparison of these high resolution (0.16 nm) structures showed four related sets of changes between the two pH values. First, the difference in protonation state of the active-site histidine leads to a change in conformation of the active-site triad. Secondly, there are resulting changes in the water structure around this histidine residue with much increased mobility of the water in the active-site at pH 10.5. Thirdly, the two substrate-binding loops on either side of the binding site are less well linked by ordered water molecules at pH 10.5 and show substantially increased flexibility. Finally, the first substrate-binding loop, residues at positions 99-104, moves slightly so as to widen the substrate channel.

The crystal structure of a transition state/product complex formed by the interaction between proteinase K and the substrate analogue N-Ac-L-Pro-L-Ala-L-Pro-L-Phe-D-Ala-L-Ala-NH2 has been determined at a resolution of 2.2 A and refined to an R-factor of 0.165 for 12,725 reflections. The inhibitor forms a stable complex through a series of hydrogen bonds with protein atoms and water molecules. The inhibitor is hydrolyzed between Phe 4I and D-Ala5I (I indicates inhibitor). The two fragments are separated by a distance of 3.07 A between the carbonyl carbon and the main chain nitrogen. Both fragments remain bound to the protein. The N-terminal fragment occupies subsites S5 to S1, whereas the C-terminal part is bound in S1' and S2', the first time that electron density for a substrate analogue has been observed in the P1' and P2' sites of a subtilisin-like enzyme. The flexible segments of the substrate recognition sites Gly100-Tyr104 and Ser132-Gly136 move appreciably to accommodate the inhibitor. Biochemical results indicate an inhibition by this specifically designed peptide of 95%.

Circular dichroic spectroscopy has been used to study the effect of pH, guanidinium hydrochloride concentration and temperature on the conformation of the fungal subtilisin-like proteinase K and the bacterial DY. The ellipticity of the bands in the far ultraviolet region remains almost unchanged in the pH range 3.0-11.0 (PMS-proteinase K) and 5.0-10.0 (PMS-subtilisin DY). The same ranges of pH stability were determined from the pH dependence of the near ultraviolet dichroic spectra. Hence the changes in the tertiary and secondary structure occur in parallel. Proteinase K is considerably more stable at acidic and somewhat more stable at alkaline pH than subtilisin DY. At neutral pH proteinase K is more resistant to denaturation by guanidinium hydrochloride than is subtilisin DY. The midpoints of the denaturation curves were 6.2 M and 3.2 M guanidinium, respectively. The thermal unfolding of proteinase K occurred at a higher temperature than for subtilisin DY, the transition midpoints being 65 degrees and 48 degrees, respectively. Thus proteinase K is overall a much more robust molecule than subtilisin DY, showing greater resistance to all three forms of denaturation. The differences in the stability of the two proteinases can be partly explained by differences in their calcium binding sites.

The fluorescence properties of proteinase K are described and related to the X-ray model refined at 1.48 A resolution. Upon excitation of proteinase K at 295 nm the fluorescence is determined by the two tryptophan residues, Trp-8 and Trp-212. The tryptophans are partly buried just below the surface of the molecule. Neither Trp is in a highly hydrophobic environment, suggesting that this cannot be the explanation for the fluorescence at 330 nm: formation of exiplexes with adjacent peptide bonds would seem to be the more likely cause. Trp-8 is located in a 'cavity', close to an internal cluster of water molecules. The contribution of Trp-8 to the total indole emission is 60% and that of Trp-212 is 40%. The tryptophan fluorescence quantum yield is constant in the pH range 3-9. The fluorescence spectrum resulting from the simultaneous excitation of the tyrosyl and tryptophyl residues at 280 nm is dominated by the indole fluorophores: 61% of the light absorbed by the tyrosyl side chains is transferred to the two indole rings. Iodide and caesium are not efficient quenchers of the proteinase K tryptophan fluorescence, which is explained by restricted access of the ions to the somewhat buried Trp side chains and by electrostatic repulsion of caesium ions. Acrylamide quenching proceeds via both a dynamic and a static process and the data show homogeneity of the indole fluorescence arising from fluorophores in similar environments. The activation energy for the thermal deactivation of the excited tryptophans is 54 kJ mol-1. This value is substantially higher than those found for other proteinases from microorganisms and arises from the thermostability of proteinase K. Photooxidation of proteinase K in the presence of proflavine follows the kinetics of a first order reaction. The two tryptophans differ in their photoreactivity, Trp-212 being considerably more reactive.

Savinase (EC3.4.21.14) is secreted by the alkalophilic bacterium Bacillus lentus and is a representative of that subgroup of subtilisin enzymes with maximum stability in the pH range 7 to 10 and high activity in the range 8 to 12. It is therefore of major industrial importance for use in detergents. The crystal structure of the native form of Savinase has been refined using X-ray diffraction data to 1.4 A resolution. The starting model was that of subtilisin Carlsberg. A comparison to the structures of the closely related subtilisins Carlsberg and BPN' and to the more distant thermitase and proteinase K is presented. The structure of Savinase is very similar to those of homologous Bacillus subtilisins. There are two calcium ions in the structure, equivalent to the strong and the weak calcium-binding sites in subtilisin Carlsberg and subtilisin BPN', well known for their stabilizing effect on the subtilisins. The structure of Savinase shows novel features that can be related to its stability and activity. The relatively high number of salt bridges in Savinase is likely to contribute to its high thermal stability. The non-conservative substitutions and deletions in the hydrophobic binding pocket S1 result in the most significant structural differences from the other subtilisins. The different composition of the S1 binding loop as well as the more hydrophobic character of the substrate-binding region probably contribute to the alkaline activity profile of the enzyme. The model of Savinase contains 1880 protein atoms, 159 water molecules and two calcium ions. The crystallographic R-factor [formula; see text].

The alkaline proteinase from the mesophilic bacterium Bacillus mesentericus has been crystallized in a 1:1 complex with the inhibitor eglin-C from the medical leech. The crystals have cell dimensions of a = 43.0, b = 71.9, c = 48.3 A and beta = 110.0 degrees and are in the space group P2(1). Three-dimensional data to 2.0 A have been recorded on film from a single crystal. The orientation and position of the complex in the unit cell have been established using the refined coordinates of subtilisin Carlsberg and of eglin-C as independent models. The structure of the complex has been refined by restrained least-squares minimization. The crystallographic R factor (= sigma[magnitude of Fo - magnitude of Fc[/sigma magnitude of Fo) is 15.1% including two Ca2+ ions and 312 water molecules. The structure is discussed in terms of its physicochemical properties in solution and its relation to other Bacillus subtilisins.

We compare the three-dimensional structures of thermitase and of proteinase K determined by X-ray crystallography to a resolution of 1.4 and 1.48 A respectively. Both enzymes are relatively stable towards heat and denaturating agents and are representative of a subgroup of subtilisins characterized by a free SH group close to the active site histidine. Even though they have low sequence homology, the overall tertiary structures are highly conserved. The high resolution structures are compared in terms of the overall fold of the molecules, the active sites, the calcium binding sites, disulphide bridge positions, the positions of the charged residues and the solvent structure. Most subtilisins such as thermitase are of prokaryotic origin and proteinase K is up to now the only known eukaryotic structure.

Thermitase, the thermostable alkaline protease from Thermoactinomyces vulgaris, has been crystallised in a 1:1 complex with eglin, the inhibitor from the medical leech. Two large crystals were grown, with cell dimensions of a = 49.3 A, b = 67.3 A, c = 90.5 A and space group P2(1)2(1)2(1). The crystals are relatively tightly packed with Vm = 2.1 A3/Da. Three-dimensional data to 1.9 A have been recorded from one of these crystals. The orientation and position of the complex in the unit cell have been established using the subtilisin Carlsberg-eglin structure as a model. The structure of the complex is being refined by restrained least-squares. The present crystallographic R factor (= sigma parallel Fo - Fc parallel/sigma/Fo parallel) is 26% at 2.5 A resolution.

The crystal and molecular structure of proteinase K was determined by X-ray diffraction data to 0.15-nm resolution. The enzyme belongs to the subtilisin family with an active-site catalytic triad Asp39--His69--Ser224 but is a representative of a subgroup with a free Cys73 close to and 'below' the active His69. Besides this Cys72, proteinase K has two disulfide bonds, Cys34--Cys123 and Cys178--Cys249, which contribute to the stability of the tertiary structure consisting of an extended central parallel beta-sheet decorated by six alpha-helices, three short antiparallel beta-sheets, 18 beta-turns and involving several internal, structurally important water molecules. Proteinase K exhibits two Ca2+-binding sites, one very strong and the other weak, which were the sites of the heavy atoms (Pb2+, Sm3+) used to solve the crystal structure. The weak binding site is liganded to the N and C termini, Thr16 and Asp260, and is only incompletely coordinated by oxygen ligands. The strong binding site is coordinated in the form of a pentagonal bipyramid with the side chain carboxylate of Asp200 and the C = O of Pro175 as apex, and C = O of Val177 and four water molecules in the equatorial plane. Upon removal of this Ca2+, proteinase K loses activity which is interpreted in terms of a local structural deformation involving the substrate-recognition site (Ser132--Gly136), probably associated with a cis----trans isomerization of cis Pro171. Several water molecules are located in the active site. One, W335, is positioned in the 'oxyanion hole' and is displaced by the C = O of the scissile peptide bond of the substrate, as indicated by crystallographic studies with peptide chloromethane inhibitors. Based on these experiments, a reaction mechanism is proposed where the peptide substrate forms a three-stranded antiparallel pleated sheet with the recognition site of proteinase K consisting of Ser132--Leu133--Gly134 on one side and Gly100--Ser101 on the other, followed by expulsion of the oxyanion hole water W335 and hydrolytic cleavage by the Asp39--His69--Serr224 triad. These latter residues display low thermal motion corresponding to well-defined geometry and are hardly accessible to solvent molecules, whereas the recognition-site amino acids are more flexible and partially exposed to solvent.

Proteinase K, the extracellular serine endopeptidase (E.C.3.4.21.14) from the fungus Tritirachium album limber, is homologous to the bacterial subtilisin proteases. The binding geometry of the synthetic inhibitor carbobenzoxy-Ala-Phechloromethyl ketone to the active site of proteinase K was first determined from a Fourier synthesis based on synchrotron X-ray diffraction data between 1.8 A and 5.0 A resolution. The protein inhibitor complex was refined by restrained least-squares minimization with the data between 10.0 and 1.8 A. The final R factor was 19.1%, and the model contained 2,018 protein atoms, 28 inhibitor atoms, 125 water molecules, and two Ca2+ ions. The peptide portion of the inhibitor is bound to the active center of proteinase K by means of a three-stranded antiparallel pleated sheet, with the side chain of the phenylalanine located in the P1 site. Model building studies, with lysine replacing phenylalanine in the inhibitor, explain the relatively unspecific catalytic activity of the enzyme.

The crystal structure of the complex of thermitase with eglin-c in crystal form II, obtained in the presence of 5 mM-CaCl2, has been determined at 1.98 A resolution. The structure was solved by a molecular replacement method, then molecular dynamics crystallographic refinement was started using the thermitase-eglin-c structure as determined for crystal form I. A ten degrees rigid body misplacement of the core of eglin-c was corrected by the molecular dynamics crystallographic refinement without any need for manual rebuilding on a graphics system. A final crystallographic R-factor of 16.5% was obtained for crystal form II. The comparison of the complexes of thermitase with eglin-c in the two crystal forms shows that the eglin-c cores are differently oriented with respect to the protease. The inhibiting loop of eglin binds in a similar way to thermitase as to subtilisin Carlsberg. A tryptophanyl residue at the S4 site explains the preference of thermitase for aromatic residues of the substrate at the P4 site. The difference in the P1 binding pocket, asparagine in thermitase instead of glycine in subtilisin Carlsberg, does not change the binding of eglin-c. The preference for an arginyl residue at the P1 site of thermitase can be explained by the hydrogen bonding with Asn170 in thermitase. Three ion-binding sites of thermitase have been identified. The strong and weak calcium-binding sites resemble the equivalent sites of subtilisin Carlsberg and subtilisin BPN', though there are important amino acid differences at the calcium-binding sites. The medium-strength calcium-binding site of thermitase is observed in the subtilisin family for the first time. The calcium is bound to residues from the loop 57 to 66. A difference in chelation is observed at this site between the two crystal forms of thermitase, which differ in calcium concentration. Additional electron density is observed near Asp60 in crystal form II, which has more calcium bound than form I. This density is possibly due to a water molecule ligating the calcium ion or the result of Asp60 assuming two significantly different conformations.

The structure of the serine endopeptidase proteinase K (279 amino acid residues; 28,790 daltons) has been refined by restrained least-squares methods to a conventional R value of 16.7% employing synchrotron film data of 30,812 reflections greater than 3 sigma in the 5.0 to 1.5 A resolution range. During refinement, the molecular structure was restrained to known stereochemistry, with root-mean-square (r.m.s.) deviation of 0.015 A from ideal bond lengths. The average atomic temperature factor, B, is 11.1 A2 for all atoms. The final model comprises 2020 protein atoms and 174 solvent molecules (which were given unit occupancies). Four corrections to the amino acid sequence were made, which were confirmed later by sequence analysis of the proteinase K gene: a deletion of one glycine in position 80; a change of sequence in position 207-208 and insertions of the dipeptide 210-211 and of residue 270. The r.m.s. deviation in the alpha-C atomic positions between the final refined model and the initial model built on the basis of a 3.3 A mini-map is 1.72 A for 227 out of 266 residues, which were originally traced in the mini-map without sequence information. The positions of the remaining 39 residues deviate by more than 8 A from the original ones and are located in regions where extensive revision of the structural model was necessary.