Crystal structure of a nonsymbiotic plant hemoglobin.

BACKGROUND: Nonsymbiotic hemoglobins (nsHbs) form a new class of plant proteins that is distinct genetically and structurally from leghemoglobins. They are found ubiquitously in plants and are expressed in low concentrations in a variety of tissues including roots and leaves. Their function involves a biochemical response to growth under limited O(2) conditions. RESULTS: The first X-ray crystal structure of a member of this class of proteins, riceHb1, has been determined to 2.4 A resolution using a combination of phasing techniques. The active site of ferric riceHb1 differs significantly from those of traditional hemoglobins and myoglobins. The proximal and distal histidine sidechains coordinate directly to the heme iron, forming a hemichrome with spectral properties similar to those of cytochrome b(5). The crystal structure also shows that riceHb1 is a dimer with a novel interface formed by close contacts between the G helix and the region between the B and C helices of the partner subunit. CONCLUSIONS: The bis-histidyl heme coordination found in riceHb1 is unusual for a protein that binds O(2) reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O(2) affinity is ultra-high (K(D) approximately 1 nM). Our crystallographic model suggests that ligand binding occurs by an upward and outward movement of the E helix, concomitant dissociation of the distal histidine, possible repacking of the CD corner and folding of the D helix. Although the functional relevance of quaternary structure in nsHbs is unclear, the role of two conserved residues in stabilizing the dimer interface has been identified.

Refined structure of yeast apo-enolase at 2.25 A resolution.

The crystal structure of apo-enolase from baker's yeast (Saccharomyces cerevisiae) was established at 2.25 A resolution using a restrained least-squares refinement method. Based on 21,077 independent reflections of better than 8 A resolution, a final R-factor of 15.4% was obtained with a model obeying standard geometry within 0.017 A in bond length and 3.5 degrees in bond angles. The upper limit for the co-ordinate accuracy of the atoms was estimated to be 0.18 A. The refinement confirmed the heterodox, non-parallel character of the 8-fold beta alpha-barrel domain with beta beta alpha alpha(beta alpha)6 topology. The reported structure for which the data were collected at pH 5.0 represents an apo-form of the enzyme. Of the three carboxylic ligands that form the conformational metal ion binding site two, Glu295 and Asp320, are very close and presumably form a strong acidic type hydrogen bond with the proton partially replacing the electric charge of the physiological cofactor Mg2+. The single sulfate ion found in the structure is in the active site cavity, co-ordinated to the side-chains of Lys345 and Arg374, and to the N atom of Ser375. It is located about 7.4 A from the conformational metal ion binding site. It occupies the site in which the phosphate group of the substrate binds.

A revised mechanism for the alkaline phosphatase reaction involving three metal ions.

Here, X-ray crystallography has been used to investigate the proposed double in-line displacement mechanism of Escherichia coli alkaline phosphatase in which two of the three active-site metal ions have a direct role in catalysis. Two new X-ray crystal structures of the wild-type enzyme in the absence and presence of inorganic phosphate have been refined at 1.75 A to final working R-factors of 15.4% and 16.4%, respectively. In the refinement of both structures, residues in the active sites were treated anisotropically. The ellipsoids resulting from the partial anisotropic refinement show a clear route for the binding and release of substrate/product. In addition, a direct comparison of the refined structures with and without phosphate reveal a strong correlation between the occupancy of the third metal-binding site and the conformation of the Ser102 nucleophile. These findings clarify two important and unresolved aspects of the previously proposed catalytic mechanism, how Ser102 is activated for nucleophilic attack and why a magnesium ion in the third metal site is required for catalysis. Analysis of these results suggest that three metal-ion assisted catalysis is a more accurate description of the mechanism of the alkaline phosphatase reaction. A revised mechanism for the catalytic reaction of alkaline phosphatase is proposed on the basis of the two new X-ray crystal structures reported.

A single mutation in the regulatory chain of Escherichia coli aspartate transcarbamoylase results in an extreme T-state structure.

Kinetic analysis of a mutant version of Escherichia coli aspartate transcarbamoylase in which Thr82 in the regulatory chain (Thr82r) was replaced by Ala results in a shift in the T <==> R equilibrium towards the T-state. In order to understand the structural determinants of this T-state stabilization, the X-ray structure of the unliganded Thr82r-->Ala enzyme was determined at 2. 6 A resolution and refined to a crystallographic residual of 0.175. The structure of the mutant r1 regulatory chain is more similar to that of the r6 regulatory chain than observed for the wild-type enzyme, resulting in a more symmetric structure. Furthermore, the structural changes in the mutant enzyme appears to occur only in the r1 chain, while the r6 chain is almost identical in structure to that of the r6 chain of the wild-type enzyme. The structure of the mutant enzyme exhibits alterations in the subunit interfaces between the regulatory and catalytic chains, as well as in the interface between the allosteric and zinc domains within the regulatory chain. Moreover, the regulatory dimers are rotated around their respective 2-fold axes approximately 1 degrees beyond the rotation which occurs in the wild-type T-state enzyme. The structural analysis indicates that the enzyme is an "extreme" T-state, in which a larger rotation of the regulatory dimers is required for the T to R transition compared to the wild-type enzyme. This extreme T-state structure correlates well with the kinetic parameters determined for the mutant enzyme, showing a stabilized T-state. Furthermore, the structural analysis of the mutant enzyme suggests that replacement of Thr82r with Ala alters the local conformation of the nucleotide binding pocket and therefore offers a plausible explanation for the reduced affinity of the enzyme for nucleotides.

Kinetic and X-ray structural studies of three mutant E. coli alkaline phosphatases: insights into the catalytic mechanism without the nucleophile Ser102.

Escherichia coli alkaline phosphatase (EC 3.1.3.1) is a non-specific phosphomonoesterase that catalyzes the hydrolysis reaction via a phosphoseryl intermediate to produce inorganic phosphate and the corresponding alcohol. We investigated the nature of the primary nucleophile, fulfilled by the deprotonated Ser102, in the catalytic mechanism by mutating this residue to glycine, alanine and cysteine. The efficiencies of the S102G, S102A and S102C enzymes were 6 x 10(5)-fold, 10(5)-fold and 10(4)-fold lower than the wild-type enzyme, respectively, as measured by the kcat/Km ratio, still substantially higher than the non-catalyzed reaction. In order to investigate the structural details of the altered active site, the enzymes were crystallized and their structures determined. The enzymes crystallized in a new crystal form corresponding to the space group P6322. Each structure has phosphate at each active site and shows little departure from the wild-type model. For the S102G and S102A enzymes, the phosphate occupies the same position as in the wild-type enzyme, while in the S102C enzyme it is displaced by 2.5 A. This kinetic and structural study suggests an explanation for differences in catalytic efficiency of the mutant enzymes and provides a means to study the nature and strength of different nucleophiles in the same environment. The analysis of these results provides insight into the mechanisms of other classes of phosphatases that do not utilize a serine nucleophile.

The road less travelled: taming phosphatases.

Phosphatases are important in signal transduction, bacterial pathogenesis and several human diseases. So far, however, it is their opposite numbers, the kinases, that have received more attention from chemists. Recent progress in inhibitor development offers hope that new probes of cellular processes, and perhaps novel therapeutic agents, may soon become available.

Crystal structures of the active site mutant (Arg-243-->Ala) in the T and R allosteric states of pig kidney fructose-1,6-bisphosphatase expressed in Escherichia coli.

The active site of pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-1,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain. In this study, we present the X-ray structures of the mutant version of the enzyme with Arg-243 replaced by alanine, crystallized in both T and R allosteric states. Kinetic characteristics of the altered enzyme showed the magnesium binding and inhibition by AMP differed slightly; affinity for the substrate fructose-1,6-bisphosphate was reduced 10-fold and affinity for the inhibitor fructose-2,6-bisphosphate was reduced 1,000-fold (Giroux E, Williams MK, Kantrowitz ER, 1994, J Biol Chem 269:31404-31409). The X-ray structures show no major changes in the organization of the active site compared with wild-type enzyme, and the structures confirm predictions of molecular dynamics simulations involving Lys-269 and Lys-274. Comparison of two independent models of the T form structures have revealed small but significant changes in the conformation of the bound AMP molecules and small reorganization of the active site correlated with the presence of the inhibitor. The differences in kinetic properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase. Calculations using the X-ray structures of the Arg-243-->Ala enzyme suggest that the role of Arg-243 in the wild-type enzyme is predominantly electrostatic in nature.

Evidence for an active T-state pig kidney fructose 1,6-bisphosphatase: interface residue Lys-42 is important for allosteric inhibition and AMP cooperativity.

During the R-->T transition in the tetrameric pig kidney fructose-1,6-bisphosphatase (Fru-1,6-P2ase, EC 3.1.3.11) a major change in the quaternary structure of the enzyme occurs that is induced by the binding of the allosteric inhibitor AMP (Ke HM, Liang JY, Zhang Y, Lipscomb WN, 1991, Biochemistry 30:4412-4420). The change in quaternary structure involving the rotation of the upper dimer by 17 degrees relative to the lower dimer is coupled to a series of structural changes on the secondary and tertiary levels. The structural data indicate that Lys-42 is involved in a complex set of intersubunit interactions across the dimer-dimer interface with residues of the 190's loop, a loop located at the pivot of the allosteric rotation. In order to test the function of Lys-42, we have replaced it with alanine using site-specific mutagenesis. The kcat and K(m) values for Lys-42-->Ala Fru-1,6-P2ase were 11 s-1 and 3.3 microM, respectively, resulting in a mutant enzyme that was slightly less efficient catalytically than the normal pig kidney enzyme. Although the Lys-42-->Ala Fru-1,6-P2ase was similar kinetically in terms of K(m) and kcat, the response to inhibition by AMP was significantly different than that of the normal pig kidney enzyme. Not only was AMP inhibition no longer cooperative, but also it occurred in two stages, corresponding to high- and low-affinity binding sites. Saturation of the high-affinity sites only reduced the activity by 30%, compared to 100% for the wild-type enzyme. In order to determine in what structural state the enzyme was after saturation of the high-affinity sites, the Lys-42-->Ala enzyme was crystallized in the presence of Mn2+, fructose-6-phosphate (Fru-6-P), and 100 microM AMP and the data collected to 2.3 A resolution. The X-ray structure showed the T state with AMP binding with full occupancy to the four regulatory sites and the inhibitor Fru-6-P bound at the active sites. The results reported here suggest that, in the normal pig kidney enzyme, the interactions between Lys-42 and residues of the 190's loop, are important for propagation of AMP cooperativity to the adjacent subunit across the dimer-dimer interface as opposed to the monomer-monomer interface, and suggest that AMP cooperativity is necessary for full allosteric inhibition by AMP.

The 80s loop of the catalytic chain of Escherichia coli aspartate transcarbamoylase is critical for catalysis and homotropic cooperativity.

The X-ray structure of the Escherichia coli aspartate transcarbamoylase with the bisubstrate analog phosphonacetyl-L-aspartate (PALA) bound shows that PALA interacts with Lys84 from an adjacent catalytic chain. To probe the function of Lys84, site-specific mutagenesis was used to convert Lys84 to alanine, threonine, and asparagine. The K84N and K84T enzymes exhibited 0.08 and 0.29% of the activity of the wild-type enzyme, respectively. However, the K84A enzyme retained 12% of the activity of the wild-type enzyme. For each of these enzymes, the affinity for aspartate was reduced 5- to 10-fold, and the affinity for carbamoyl phosphate was reduced 10- to 30-fold. The enzymes K84N and K84T exhibited no appreciable cooperativity, whereas the K84A enzyme exhibited a Hill coefficient of 1.8. The residual cooperativity and enhanced activity of the K84A enzyme suggest that in this enzyme another mechanism functions to restore catalytic activity. Modeling studies as well as molecular dynamics simulations suggest that in the case of only the K84A enzyme, the lysine residue at position 83 can reorient into the active site and complement for the loss of Lys84. This hypothesis was tested by the creation and analysis of the K83A enzyme and a double mutant enzyme (DM) that has both Lys83 and Lys84 replaced by alanine. The DM enzyme has no cooperativity and exhibited 0.18% of wild-type activity, while the K83A enzyme exhibited 61% of wild-type activity. These data suggest that Lys84 is not only catalytically important, but is also essential for binding both substrates and creation of the high-activity, high-affinity active site. Since low-angle X-ray scattering demonstrated that the mutant enzymes can be converted to the R-structural state, the loss of cooperativity must be related to the inability of these mutant enzymes to form the high-activity, high-affinity active site characteristic of the R-functional state of the enzyme.

Alternate modes of binding in two crystal structures of alkaline phosphatase-inhibitor complexes.

Two high resolution crystal structures of Escherichia coli alkaline phosphatase (AP) in the presence of phosphonate inhibitors are reported. The phosphonate compounds, phosphonoacetic acid (PAA) and mercaptomethylphosphonic acid (MMP), bind competitively to AP with dissociation constants of 5.5 and 0.6 mM, respectively. The structures of the complexes of AP with PAA and MMP were refined at high resolution to crystallographic R-values of 19.0 and 17.5%, respectively. Refinement of the AP-inhibitor complexes was carried out using X-PLOR. The final round of refinement was done using SHELXL-97. Crystallographic analyses of the inhibitor complexes reveal different binding modes for the two phosphonate compounds. The significant difference in binding constants can be attributed to these alternative binding modes observed in the high resolution X-ray structures. The phosphinyl group of PAA coordinates to the active site zinc ions in a manner similar to the competitive inhibitor and product inorganic phosphate. In contrast, MMP binds with its phosphonate moiety directed toward solvent. Both enzyme-inhibitor complexes exhibit close contacts, one of which has the chemical and geometrical potential to be considered an unconventional hydrogen bond of the type C-H...X.

Homology modeling of the dopamine D2 receptor and its testing by docking of agonists and tricyclic antagonists.

We present the first model of dopamine D2 receptor transmembrane helices constructed directly from the bacteriorhodopsin (bR) coordinates derived from two-dimensional electron diffraction experiments. We have tested this model by its ability to accommodate rigid agonist and semirigid antagonist molecules which were docked into the putative binding pocket with stabilizing interactions. The model is consistent with structure-activity relationships of agonists and antagonists that interact with the receptor. It also illuminates data on a Na+ site for regulation of receptor function. The plausibility of the model is increased by its consistency with many mutagenesis studies on G protein-coupled receptors. Further, this model provides a basis to suggest testable molecular mechanisms for changes in the D2 conformational states for high- and low-affinity binding and signal transduction. Changes in the conformational state of the receptor are hypothesized to be due partly to movement of helix 7. In contrast to the model presented here, other published models were built using ideal helical structures or following the sense of the bacteriorhodopsin structure rather than the actual available coordinates. The presented model for the dopamine G protein-coupled receptor can be reconciled with the recent rhodopsin projection structure (Schertler, G. F. X.; Villa, C.; Henderson, R. Projection Structure of Rhodopsin.

Mapping of isozymic differences in enolase.

The existence of the isozymes of non-regulatory enzymes often has been linked to their interaction with other macromolecules. Enolase, a non-regulatory enzyme, has three isozymes for which sequences have been determined in two or more vertebrate species. The positions in the enolase sequences that differ between the isozymes were mapped in the 3-D structure of the enzyme. The positions in a given isozymic form which were not conserved in different species were considered to be resulting from the neutral drift of sequences and rejected. Also, the residues with no accessible surface were rejected. Three areas with relatively high densities of isozymic substitutions were found. We consider them as the likely sites of contact with other macromolecules.

Plant thionins--the structural perspective.

Thionins belong to a rapidly growing family of biologically active peptides in the plant kingdom. Thionins are small ( approximately 5 kDA), cysteine-rich peptides with toxic and antimicrobial properties. They show a broad cellular toxicity against wide range of organisms and eukaryotic cell lines; while possessing some selectivity. Thionins are believed to be involved in protection against plant pathogens, including bacteria and fungi, by working directly at the membrane. The direct mechanism of action is still surrounded by controversy. Here the results of structural studies are reviewed and confronted with recent results of biophysical studies aimed at defining the function of thionins. The proposed toxicity mechanisms are reviewed and the attempt to reconcile competing hypotheses with a wealth of structural and functional studies is made.

Crystal structure of enolase indicates that enolase and pyruvate kinase evolved from a common ancestor.

Enolase or 2-phospho-D-glycerate hydrolase catalyses the dehydration of 2-phosphoglycerate to phosphoenolpyruvate, which in turn is converted by pyruvate kinase to pyruvate. We describe here the crystallographic determination of the structure of yeast enolase at high resolution (2.25 A) and an analysis of the structural homology between enolase, pyruvate kinase and triose phosphate isomerase. Each of the two subunits of enolase forms two distinctive domains. The larger domain (residues 143-420) is a regular 8-fold beta/alpha-barrel, as first found in triose phosphate isomerase, and later in pyruvate kinase and 11 other functionally different enzymes. An analysis of the molecular geometries of enolase and pyruvate kinase based on the roughly 8-fold symmetry of the barrel showed a structural homology better than expected for proteins related by convergent evolution. We argue that enolase and pyruvate kinase have evolved from a common ancestral multifunctional enzyme which could process phosphoenolpyruvate in both directions along the glycolytic pathway. There is structural and sequence evidence that muconate lactonizing enzyme later evolved from enolase.

Mechanism of enolase: the crystal structure of enolase-Mg2(+)-2-phosphoglycerate/phosphoenolpyruvate complex at 2.2-A resolution.

Enolase in the presence of Mg2+ catalyzes the elimination of H2O from 2-phosphoglyceric acid (PGA) to form phosphoenolpyruvate (PEP) and the reverse reaction, the hydration of PEP to PGA. The structure of the ternary complex yeast enolase-Mg2(+)-PGA/PEP has been determined by X-ray diffraction and refined by crystallographic restrained least-squares to an R = 16.9% for those data with I/sigma (I) greater than or equal to 2 to 2.2-A resolution with a good geometry of the model. The structure indicates the substrate molecule in the active site has its hydroxyl group coordinated to the Mg2+ ion. The carboxylic group interacts with the side chains of His373 and Lys396. The phosphate group is H-bonded to the guanidinium group of Arg374. A water molecule H-bonded to the carboxylic groups of Glu168 and Glu211 is located at a 2.6-A distance from carbon-2 of the substrate in the direction of its proton. We propose that this cluster functions as the base abstracting the proton in the catalytic process. The proton is probably transferred, first to the water molecule, then to Glu168, and further to the substrate hydroxyl to form a water molecule. Some analogy is apparent between the initial stages of the enolase reverse reaction, the hydration of PEP, and the proteolytic mechanism of the metallohydrolases carboxypeptidase A and thermolysin. The substrate/product binding is accompanied by large movements of loops Ser36-His43 and Ser158-Gly162. The role of these conformational changes is not clear at this time.

How the CO in myoglobin acquired its bend: lessons in interpretation of crystallographic data.

Contrary to the expectation of chemists, the first X-ray structures of carbon monoxide bound to myoglobin (Mb) showed a highly distorted Fe-C-O bond system. These results appeared to support the idea of a largely steric mechanism for discrimination by the protein against CO binding, a lethal act for the protein in terms of its physiological function. The most recent independently determined high-resolution structures of Mb-CO have allowed the 25 year old controversy concerning the mode of CO binding to be resolved. The CO is now seen to bind in a roughly linear fashion without substantial bending, consistent with chemical expectations and spectroscopic measurements. Access to deposited diffraction data prompted a reevaluation of the sources of the original misinterpretation. A series of careful refinements of models against the data at high (1.1 A) and modest resolutions (1.5 A) have been performed in anisotropic versus isotropic modes. The results suggest that the original artifact was a result of lower quality crystals combined with anisotropic motion and limited resolution of the diffraction data sets. This retrospective analysis should serve as a caution for all researchers using structural tools to draw far-reaching biochemical conclusions.

The structure of yeast enolase at 2.25-A resolution. An 8-fold beta + alpha-barrel with a novel beta beta alpha alpha (beta alpha)6 topology.

The three-dimensional structure of yeast enolase has been determined by the multiple isomorphous replacement method followed by the solvent flattening technique. A polypeptide model, corresponding with the known amino acid sequence, has been fitted to the electron density map. Crystallographic restrained least-squares refinement of the model without solvent gave R = 20.0% for 6-2.25-A resolution with good geometry. A model with 182 water molecules and 1 sulfate which is still being refined has presently R = 17.0%. The molecule is a dimer with subunits related by 2-fold crystallographic symmetry. The subunit has dimensions 60 X 55 X 45 A and is built from two domains. The smaller N-terminal domain has an alpha + beta structure based on a three-stranded antiparallel meander and four helices. The main domain is an 8-fold beta + alpha-barrel. The enolase barrel is, however, different from the triose phosphate isomerase barrel; its topology is beta beta alpha alpha (beta alpha)6 rather than (beta alpha)8 as found in triose phosphate isomerase. The inner beta-barrel is not entirely parallel, the second strand is antiparallel to the other strands, and the direction of the first helix is also reversed with respect to the other helices. This supports the hypothesis that some enzymes evolved independently producing the stable structure of beta alpha barrels with either enolase or triose phosphate isomerase topology. The active site of enolase is located at the carboxylic end of the barrel. A fragment of the N-terminal domain and two long loops protruding from the barrel domain form a wide crevice leading to the active site region. Asp246, Glu295, and Asp320 are the ligands of the conformational cation. Other residues in the active site region are Glu168, Asp321, Lys345, and Lys396.

Inhibition of enolase: the crystal structures of enolase-Ca2(+)- 2-phosphoglycerate and enolase-Zn2(+)-phosphoglycolate complexes at 2.2-A resolution.

Enolase is a metalloenzyme which catalyzes the elimination of H2O from 2-phosphoglyceric acid (PGA) to form phosphoenolpyruvate (PEP). Mg2+ and Zn2+ are cofactors which strongly bind and activate the enzyme. Ca2+ also binds strongly but does not produce activity. Phosphoglycolate (PG) is a competitive inhibitor of enolase. The structures of two inhibitory ternary complexes: yeast enolase-Ca2(+)-PGA and yeast enolase-Zn2(+)-PG, were determined by X-ray diffraction to 2.2-A resolution and were refined by crystallographic least-squares to R = 14.8% and 15.7%, respectively, with good geometries of the models. These structures are compared with the structure of the precatalytic ternary complex enolase-Mg2(+)-PGA/PEP (Lebioda & Stec, 1991). In the complex enolase-Ca2(+)-PGA, the PGA molecule coordinates to the Ca2+ ion with the hydroxyl group, as in the precatalytic complex. The conformation of the PGA molecule is however different. In the active complex, the organic part of the PGA molecule is planar, similar to the product. In the inhibitory complex, the carboxylic group is in an orthonormal conformation. In the inhibitory complex enolase-Zn2(+)-PG, the PG molecule coordinates with the carboxylic group in a monodentate mode. In both inhibitory complexes, the conformational changes in flexible loops, which were observed in the precatalytic complex, do not take place. The lack of catalytic metal ion binding suggests that these conformational changes are necessary for the formation of the catalytic metal ion binding site.

Structure of human plasminogen kringle 4 at 1.68 a and 277 K. A possible structural role of disordered residues.

Despite considerable effort to elucidate the functional role of the kringle domains, relatively little is known about interactions with other protein domains. Most of the crystal structures describe the interactions at the kringle active site. This study suggests a novel way to interpret structural results such as disorder located away from an active site. The crystal structure of human plasminogen kringle 4 (PGK4) has been refined against 10-1.68 A resolution X-ray data (R(merge) = 3.7%) to the standard crystallographic R = 14.7% using the program X-PLOR. The crystals of PGK4 showed significant instability in cell dimensions (changes more than 1.5 A) even at 277 K. The refinement revealed structural details not observed before [Mulichak, Tulinsky & Ravichandran (1991). Biochemistry, 30, 10576-10588], such as clear density for additional side chains and more extensive disorder. Discrete disorder was detected for residues S73, S78, T80, S89, S91, S92, Ml12, S132, C138 and K142. Most of the disordered residues form two patches on the surface of the protein. This localized disorder suggests that these residues may play a role in quaternary interactions and possibly form an interface with the other domains of proteins that contain kringles, such as plasminogen. Although, an additional residue D65 was refined at the beginning of the sequence, still more residues near the peptide cleavage site must be disordered in the crystal.

On the nature of a glassy state of matter in a hydrated protein: Relation to protein function.

Diverse biochemical and biophysical experiments indicate that all proteins, regardless of size or origin, undergo a dynamic transition near 200 K. The cause of this shift in dynamic behavior, termed a "glass transition," and its relation to protein function are important open questions. One explanation postulated for the transition is solidification of correlated motions in proteins below the transition. We verified this conjecture by showing that crambin's radius of gyration (Rg) remains constant below approximately 180 K. We show that both atom position and dynamics of protein and solvent are physically coupled, leading to a novel cooperative state. This glassy state is identified by negative slopes of the Debye-Waller (B) factor vs. temperature. It is composed of multisubstate side chains and solvent. Based on generalization of Adam-Gibbs' notion of a cooperatively rearranging region and decrease of the total entropy with temperature, we calculate the slope of the Debye-Waller factor. The results are in accord with experiment.