Macromolecular crystallization in microgravity generated by a superconducting magnet.

About 30% of the protein crystals grown in space yield better X-ray diffraction data than the best crystals grown on the earth. The microgravity environments provided by the application of an upward magnetic force constitute excellent candidates for simulating the microgravity conditions in space. Here, we describe a method to control effective gravity and formation of protein crystals in various levels of effective gravity. Since 2002, the stable and long-time durable microgravity generated by a convenient type of superconducting magnet has been available for protein crystal growth. For the first time, protein crystals, orthorhombic lysozyme, were grown at microgravity on the earth, and it was proved that this microgravity improved the crystal quality effectively and reproducibly. The present method always accompanies a strong magnetic field, and the magnetic field itself seems to improve crystal quality. Microgravity is not always effective for improving crystal quality. When we applied this microgravity to the formation of cubic porcine insulin and tetragonal lysozyme crystals, we observed no dependence of effective gravity on crystal quality. Thus, this kind of test will be useful for selecting promising proteins prior to the space experiments. Finally, the microgravity generated by the magnet is compared with that in space, considering the cost, the quality of microgravity, experimental convenience, etc., and the future use of this microgravity for macromolecular crystal growth is discussed.

Effect of peracylation of beta-cyclodextrin on the molecular structure and on the formation of inclusion complexes: an X-ray study.

The molecular structures of peracylated beta-cyclodextrins (CDs)--heptakis(2,3,6-tri-O-acetyl)-beta-CD (TA), heptakis(2,3,6-tri-O-propanoyl)-beta-CD (TP), and heptakis(2,3,6-tri-O-butanoyl)-beta-CD (TB)--have been determined by single crystal X-ray structure analysis. Due to the lack of O2...O3' hydrogen bonds between adjacent glucose units of the peracylated CDs, the macrocycles are elliptically distorted into nonplanar boat-shaped structures. The glucose units are tilted with respect to the O4 plane to relieve steric hindrance between adjacent acyl chains. In TB, all glucose units adopt the common (4)C(1)-chair conformation and one butanoyl chain intramolecularly penetrates the cavity, whereas, in TA and TP, one glucose unit each occurs in (O)S(2)-skew-boat conformation and one acyl chain closes the O6 side like a lid. In each of the three homologous molecules the intramolecular self-inclusion and lidlike orientation of acyl chains forces the associated O5-C5-C6-O6 torsion angle into a trans-conformation never observed before for unsubstituted CD; the inclusion behavior of TA, TP, and TB in solution has been studied by circular dichroism spectroscopy with the drug molsidomine and several organic compounds. No inclusion complexes are formed, which is attributed to the intramolecular closure of the molecular cavity by one of the acyl chains.

Structure of Urtica dioica agglutinin isolectin I: dimer formation mediated by two zinc ions bound at the sugar-binding site.

Ultica dioica agglutinin, a plant lectin from the stinging nettle, consists of a total of seven individual isolectins. One of these structures, isolectin I, was determined at 1.9 A resolution by the X-ray method. The crystals belong to the space group P2(1) and the asymmetric unit contains two molecules related by local twofold symmetry. The molecule consists of two hevein-like chitin-binding domains lacking distinct secondary structure, but four disulfide bonds in each domain maintain the tertiary structure. The backbone structure of the two independent molecules is essentially identical and this is similarly true of the sugar-binding sites. In the crystal, the C-terminal domains bind Zn(2+) ions at the sugar-binding site. Owing to their location near a pseudo-twofold axis, the two zinc ions link the two independent molecules in a tail-to-tail arrangement: thus, His47 of molecule 1 and His67 of molecule 2 coordinate the first zinc ion, while the second zinc ion links Asp75 of molecule 1 and His47 of molecule 2.

Crystallization and preliminary X-ray studies of V(1)-ATPase of Thermus thermophilus HB8 complexed with Mg-ADP.

Crystals have been grown of the V(1)-ATPase sector of the V-type ATP synthase complex (V(0)V(1)) from the thermophilic eubacterium Thermus thermophilus HB8. These crystals are grown by the vapor diffusion method in the presence of 5 mM Mg-ADP, from solutions containing 100 mM sodium acetate and 2 M sodium formate, pH 5.5. The crystals diffracted X rays beyond 3.4 A in resolution on a synchrotron radiation source. The crystals belong to the trigonal space group P3, with unit cell dimensions of a = b = 89.0 A, c = 179.2 A, and gamma = 120 degrees. The unit cell presumably contains one molecule of V(1)-ATPase and the V(m) value is calculated as 3.0 A(3)/Da.

The novel function of a short region K253xRxxxD259 conserved in the exonuclease domain of hyperthermostable DNA polymerase I from Pyrococcus horikoshii.

The DNA polymerase gene of the hyperthermophile Pyrococcus horikoshii was successfully overexpressed after removing an intein. The importance of an amino acid sequence around a highly conserved Asp was studied by site-directed mutagenesis. The results indicated that Lys253, Arg255, and Asp259 form a novel functional motif, K253xRxxxD259 (outside known motifs Exo I, II, and III), that is important not only for exonuclease activity but also for polymerizing activity, confirming functional interdependence between the polymerase and exonuclease domains. The short loop region, K253G254R255, probably contributes to binding to DNA substrates. Moreover, the negative charge and the side-chain length of D259 might play a supporting role in coordinating the conserved Mg2+ to the correct position at the active center in the exonuclease domain.

Crystal structure of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 complexed with 1-deoxynojirimycin at 2.0 A resolution.

1-Deoxynojirimycin, a pseudo-monosaccharide, is a strong inhibitor of glucoamylase but a relatively weak inhibitor of cyclodextrin glucanotransferase (CGTase). To elucidate this difference, the crystal structure of the CGTase from alkalophilic Bacillus sp. 1011 complexed with 1-deoxynojirimycin was determined at 2.0 A resolution with the crystallographic R value of 0.154 (R(free) = 0.214). The asymmetric unit of the crystal contains two CGTase molecules and each molecule binds two 1-deoxynojirimycins. One 1-deoxynojirimycin molecule is bound to the active center by hydrogen bonds with catalytic residues and water molecules, but its binding mode differs from that expected in the substrate binding. Another 1-deoxynojirimycin found at the maltose-binding site 1 is bound to Asn-667 with a hydrogen bond and by stacking interaction with the indole moiety of Trp-662 of molecule 1 or Trp-616 of molecule 2. Comparison of this structure with that of the acarbose-CGTase complex suggested that the lack of stacking interaction with the aromatic side chain of Tyr-100 is responsible for the weak inhibition by 1-deoxynojirimycin of the enzymatic action of CGTase.

Temperature dependence of the enzyme-substrate recognition mechanism.

We determined the crystal structure of the liganded form of alpha-aminotransferase from a hyperthermophile, Pyrococcus horikoshii. This hyperthermophilic enzyme did not show domain movement upon binding of an acidic substrate, glutamate, except for a small movement of the alpha-helix from Glu16 to Ala25. The omega-carboxyl group of the acidic substrate was recognized by Tyr70* without its side-chain movement, but not by positively charged Arg or Lys. Compared with the homologous enzymes from Thermus thermophilus HB8 and Escherichia coli, it was suggested that the more thermophilic the enzyme is, the smaller the domain movement is. This rule seems to be applicable to many other enzymes already reported.

Crystal structures of heptakis(2,6-di-O-ethyl)cyclomaltoheptaose.

Heptakis(2,6-di-O-ethyl)-beta-cyclodextrin (DE-beta-CD) was crystallized in two forms from hexane and 95% aqueous methanol, respectively: A form I crystal with the space group P2(1)2(1)2(1) and a form II crystal with the space group P3(1). In both crystals, DE-beta-CD molecules are in a round shape with intramolecular O-3-H...O-2 hydrogen bonds. In the form I crystal, the DE-beta-CD molecules are arranged along the twofold screw axis to form a helically extended polymeric chain by including the 6-O-ethyl groups of the adjacent molecule. One hexane molecule with twofold disorder is located in the intermolecular channel along the a-axis. In contrast, the DE-beta-CD molecules in the form II crystal form a helical arrangement along the threefold screw axis. One methanol and one water molecule are included on the O-6 side of the molecular cavity. The water molecule links the methanol molecule and two ethoxy groups of the adjacent DE-beta-CD molecule with hydrogen bonds. The result suggests the important role of solvent in the formation of helical arrangement of DE-beta-CD molecules.

Chemically prepared hevein domains: effect of C-terminal truncation and the mutagenesis of aromatic residues on the affinity for chitin.

Chemically prepared hevein domains (HDs), N-terminal domain of an antifungal protein from Nicotiana tabacum (CBP20-N) and an antimicrobial peptide from Amaranthus caudatus (Ac-AMP2), were examined for their affinity for chitin, a beta-1,4-linked polymer of N-acetylglucosamine. An intact binding domain, CBP20-N, showed a higher affinity than a C-terminal truncated domain, Ac-AMP2. The formation of a pyroglutamate residue from N-terminal Gln of CBP20-N increased the affinity. The single replacement of any aromatic residue of Ac-AMP2 with Ala resulted in a significant reduction in affinity, suggesting the importance of the complete set of three aromatic residues in the ligand binding site. The mutations of Phe18 of Ac-AMP2 to the residues with larger aromatic rings, i.e. Trp, beta-(1-naphthyl)alanine or beta-(2-naphthyl)alanine, enhanced the affinity, whereas the mutation of Tyr20 to Trp reduced the affinity. The affinity of an HD for chitin might be improved by adjusting the size and substituent group of stacking aromatic rings.

Crystallization and preliminary X-ray study of Pk--REC from a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1.

Pk-REC is a protein which binds to DNA and catalyzes the central step of recombination and repair. The protein was crystallized using the hanging-drop vapour-diffusion method with PEG as a precipitant. Two orthorhombic crystal forms I and II with the same space group P2(1)2(1)2(1) were obtained at pH 8.0 using PEG 3000 and PEG 550 monomethylether, respectively. The unit-cell parameters were a = 151, b = 174, c = 241 A for form I and a = 151, b = 176, c = 300 A for form II, indicating that the asymmetric unit contains more than 20 molecules.

Crystal structures of Urtica dioica agglutinin and its complex with tri-N-acetylchitotriose.

Urtica dioica agglutinin is a small plant lectin that binds chitin. We purified the isolectin VI (UDA-VI) and crystal structures of the isolectin and its complex with tri-N-acetylchitotriose (NAG3) were determined by X-ray analysis. The UDA-VI consists of two domains analogous to hevein and the backbone folding of each domain is maintained by four disulfide bridges. The sequence similarity of the two domains is not high (42 %) but their backbone structures are well superimposed except some loop regions. The chitin binding sites are located on the molecular surface at both ends of the dumbbell-shape molecule. The crystal of the NAG3 complex contains two independent molecules forming a protein-sugar 2:2 complex. One NAG3 molecule is sandwiched between two independent UDA-VI molecules and the other sugar molecule is also sandwiched by one UDA-VI molecule and symmetry-related another one. The sugar binding site of N-terminal domain consists of three subsites accommodating NAG3 while two NAG residues are bound to the C-terminal domain. In each sugar-binding site, three aromatic amino acid residues and one serine residue participate to the NAG3 binding. The sugar rings bound to two subsites are stacked to the side-chain groups of tryptophan or histidine and a tyrosine residue is in face-to-face contact with an acetylamino group, to which the hydroxyl group of a serine residue is hydrogen-bonded. The third subsite of the N-terminal domain binds a NAG moiety with hydrogen bonds. The results suggest that the triad of aromatic amino acid residues is intrinsic in sugar binding of hevein-like domains.

Crystal structure of alkalophilic asparagine 233-replaced cyclodextrin glucanotransferase complexed with an inhibitor, acarbose, at 2.0 A resolution.

The product specificity of cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. #1011 is improved to near-uniformity by mutation of histidine-233 to asparagine. Asparagine 233-replaced CGTase (H233N-CGTase) no longer produces alpha-cyclodextrin, while the wild-type CGTase from the same bacterium produces a mixture of predominantly alpha-, beta-, and gamma-cyclodextrins, catalyzing the conversion of starch into cyclic or linear alpha-1,4-linked glucopyranosyl chains. In order to better understand the protein engineering of H233N-CGTase, the crystal structure of the mutant enzyme complexed with a maltotetraose analog, acarbose, was determined at 2.0 A resolution with a final crystallographic R value of 0.163 for all data. Taking a close look at the active site cleft in which the acarbose molecule is bound, the most probable reason for the improved specificity of H233N-CGTase is the removal of interactions needed to form a compact ring like a-cyclodextrin.

Crystal structure of asparagine 233-replaced cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 determined at 1.9 A resolution.

The crystal structure of asparagine 233-replaced cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 was determined at 1.9 A resolution. While the wild-type CGTase from the same bacterium produces a mixture of mainly alpha-, beta- and gamma-cyclodextrins, catalyzing the conversion of starch into cyclic or linear alpha-1,4-linked glucopyranosyl chains, site-directed mutation of histidine-233 to asparagine changed the nature of the enzyme such that it no longer produced alpha-cyclodextrin. This is a promising step towards an industrial requirement, i.e. unification of the products from the enzyme. Two independent molecules were found in an asymmetric unit, related by pseudo two-fold symmetry. The backbone structure of the mutant enzyme was very similar to that of the wild-type CGTase except that the position of the side chain of residue 233 was such that it is not likely to participate in the catalytic function. The active site cleft was filled with several water molecules, forming a hydrogen bond network with various polar side chains of the enzyme, but not with asparagine-233. The differences in hydrogen bonds in the neighborhood of asparagine-233, maintaining the architecture of the active site cleft, seem to be responsible for the change in molecular recognition of both substrate and product of the mutant CGTase.

The molecular structure of hyperthermostable aromatic aminotransferase with novel substrate specificity from Pyrococcus horikoshii.

Aromatic amino acid aminotransferase (ArATPh), which has a melting temperature of 120 degrees C, is one of the most thermostable aminotransferases yet to be discovered. The crystal structure of this aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii was determined to a resolution of 2.1 A. ArATPh has a homodimer structure in which each subunit is composed of two domains, in a manner similar to other well characterized aminotransferases. By the least square fit after superposing on a mesophilic ArAT, the ArATPh molecule exhibits a large deviation of the main chain coordinates, three shortened alpha-helices, an elongated loop connecting two domains, and a long loop transformed from an alpha-helix, which are all factors that are likely to contribute to its hyperthermostability. The pyridine ring of the cofactor pyridoxal 5'-phosphate covalently binding to Lys(233) is stacked parallel to F121 on one side and interacts with the geminal dimethyl-CH/pi groups of Val(201) on the other side. This tight stacking against the pyridine ring probably contributes to the hyperthermostability of ArATPh. Compared with other ArATs, ArATPh has a novel substrate specificity, the order of preference being Tyr > Phe > Glu > Trp > His>> Met > Leu > Asp > Asn. Its relatively weak activity against Asp is due to lack of an arginine residue corresponding to Arg(292)* (where the asterisk indicates that this is a residues supplied by the other subunit of the dimer) in pig cytosolic aspartate aminotransferase. The enzyme recognizes the aromatic substrate by hydrophobic interaction with aromatic rings (Phe(121) and Tyr(59)*) and probably recognizes acidic substrates by a hydrophilic interaction involving a hydrogen bond network with Thr(264)*.

Protein-carbohydrate interactions in human lysozyme probed by combining site-directed mutagenesis and affinity labeling.

The synergism between apolar and polar interactions in the carbohydrate recognition by human lysozyme (HL) was probed by site-directed mutagenesis and affinity labeling. The three-dimensional structures of the Tyr63-->Leu mutant HL labeled with 2',3'-epoxypropyl beta-glycoside of N,N'-diacetylchitobiose (L63-HL/NAG-NAG-EPO complex) and the Asp102-->Glu mutant HL labeled with the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine were revealed by X-ray diffraction at 2.23 and 1.96 A resolution, respectively. Compared to the wild-type HL labeled with the 2', 3'-epoxypropyl beta-glycoside of N,N'-diacetylchitobiose, the N-acetylglucosamine residue at subsite B of the L63-HL/NAG-NAG-EPO complex markedly moved away from the 63rd residue, with substantial loss of hydrogen-bonding interactions. Evidently, the stacking interaction with the aromatic side chain of Tyr63 is essential in positioning the N-acetylglucosamine residue in the productive binding mode. On the other hand, the position of the galactose residue in subsite B of HL is almost unchanged by the mutation of Asp102 to Glu. Most hydrogen bonds, including the one between the carboxylate group of Glu102 and the axial 4-OH group of the galactose residue, were maintained by local movement of the backbone from residues 102-104. In both structures, the conformation of the disaccharide was conserved, reflecting an intrinsic conformational rigidity of the disaccharides. The structural analysis suggested that CH-pi interactions played an important role in the recognition of the carbohydrate residue at subsite B of HL.

Crystallographic evaluation of internal motion of human alpha-lactalbumin refined by full-matrix least-squares method.

The low temperature form of human alpha-lactalbumin (HAL) was crystallized from a 2H2O solution and its structure was refined to the R value of 0.119 at 1.15 A resolution by the full-matrix least-squares method. Average estimated standard deviations of atomic parameters for non-hydrogen atoms were 0.038 A for coordinates and 0.044 A2 for anisotropic temperature factors (Uij). The magnitude of equivalent isotropic temperature factors (Ueqv) was highly correlated with the distance from the molecular centroid and fitted to a quadratic equation as a function of atomic coordinates. The atomic thermal motion was rather isotropic in the core region and the anisotropy increased towards the molecular surface. The statistical analysis revealed the out-of-plane motion of main-chain oxygen atoms, indicating that peptide groups are in rotational vibration around a Calpha.Calpha axis. The TLS model, which describes the rigid-body motion in terms of translation, libration, and screw motions, was adopted for the evaluation of the molecular motion and the TLS parameters were determined by the least-squares fit to Uij. The reproduced Ueqvcal from the TLS parameters was in fair agreement with observed Ueqv, but differences were found in regions of residues, 5-22, 44-48, 70-75, and 121-123, where Ueqv was larger than Ueqvcal because of large local motions. To evaluate the internal motion of HAL, the contribution of the rigid-body motion was determined to be 42.4 % of Ueqv in magnitude, which was the highest estimation to satisfy the condition that the Uijint tensors of the internal motion have positive eigen values. The internal motion represented with atomic thermal ellipsoids clearly showed local motions different from those observed in chicken-type lysozymes which have a backbone structure very similar to HAL. The result indicates that the internal motion is closely related to biological function of proteins.

Dual affinity labeling of the active site of human lysozyme with an N-acetyllactosamine derivative: first ligand assisted recognition of the second ligand.

Among the three kinds of the 2',3'-epoxypropyl beta-glycoside of disaccharides (GlcNAc-beta1,4-GlcNAc, Gal-beta1,4-GlcNAc, and Man-beta1,4-GlcNAc), the derivative of N-acetyllactosamine (Gal-beta1,4-GlcNAc-Epo) caused the dual labeling of human lysozyme (HL) most efficiently. The labeled HL was crystallized and analyzed by X-ray diffraction methodology. The X-ray analysis located the two Gal-beta1,4-GlcNAc-Epo moieties inside the catalytic cleft of HL. The attachment sites were the side-chain carboxylate groups of the catalytic residues Glu35 and Asp53 in HL. The first Gal-beta1, 4-GlcNAc-Epo moiety occupied virtually the same position as observed in the HL labeled with single Gal-beta1,4-GlcNAc-Epo molecule. The second Gal-beta1,4-GlcNAc-Epo moiety was recognized via the carbohydrate-carbohydrate interaction with the first Gal-beta1, 4-GlcNAc-Epo moiety in addition to the protein-carbohydrate interaction with the "right-side" catalytic cleft of HL through a number of hydrogen bonds including water-mediated ones as well as many van der Waals contacts. The two N-acetylglucosamine residues stacked with each other, while the two rings of galactose residues approximately shared the same plane. The dual labeling with two Gal-beta1,4-GlcNAc-Epo molecules was supposed to have occurred sequentially, which was accompanied with the alteration to the pKa of Glu35 derived from the esterification of Asp53 in the first labeling. Both asymmetric carbons in the connection parts between HL and N-acetyllactosamine moieties showed the same stereoconfiguration derived from the reaction with (2'R) stereoisomer concerning the epoxide group in the labeling reagent. The results demonstrated that the HL labeled with single Gal-beta1,4-GlcNAc-Epo was functional as a novel N-acetyllactosamine-binding protein, and the second labeling was performed by way of the first-ligand assisted recognition of the second ligand.

Human lysozyme secretion increased by alpha-factor pro-sequence in Pichia pastoris.

To get high level secretion of human lysozyme in Pichia pastoris, the following three signal sequences and one prepro sequence were evaluated: chicken lysozyme signal peptide, leucine-rich artificial signal peptide, Saccharomyces invertase signal peptide, and Saccharomyces prepro sequence of alpha factor (MF-alpha Prepro). Transformants harboring a lysozyme gene with MF-alpha Prepro secreted 20-fold more lysozyme than those harboring the lysozyme gene with any one of the other three signal sequences. Three mutant leader sequences derived from MF-alpha Prepro were constructed to discover the function of the pro region. The secretion was dramatically decreased by eliminating the pro region of MF-alpha Prepro. In contrast, MF-alpha Prepro with the EAEAEA sequence directed the secretion of an equivalent level of lysozyme having the extra amino acids (EAEAEA) in its N-terminus. For the effective secretion of native human lysozyme, MF-alpha Prepro without any spacer sequences was most suitable. The secreted protein by MF-alpha Prepro construct was identical with the authentic human lysozyme, judging from N-terminal amino acid sequencing and molecular mass spectrometric and crystallographic analysis.

X-ray structure of human lysozyme labelled with 2',3'-epoxypropyl beta-glycoside of man-beta1,4-GlcNAc. Structural change and recognition specificity at subsite B.

Human lysozyme (HL) labelled with the 2',3'-epoxypropyl beta-glycoside of Man-beta1,4-GlcNAc was crystallized at pH 4.5. The cell dimensions were a = 36.39, b = 116.38, c = 30.91 A and the space group was P212121. The unit cell contained four molecules (Vm = 2.18 A3 Da-1). The crystal structure was determined by molecular replacement and refined to an R value of 0.168 for 7060 reflections [|Fo| > 3sigma(F)] in the resolution range 8.0-2.1 A. A prominent shift of the Calpha-atom positions by up to 3.8 A in the region of residues 45-50 was observed compared with wild-type HL. Owing to the conformational change in this region the intermolecular contacts were altered remarkably compared to wild-type HL, explaining the difference in molecular packing. The Man-beta1,4-GlcNAc moiety occupied subsites B and C in the substrate-binding site of HL. Several differences in the hydrogen-bonded contacts between the ligand part and the protein part were observed for HL labelled with the 2',3'-epoxypropyl beta-glycoside of Man-beta1,4-GlcNAc compared with HL labelled with the corresponding derivatives of GlcNAc-beta1, 4-GlcNAc and Gal-beta1,4-GlcNAc. In contrast to the replacement of GlcNAc with Gal, the replacement of GlcNAc with Man did not sacrifice the stacking interactions with the side-chain group of Tyr63 as determined by the parallelism of the apolar face of the carbohydrate residue and the aromatic plane of the Tyr63 side chain. The 2',3'-epoxypropyl beta-glycoside of Man-beta1,4-GlcNAc exhibited almost the same affinity towards HL as Gal-beta1,4-GlcNAc, a much lower affinity than that of GlcNAc-beta1,4-GlcNAc. The difference in the protein-ligand interactions was discussed in relation to the carbo-hydrate-residue recognition specificity at subsite B of HL. The results suggested that Gln104 was a determinant for the strong recognition of GlcNAc residue at subsite B in HL.

Full-matrix least-squares refinement of lysozymes and analysis of anisotropic thermal motion.

Crystal structures of turkey egg lysozyme (TEL) and human lysozyme (HL) were refined by full-matrix least-squares method using anisotropic temperature factors. The refinement converged at the conventional R-values of 0.104 (TEL) and 0.115 (HL) for reflections with Fo > 0 to the resolution of 1.12 A and 1.15 A, respectively. The estimated r.m.s. coordinate errors for protein atoms were 0.031 A (TEL) and 0.034 A (HL). The introduction of anisotropic temperature factors markedly reduced the R-value but did not significantly affect the main chain coordinates. The degree of anisotropy of atomic thermal motion has strong positive correlation with the square of distance from the molecular centroid. The ratio of the radial component of thermal ellipsoid to the r.m.s. magnitude of three principal components has negative correlation with the distance from the molecular centroid, suggesting the domination of libration rather than breathing motion. The TLS model was applied to elucidate the characteristics of the rigid-body motion. The TLS tensors were determined by the least-squares fit to observed temperature factors. The profile of the magnitude of reproduced temperature factors by the TLS method well fitted to that of observed B(eqv). However, considerable disagreement was observed in the shape and orientation of thermal ellipsoid for atoms with large temperature factors, indicating the large contribution of local motion. The upper estimate of the external motion, 67% (TEL) and 61% (HL) of B(eqv), was deduced from the plot of the magnitude of TLS tensors determined for main chain atoms which were grouped into shells according to the distance from the center of libration. In the external motion, the translational portion is predominant and the contribution of libration and screw motion is relatively small. The internal motion, estimated by subtracting the upper estimate of the external motion from the observed temperature factor, is very similar between TEL and HL in spite of the difference in 54 of 130 amino acid residues and in crystal packing, being suggested to reflect the intrinsic internal motion of chicken-type lysozymes.