J. Mol. Biol. (1975) 91, 293-300
Crystals of Glutamine Synthetase from Escherichia coli R. P. BYw&rERt,
C. H. CARLISLE, R. B. JACKSONS A. L. MACKAY AND P. A. TIMMINS~
Department of Crystallography Birkbeck College, University of London Malet &beet, London, W.C.1 (Received 30 May 1974, and in revised form 2 October 1974) Further details are given of crystals of glutamine synthetaae prepared from Eecherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et a2. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and ptxking considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetaae preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are a = 140 & 2 A, and c = 148 & 2 b. The Laue symmetry group is 3m P31.
1. Introduction Since work on Escherichia coli glutamine synthetase has been fully reviewed very recently by Ginsburg (1972) it is necessary to mention only details pertinent to our investigation which was continued from that reported in an earlier paper (Bywater et al., 1969). Crystals of glutamine synthetase were grown from a solution of ammonium sulphate, 40% saturated, at pH 6-l and 18°C. Although some dozen tubes were kept under nearly identical conditions, only one tube produced crystals large enough for X-ray work. Further attempts to repeat the crystallization also failed.
2. Optical examination Crystals were optically almost isotropic. Most of them were rhombohedra with maximum dimensions O-07 mm and an apex angle a = 95.5” f l”, although the three apex angles could not be checked simultaneously. t Present address: Ph armaoia Fine Chemicals AB, Box 176, S-76104 Uppaala 1, Sweden. $ Present address: Rhode Island Junior College, Knight Campus, 400 East Ave., Warwick, Rhode Island 02886, U.S.A. 0 Present address: Institut Max von Laue-Paul Langevin, BP166 Centre de tri, 38042 Grenoble Cedex, France. 293
294
R. P. BYWATER
ET
AL.
Indexed with respect to the hexagonal axes (see next section) the majority of faces appear to be of the form (loll>. Other crystals with the form of rhombic dodecahedra, extended along one cube axis, were also seen.
3. Crystal handling techniques In order to get these extremely small crystals into tubes for X-ray examination a special micromanipulator was used for bringing up the tube to the crystal which was then sucked in (Timmins, 1972). Radiation damage was particularly acute because of the sma,ll size of the crystals, and X-ray setting had to be kept to a minimum so that crystals were studied in the first rational orientation encountered when the orientation could not be recognized from the external form. X-ray pictures were taken only from the rhombohedral crystals. 4. Unit cell dimensions X-ray precession photographs (using a fine-focus rotating anode tube) showed the unit cell to be hexagonal with dimensions a=b=140&2A c = 148 f 2 A. In our earlier paper (Bywater et al., 1969) the cell was reported as being monoclinic (a = 242, b = 140, c = 148 8, with p = 90’) the dimensions of which correspond to an orthohexagonal cell equivalent to the above hexagonal cell (since 1402/3 = 242). Subsequently an [OO*l] axis precession photograph (Plate I) showed clearly the 6 mm symmetry of the h&O layer. A number of zero layer photographs in ten different directions were obtained and collated. In the best example reflections extended to 8 A. The weighted reciprocal lattice appeared roughly cubic in general intensity distribution but did not even approximately show rhombohedral extinctions. For the above hexagonal cell the angle u between possible rhombohedral axes is 95.33” (correspondu = 2 arcsin (a/2)/[(c/3)” + (a/3)“]* and arh = 94.7 A). (The ing to the form {liol}, vertex angle of the form (2132) with the same cell dimensions is 95*27”, indistinguishable from the above.)
5. Space group The diffraction symmetry is 3m, the presence of a 6-fold axis being excluded by the difference between the intensities of 1On and liO2 reflections in the [20*1] zone (allowing for different Lorentz polarization factors). 001 reflections were only present for 1 = 3n. There were no systematic rhombohedral extinctions. Taking into account the absence of improper symmetry operations from cheiral structures, the possible space groups are thus P3121, P3,21, P3112 and P3,12. Intensities were estimated by eye and the Patterson function P (U, V, W) was calculated. There were two notable features in the vector distribution: (i) no significant disposition of vectors on W = 0 and (ii) a heavy vector of length about 50 A lying approximately 33” from c and arising chiefly from the 20’21 reflection.
/ ,‘,~‘ti”,
,’
‘!!!,
PLATE II. (a) (dimensions about (b) Crystals of the rhombohedral (c) Photograph reproduced from
Rhombohedral crystals of glutamine synthetasr as uwtl in the pwscnt SI u(I? 0.1 mm). elongated rhombic dodecahodral form also ohwrwd in thv same pr~~paratio~l as crystals (dimensions also about 0.1 mm). of glutamine synthetaw crystals of the olortgated rhombic ~lotlrcah~~lral ~OIY~ Eisanberg et 02. (1972).
PLATE III. Electron micrographs of various aggregations, not necessarily Ibnra represent 1000 A. (a) Isolated dodecamers wcur as eclipsed hexagons 130 A& overall. Nnrron(h) Thr dodcoamws we frequently associated into columns.
synthetase, rotl~ are also ohserve~l.
of glntamine
found
in preparations
of glutamine
synthetasr.
SC~III.
CRYSTALS
OF GLUTAMINE
SYNTHETASE
295
6. Unit cell contents Using the molecular weight of 592,000, a value of 0.74 ml/g for the protein specific volume and a solvent content of 45% (Matthews, 1968) there appear to be 24 subunits/ unit cell. Assuming that the crystals belong to any one of the space-groups mentioned above, where the general position is 6-fold, the asymmetric unit must contain four protein subunits. These could be paired to yield three octomers per unit cell, with the octomer containing a S-fold axis. Whatever the manner of the clustering of the subunits this would be in keeping with the volume of our unit cell, 2.51 x IO6 A3, lower than that found by Eisenberg et al. (1971) which was 2.89 x IO6 a3 for the tetragonal unit cell of their crystal.
7. Electron microscope observations The electron microscopic examination of the solutions of glutamine synthetase which produced crystals of the form we describe showed a wide variety of configurations. (We were not able to examine an actual crystal broken down on to an electron microscope grid.) Looking back critically at some of Valentine’s photographs of the dodecamers, through the courtesy of Dr N. Wrigley, we see that small numbers of other particles are present there also.
FIG. 1. The T = 7 structure which is a possible packing for dodeoamers in Valentine’s wheatsheaf assemblies of glut&mine s.ynthetase particles. The hexagonal cell (with a = 140 A) is seen sectioned perpendicular to c. Circles represeut subunits.
296
R.
P. BYWATER
ET
AL.
In summary, we find (Plate III) : (1) Dodecamers in the form of eclipsed hexagons corresponding closely to the particles described by Valentine et al. (1968) and by Eisenberg et al. (1971) and composed of subunits of 45 to 50 A diameter, although the subunits appear to have substructures leading them to be non-spherical. When seen end-on they often have the symmetry 6 and not 6 mm. (2) These dodecamers are frequently common).
associated into columns (hexagonal axes in
(3) The columns tend to associate further side by side parallel to each other. Mutual contacts correspond to a “mirror plane”, that is, protuberances do not interdigitate. This leads us to suggest that Valentine’s wheatsheaf structure may be parallel stacking AA AA with a T = 7 cross-section (Fig. 1). (4) Large open square tetramers (or possibly cubic octomers) of overall dimensions some 120 A. These are observed to associate in squarely-packed arrays.
FIQ. 2. Plan of the tetragonel structure of glutamine synthetase proposed by Eisenberg e-6al. (1971) for their crystals of elongated rhombic dodeoahedml form. The oell ia men seotioned perpendicular to c. Circles represent subunits and a = 129 8.
CRYSTALS
OF
GLUTAMINE
297
SYNTHETASE
(5) Narrow rods some 60 A across. Substructure showing the rod to be a double row of particles at intervals of about 40 A is evident. These particles appear in parallel association and tend to lie across each other at close to 4.5”. (6) Other sporadic configurations such as pentamers, larger rings, globules. We conclude that: (i) the subunits are by no means spherical, and (ii) there is no clear correspondence between any one form and the crystallographic data of our crystals.
8. Comparison with crystals from Eisenberg et al. (1971) Since our earlier paper, Eisenberg et at. (1971) have described crystals of glutamine synthetase from E. coli. Their unit cell is tetragonal with a = 129, c = 174 A and appears to contain two dodecamers of glutamine synthetase with dimensions close to those described by Valentine et a.l. (1968) from electron microscopic examination. The Eisenberg structure (Fig. 2) can be described topologically as being either a body-centred cubic arrangement of dodecamers (parallel to each other) with cell dimensions as given, or as a face-centred cubic arrangement with cell dimensions a = 2/2a = 182, c = 174 A. A face-cent& cell could also correspond to a rhombohedral lattice with a M 60” (or 104”). The vertex angle of a 110 face would be 92.7” from Eisenberg’s cell dimensions, which agrees only roughly with that in the photograph of his crystals which appear to be rhombic dodecahedra, extended somewhat along c, exactly like those visible as a minorit,y component in our preparation (Bywater et al., 1969). As Eisenberg’s cell explains the form of his crystals but not the rhombohedral form, whereas our cell explains the rhombohedral form but not the tetragonal form, we must tentatively conclude that two crystal structures coexist. This can be seen in the photograph of the crystals reproduced by Bywater et al. (1969) and repeated in TAEZLE~ Conditions for crystallizution Solvent/solute parameters
40% saturated %2*3 aa
W&)sSO~
(1VC)
None in crystallizing solution (enzyme was prepared in presence of excess imidazole/MxP + /etc.)
Buffers PH Ionic strength Enzyme
Birkbeck conditions
concn
State of enzyme Temperature
6.1 -6-9 10 mg/ml Adenylylated 18°C
Eisenberg’s conditions 1.86 M N 31% saturated (4’C) 0.02 rd-oitrate/imidazole 0.001 wMnCl, 0.01 rd-histidine 0.001 M-glutathione 6.9 ~6.8 7 mg/ml A deny1 ylatcd 4°C
298
R. P. BYWATER
ET
AL.
Plate II(a) and (b). This photograph shows crystals looking very much like those in the photograph (Plate II(c)) reproduced by Eisenberg et al. co-existing with rhombohedral crystals. Table 1 compares the growth conditions for the two cases.
9. Model structures Only two model structures appeared as a result of considering the packing of 24 subunits of about 60 A diameter into our unit cell. One (referred to as the B.C.C. model) consists of octonaers (8 spheres at the vertices of a cube) and the other (referred to as the T = 7 model) consists of dodecamers (12 spheres arranged as two hexagonal rings in the eclipsed position). We now consider the geometry of the models.
10. B.C.C. (body-centred cubic) model (Fig. 3) If octomers in the form of cubes are assembled, so that one sphere lies in the cavity between the four spheres making the face of the adjacent cube, an interesting rhombohedral structure results where the hexagonal unit cell contains three octomers. If the diameter of the spherical subunit is D, then the dimensions of this cell are a = (912 + 22/2)+
D = 2.707 D and
c = 2/3 (1 + l/d2)
D = 2.957 D.
The ratio c/a is thus 1992 corresponding to a = 94.2”. Our own dimensions thus give (for a = 140) D = 51.7 A and (for c = 148) D = 50.0 it. It might be noted that this is a dense packing of spheres (not all in equivalent positions but with two subunits on trigonal axes and 6 off them). If further subunits were inserted, one at the centre of each cube of eight subunits, and the structure were
GS
PRCKINC
OF
OCIOHERS
FIG. 3. Perspective view of the B.C.C. structure proposed for the packing of octomers of glutamine synthetsse (GS). The dimensions of the hexagonal call sre a = 140 and c = 148 A. Each subunit is represented as a dumbell to lower its symmetry.
CRYSTALS
OF
GLUTAMINE
SYNTHETASE
299
expanded in compensation, then the resulting structure would simply be body-centredcubic (as a-Fe). Parameters are: 2 spheres at (0, 0, O-293) 6 spheres at (0.072, O-317, 0.097). If the subunits are not spheres but, as seems clear from the electron micrographs, much less regular, then according to the a,ssembly into octomers, the symmetry will be reduced from rhombohedral R 32. In the computer-produced drawing of Figure 3, each of the eight subunits making a cubic octomer has been represented as a dumbell in order to reduce the local symmetry. In particular, if the octomers are each composed of two tetramers, then the structure cannot be rhombohedral. Tests were made with pear-shaped units (two tetramers related by a local dyad) but no decisive agreement with observed intensities was found.
11. T = 7 model (Fig. 1) A possible model for the packing of dodecamers is based on the T = 7 tessellation of the hexagonal lattice. This would give a value D = a/d7 = 52.9 A for the diamet,er of spherical subunits. It might be noted that in one direction the units in the Eisenberg structure shown in Figure 2 are packed in this way. If the T = 7 model were sheared so that the interaxial angle of 60” were opened to 90” then the layers of the Eisenberg structure would be obtained. The density would be correspondingly reduced (to 87%). There would have to be four layers of spheres for the T = 7 model packing of dodeca,hedra and this sequence must be AABB. Using the c-dimension of 148 A this would lead to a sphere diameter of 40.7 A. (D = c/(2 + 2/8/d3).) It should be noted that very similar constellations of subunits occur in all three models (Eisenberg, B.C.C. and T = 7). As three different types of cavities appear in a T = 7 layer a number of variants of stacking are possible and might even occur simultaneously in disorder.
12. Conclusions We conclude that for the large crystals examined by X-ray diffraction the T = 7 structure is unlikely because: (a) if there were four layers of units (24 per cell) there would be extinctions for 1 # 2% instead of for 1 # 3n, (b) if there were three layers of units this would imply hexamers and (c) the structure would be too compressed, in comparison with Eisenberg’s structure, in the c direction. The B.C.C. structure is more likely because: (a) the non-sphericity of the subunits removes rhombohedral extinctions but the 3, axis remains ; (b) there is a better fit with the Patterson data. In all, the most probable configuration is that of two tetramers assembled face to face into an octomer and related by a dyad axis. We consider it likely that there are two different forms of glutamine synthetase crystals, those observed by ourselves and those observed by Eisenberg et al. (1971). The electron microscope results show dodecamers as seen by Valentine and small rods which are probably assemblies of octomers or tetramers. There is some sign that dodecamers can break down into three tetramers. 21
300
R. I’.
BYWATER
ET
AL.
The large tetramers observed seem too large to be the same tetramers we have been discussing above. Wrigley (personal communication) has pointed out their similarity with tetramers of pyruvate carboxylase, but they remain unexplained. We acknowledge the technical assistance of Sheila Lauchlan and Douglas Parry and the usual unfailing helpfulness of our other colleagues. One of us (P. A. T.) gratefully acknowledges the Gnancial assistance of Birkbeck College. Another author (R. B. J) was a post-doctoral fellow of the National Cancer Institute (U.S.A.). REFERENCES Bywater, R. P., Carlisle, C. H. & Jackson, R. B. (1969). J. Mol. Bill. 45, 429-431. Eisenberg, D., Heidner, E. C., Goodkin, P., Dastoor, M. N., Weber, B. H., Wedler, F. & Bell, J. D. (1971). Cold Spring Harbor Symp. Quant. Bill. 36, 291-294. Ginsburg, A. (1972). Advan. Prot. Chem. 26, l-72. Haschemeyer, R. H. (1970). Advan. Enzyrnol. 33, 71. IUC Abstracts Kyoto (1972), III, 13. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-496. Timmins, P. A. (1972). PhD Thesis, University of London. Valentine, R. C., Shapiro, B. M. & Stadtman, E. 12. (1968). Biochemistry, 7, 2143-2152.
Crystals of Glutamine Synthetase from Escherichia coli R. P. BYw&rERt,
C. H. CARLISLE, R. B. JACKSONS A. L. MACKAY AND P. A. TIMMINS~
Department of Crystallography Birkbeck College, University of London Malet &beet, London, W.C.1 (Received 30 May 1974, and in revised form 2 October 1974) Further details are given of crystals of glutamine synthetaae prepared from Eecherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et a2. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and ptxking considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetaae preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are a = 140 & 2 A, and c = 148 & 2 b. The Laue symmetry group is 3m P31.
1. Introduction Since work on Escherichia coli glutamine synthetase has been fully reviewed very recently by Ginsburg (1972) it is necessary to mention only details pertinent to our investigation which was continued from that reported in an earlier paper (Bywater et al., 1969). Crystals of glutamine synthetase were grown from a solution of ammonium sulphate, 40% saturated, at pH 6-l and 18°C. Although some dozen tubes were kept under nearly identical conditions, only one tube produced crystals large enough for X-ray work. Further attempts to repeat the crystallization also failed.
2. Optical examination Crystals were optically almost isotropic. Most of them were rhombohedra with maximum dimensions O-07 mm and an apex angle a = 95.5” f l”, although the three apex angles could not be checked simultaneously. t Present address: Ph armaoia Fine Chemicals AB, Box 176, S-76104 Uppaala 1, Sweden. $ Present address: Rhode Island Junior College, Knight Campus, 400 East Ave., Warwick, Rhode Island 02886, U.S.A. 0 Present address: Institut Max von Laue-Paul Langevin, BP166 Centre de tri, 38042 Grenoble Cedex, France. 293
294
R. P. BYWATER
ET
AL.
Indexed with respect to the hexagonal axes (see next section) the majority of faces appear to be of the form (loll>. Other crystals with the form of rhombic dodecahedra, extended along one cube axis, were also seen.
3. Crystal handling techniques In order to get these extremely small crystals into tubes for X-ray examination a special micromanipulator was used for bringing up the tube to the crystal which was then sucked in (Timmins, 1972). Radiation damage was particularly acute because of the sma,ll size of the crystals, and X-ray setting had to be kept to a minimum so that crystals were studied in the first rational orientation encountered when the orientation could not be recognized from the external form. X-ray pictures were taken only from the rhombohedral crystals. 4. Unit cell dimensions X-ray precession photographs (using a fine-focus rotating anode tube) showed the unit cell to be hexagonal with dimensions a=b=140&2A c = 148 f 2 A. In our earlier paper (Bywater et al., 1969) the cell was reported as being monoclinic (a = 242, b = 140, c = 148 8, with p = 90’) the dimensions of which correspond to an orthohexagonal cell equivalent to the above hexagonal cell (since 1402/3 = 242). Subsequently an [OO*l] axis precession photograph (Plate I) showed clearly the 6 mm symmetry of the h&O layer. A number of zero layer photographs in ten different directions were obtained and collated. In the best example reflections extended to 8 A. The weighted reciprocal lattice appeared roughly cubic in general intensity distribution but did not even approximately show rhombohedral extinctions. For the above hexagonal cell the angle u between possible rhombohedral axes is 95.33” (correspondu = 2 arcsin (a/2)/[(c/3)” + (a/3)“]* and arh = 94.7 A). (The ing to the form {liol}, vertex angle of the form (2132) with the same cell dimensions is 95*27”, indistinguishable from the above.)
5. Space group The diffraction symmetry is 3m, the presence of a 6-fold axis being excluded by the difference between the intensities of 1On and liO2 reflections in the [20*1] zone (allowing for different Lorentz polarization factors). 001 reflections were only present for 1 = 3n. There were no systematic rhombohedral extinctions. Taking into account the absence of improper symmetry operations from cheiral structures, the possible space groups are thus P3121, P3,21, P3112 and P3,12. Intensities were estimated by eye and the Patterson function P (U, V, W) was calculated. There were two notable features in the vector distribution: (i) no significant disposition of vectors on W = 0 and (ii) a heavy vector of length about 50 A lying approximately 33” from c and arising chiefly from the 20’21 reflection.
/ ,‘,~‘ti”,
,’
‘!!!,
PLATE II. (a) (dimensions about (b) Crystals of the rhombohedral (c) Photograph reproduced from
Rhombohedral crystals of glutamine synthetasr as uwtl in the pwscnt SI u(I? 0.1 mm). elongated rhombic dodecahodral form also ohwrwd in thv same pr~~paratio~l as crystals (dimensions also about 0.1 mm). of glutamine synthetaw crystals of the olortgated rhombic ~lotlrcah~~lral ~OIY~ Eisanberg et 02. (1972).
PLATE III. Electron micrographs of various aggregations, not necessarily Ibnra represent 1000 A. (a) Isolated dodecamers wcur as eclipsed hexagons 130 A& overall. Nnrron(h) Thr dodcoamws we frequently associated into columns.
synthetase, rotl~ are also ohserve~l.
of glntamine
found
in preparations
of glutamine
synthetasr.
SC~III.
CRYSTALS
OF GLUTAMINE
SYNTHETASE
295
6. Unit cell contents Using the molecular weight of 592,000, a value of 0.74 ml/g for the protein specific volume and a solvent content of 45% (Matthews, 1968) there appear to be 24 subunits/ unit cell. Assuming that the crystals belong to any one of the space-groups mentioned above, where the general position is 6-fold, the asymmetric unit must contain four protein subunits. These could be paired to yield three octomers per unit cell, with the octomer containing a S-fold axis. Whatever the manner of the clustering of the subunits this would be in keeping with the volume of our unit cell, 2.51 x IO6 A3, lower than that found by Eisenberg et al. (1971) which was 2.89 x IO6 a3 for the tetragonal unit cell of their crystal.
7. Electron microscope observations The electron microscopic examination of the solutions of glutamine synthetase which produced crystals of the form we describe showed a wide variety of configurations. (We were not able to examine an actual crystal broken down on to an electron microscope grid.) Looking back critically at some of Valentine’s photographs of the dodecamers, through the courtesy of Dr N. Wrigley, we see that small numbers of other particles are present there also.
FIG. 1. The T = 7 structure which is a possible packing for dodeoamers in Valentine’s wheatsheaf assemblies of glut&mine s.ynthetase particles. The hexagonal cell (with a = 140 A) is seen sectioned perpendicular to c. Circles represeut subunits.
296
R.
P. BYWATER
ET
AL.
In summary, we find (Plate III) : (1) Dodecamers in the form of eclipsed hexagons corresponding closely to the particles described by Valentine et al. (1968) and by Eisenberg et al. (1971) and composed of subunits of 45 to 50 A diameter, although the subunits appear to have substructures leading them to be non-spherical. When seen end-on they often have the symmetry 6 and not 6 mm. (2) These dodecamers are frequently common).
associated into columns (hexagonal axes in
(3) The columns tend to associate further side by side parallel to each other. Mutual contacts correspond to a “mirror plane”, that is, protuberances do not interdigitate. This leads us to suggest that Valentine’s wheatsheaf structure may be parallel stacking AA AA with a T = 7 cross-section (Fig. 1). (4) Large open square tetramers (or possibly cubic octomers) of overall dimensions some 120 A. These are observed to associate in squarely-packed arrays.
FIQ. 2. Plan of the tetragonel structure of glutamine synthetase proposed by Eisenberg e-6al. (1971) for their crystals of elongated rhombic dodeoahedml form. The oell ia men seotioned perpendicular to c. Circles represent subunits and a = 129 8.
CRYSTALS
OF
GLUTAMINE
297
SYNTHETASE
(5) Narrow rods some 60 A across. Substructure showing the rod to be a double row of particles at intervals of about 40 A is evident. These particles appear in parallel association and tend to lie across each other at close to 4.5”. (6) Other sporadic configurations such as pentamers, larger rings, globules. We conclude that: (i) the subunits are by no means spherical, and (ii) there is no clear correspondence between any one form and the crystallographic data of our crystals.
8. Comparison with crystals from Eisenberg et al. (1971) Since our earlier paper, Eisenberg et at. (1971) have described crystals of glutamine synthetase from E. coli. Their unit cell is tetragonal with a = 129, c = 174 A and appears to contain two dodecamers of glutamine synthetase with dimensions close to those described by Valentine et a.l. (1968) from electron microscopic examination. The Eisenberg structure (Fig. 2) can be described topologically as being either a body-centred cubic arrangement of dodecamers (parallel to each other) with cell dimensions as given, or as a face-centred cubic arrangement with cell dimensions a = 2/2a = 182, c = 174 A. A face-cent& cell could also correspond to a rhombohedral lattice with a M 60” (or 104”). The vertex angle of a 110 face would be 92.7” from Eisenberg’s cell dimensions, which agrees only roughly with that in the photograph of his crystals which appear to be rhombic dodecahedra, extended somewhat along c, exactly like those visible as a minorit,y component in our preparation (Bywater et al., 1969). As Eisenberg’s cell explains the form of his crystals but not the rhombohedral form, whereas our cell explains the rhombohedral form but not the tetragonal form, we must tentatively conclude that two crystal structures coexist. This can be seen in the photograph of the crystals reproduced by Bywater et al. (1969) and repeated in TAEZLE~ Conditions for crystallizution Solvent/solute parameters
40% saturated %2*3 aa
W&)sSO~
(1VC)
None in crystallizing solution (enzyme was prepared in presence of excess imidazole/MxP + /etc.)
Buffers PH Ionic strength Enzyme
Birkbeck conditions
concn
State of enzyme Temperature
6.1 -6-9 10 mg/ml Adenylylated 18°C
Eisenberg’s conditions 1.86 M N 31% saturated (4’C) 0.02 rd-oitrate/imidazole 0.001 wMnCl, 0.01 rd-histidine 0.001 M-glutathione 6.9 ~6.8 7 mg/ml A deny1 ylatcd 4°C
298
R. P. BYWATER
ET
AL.
Plate II(a) and (b). This photograph shows crystals looking very much like those in the photograph (Plate II(c)) reproduced by Eisenberg et al. co-existing with rhombohedral crystals. Table 1 compares the growth conditions for the two cases.
9. Model structures Only two model structures appeared as a result of considering the packing of 24 subunits of about 60 A diameter into our unit cell. One (referred to as the B.C.C. model) consists of octonaers (8 spheres at the vertices of a cube) and the other (referred to as the T = 7 model) consists of dodecamers (12 spheres arranged as two hexagonal rings in the eclipsed position). We now consider the geometry of the models.
10. B.C.C. (body-centred cubic) model (Fig. 3) If octomers in the form of cubes are assembled, so that one sphere lies in the cavity between the four spheres making the face of the adjacent cube, an interesting rhombohedral structure results where the hexagonal unit cell contains three octomers. If the diameter of the spherical subunit is D, then the dimensions of this cell are a = (912 + 22/2)+
D = 2.707 D and
c = 2/3 (1 + l/d2)
D = 2.957 D.
The ratio c/a is thus 1992 corresponding to a = 94.2”. Our own dimensions thus give (for a = 140) D = 51.7 A and (for c = 148) D = 50.0 it. It might be noted that this is a dense packing of spheres (not all in equivalent positions but with two subunits on trigonal axes and 6 off them). If further subunits were inserted, one at the centre of each cube of eight subunits, and the structure were
GS
PRCKINC
OF
OCIOHERS
FIG. 3. Perspective view of the B.C.C. structure proposed for the packing of octomers of glutamine synthetsse (GS). The dimensions of the hexagonal call sre a = 140 and c = 148 A. Each subunit is represented as a dumbell to lower its symmetry.
CRYSTALS
OF
GLUTAMINE
SYNTHETASE
299
expanded in compensation, then the resulting structure would simply be body-centredcubic (as a-Fe). Parameters are: 2 spheres at (0, 0, O-293) 6 spheres at (0.072, O-317, 0.097). If the subunits are not spheres but, as seems clear from the electron micrographs, much less regular, then according to the a,ssembly into octomers, the symmetry will be reduced from rhombohedral R 32. In the computer-produced drawing of Figure 3, each of the eight subunits making a cubic octomer has been represented as a dumbell in order to reduce the local symmetry. In particular, if the octomers are each composed of two tetramers, then the structure cannot be rhombohedral. Tests were made with pear-shaped units (two tetramers related by a local dyad) but no decisive agreement with observed intensities was found.
11. T = 7 model (Fig. 1) A possible model for the packing of dodecamers is based on the T = 7 tessellation of the hexagonal lattice. This would give a value D = a/d7 = 52.9 A for the diamet,er of spherical subunits. It might be noted that in one direction the units in the Eisenberg structure shown in Figure 2 are packed in this way. If the T = 7 model were sheared so that the interaxial angle of 60” were opened to 90” then the layers of the Eisenberg structure would be obtained. The density would be correspondingly reduced (to 87%). There would have to be four layers of spheres for the T = 7 model packing of dodeca,hedra and this sequence must be AABB. Using the c-dimension of 148 A this would lead to a sphere diameter of 40.7 A. (D = c/(2 + 2/8/d3).) It should be noted that very similar constellations of subunits occur in all three models (Eisenberg, B.C.C. and T = 7). As three different types of cavities appear in a T = 7 layer a number of variants of stacking are possible and might even occur simultaneously in disorder.
12. Conclusions We conclude that for the large crystals examined by X-ray diffraction the T = 7 structure is unlikely because: (a) if there were four layers of units (24 per cell) there would be extinctions for 1 # 2% instead of for 1 # 3n, (b) if there were three layers of units this would imply hexamers and (c) the structure would be too compressed, in comparison with Eisenberg’s structure, in the c direction. The B.C.C. structure is more likely because: (a) the non-sphericity of the subunits removes rhombohedral extinctions but the 3, axis remains ; (b) there is a better fit with the Patterson data. In all, the most probable configuration is that of two tetramers assembled face to face into an octomer and related by a dyad axis. We consider it likely that there are two different forms of glutamine synthetase crystals, those observed by ourselves and those observed by Eisenberg et al. (1971). The electron microscope results show dodecamers as seen by Valentine and small rods which are probably assemblies of octomers or tetramers. There is some sign that dodecamers can break down into three tetramers. 21
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The large tetramers observed seem too large to be the same tetramers we have been discussing above. Wrigley (personal communication) has pointed out their similarity with tetramers of pyruvate carboxylase, but they remain unexplained. We acknowledge the technical assistance of Sheila Lauchlan and Douglas Parry and the usual unfailing helpfulness of our other colleagues. One of us (P. A. T.) gratefully acknowledges the Gnancial assistance of Birkbeck College. Another author (R. B. J) was a post-doctoral fellow of the National Cancer Institute (U.S.A.). REFERENCES Bywater, R. P., Carlisle, C. H. & Jackson, R. B. (1969). J. Mol. Bill. 45, 429-431. Eisenberg, D., Heidner, E. C., Goodkin, P., Dastoor, M. N., Weber, B. H., Wedler, F. & Bell, J. D. (1971). Cold Spring Harbor Symp. Quant. Bill. 36, 291-294. Ginsburg, A. (1972). Advan. Prot. Chem. 26, l-72. Haschemeyer, R. H. (1970). Advan. Enzyrnol. 33, 71. IUC Abstracts Kyoto (1972), III, 13. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-496. Timmins, P. A. (1972). PhD Thesis, University of London. Valentine, R. C., Shapiro, B. M. & Stadtman, E. 12. (1968). Biochemistry, 7, 2143-2152.
