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letters Tetrameric coiled coil domain of Sendai virus phosphoprotein Nicolas Tarbouriech1, Joseph Curran2, Rob W.H. Ruigrok3 and Wilhelm P. Burmeister1,4 1
European Synchrotron Radiation Facility, Grenoble cedex, France. Department of Genetics and Microbiology, University of Geneva Medical School, CMU, 9 avenue de Champel, CH-1211 Geneva 4, Switzerland. 3European Molecular Biology Laboratory, Grenoble outstation, c/o ILL, BP 156, F-38042 Grenoble cedex 9, France. 4Forschungszentrum Jülich, BP 220, F-38043 Grenoble cedex, France.
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protein sequences are very different for the various paramyxoviruses. Only the oligomerization domain and the N-RNA binding domain show some conservation in sequence and predicted secondary structure9. It has been predicted that oligomerization occurs through a coiled coil domain8. Furthermore, limited proteolysis has defined a domain from residues 320–446 that forms a stable homotetramer in solution and adopts an elongated shape9, suggesting that it forms a four-stranded coiled coil structure. The structure of a tetrameric coiled coil has only been described for the engineered GCN4-pLI peptide10 and for the core of the heterotrimeric SNARE complex11. Here we describe the structure of a slightly shortened oligomerization domain from amino acid Glu 320–Asn 433. It crystallized in space group P4212 with one monomer in the asymmetric unit (with a solvent content of 52%) and unit cell dimensions a = 48.58 Å, c = 100.8 Å. The structure was solved by a combination of SIRAS using a mercury derivative and multiwavelength anomolous dispersion (MAD) techniques with mercury and selenomethionine labeled protein, respectively. Details of the data collection statistics are given in Table 1, and part of the experimental map is shown in Fig. 1a. The refinement (Table 1) at 1.9 Å converged (no interpretable peaks in an Fo - Fc map) at an Rcryst of 25.9% (Rfree 28.8%). The final model contains all but the first two N-terminal and the last C-terminal residues. The relatively high R-factors is probably due to a high average temperature factor (38.6 Å2) of the
The high resolution X-ray structure of the Sendai virus oligomerization domain reveals a homotetrameric coiled coil structure with many details that are different from classic coiled coils with canonical hydrophobic heptad repeats. Alternatives to the classic knobs-into-holes packing lead to differences in supercoil pitch and diameter that allow water molecules inside the core. This open and more hydrophilic structure does not seem to be destabilized by mutations that would be expected to disrupt classic coiled coils. Sendai virus (SeV) is the prototype virus of the paramyxoviruses. It infects the respiratory track of laboratory mice and causes pneumonia. This family contains human respiratory viruses, such as the human para- a influenza viruses types 1–4, that cause croup, bronchiolitis and pneumonia, but also the measles and mumps viruses. In SeV, the negative stranded viral RNA is encapsidated by nucleoprotein (N) with a stoichiometry of one N monomer for six nucleotides1. This N–RNA complex serves as the template for the RNA-dependent RNA polymerase of the virus, which is itself a complex of two virally encoded proteins, the phosphoprotein (P) and the large (L) protein2,3. The L protein contains the polymerase activity as well as the capping and polyadenylation activities 4. The P protein binds to the b C-terminal domain of N and positions the polymerase Asn 321 onto the template5,6. In the absence of P, L cannot recognize the transcription and replication signals encoded by the viral RNA and polymerization is not processive. Another role for the P protein in the replication process is to act as a chaperone for newly synthesized N, socalled N0 (ref. 7). The P protein (568 residues) seems to have a modular structure in which some domains may be altered or deleted without affecting the function of the other domains8. The domains, from the N-terminus to the Cterminus are: the N0 binding domain (residues 33–41); the oligomerization domain (320–446; see below), which includes the L binding domain (412–445), and the N-RNA binding domain (residues 479–568)8. At the mRNA level, there is an editing site at nucleotide 1,053 (before the sequence that encodes the oligomerization domain) where one or two G nucleotides are added during transcription, which leads to frame shifts and two alternative viral proteins8. P is only functional as an oligomer and the C-terminal domains (residues 320–568) are sufficient for in vitro transcription8. The P nature structural biology • volume 7 number 9 • september 2000
c
B A C D
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Fig. 1 Structure of the oligomerization domain of P protein. a, Stereo view of the solvent flattened electron density calculated with SHARP (in green, contoured at 1.5 σ) around the calcium ion (magenta) and residue Asn 389, with water molecules are shown in red. b Ribbon diagram of the tetramer. c, Ribbon diagram of the helical bundle at the N-terminus. D is the long helix forming the coiled coil (only partially shown). This figure was generated with BOBSCRIPT30.
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letters da
x
M367-T368 (da)
S382 (x)
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Fig. 2 Layers in the core of the coiled coil. The pictograms10,13 represent different types of layers observed in the core. The arrows show the direction from the N- to the C-terminus, open circles represent the Cα atoms and black circles the Cβ atoms. Ball-and-stick representations of one canonical a-layer and one d-layer, and all noncanonical layers (da- and x-layers) involved in the core of the coiled coil are shown. *Tyr 407 could not be attributed unambiguously (see text). The amino acids involved in the packing of the core (van der Waals spheres) and their first and second neighbors in sequence (for the ball and stick representations) are shown.
L393 (a)
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A378-E379 (da)
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structure and an above average proportion of mobile side chains at the protein surface. Structure description The oligomerization domain of the SeV P protein crystallized as a tetramer and each monomer is composed of three short N-terminal helices and a very long C-terminal helix (Fig. 1b). The three N-terminal helices (A, B and C; Fig. 1c) form a small helical bundle at the N-terminal end of the long helix (D). Helix D is 96 Å long, formed by 65 residues and runs over 18 turns. The tetramer is formed through a coiled coil involving the long helix of each monomer. The four-fold symmetry axis of the tetramer coincides with a four-fold crystallographic axis. A small hydrophobic core involving helices A, C and D of one monomer (Fig. 1c), and helix D from the adjacent monomer provides further stabilization of the tetramer via hydrophobic interactions. Helices A, B and C of each monomer seem to wrap around the central coiled coil. Four-stranded coiled coil Classic coiled coils have hydrophobic interfaces and a 778
hydrophilic exterior. Every first and fourth amino acid of a seven-residue repeat — residues a and d in a heptad repeat of a–g — in the helix is hydrophobic. In tetrameric coiled coils, the Cα–Cβ bonds of the side chains of residues in the a-layers are perpendicular to the Cα–Cα bonds in the holes on the adjacent helix (counterclockwise; viewed down the helix from the N-terminus)10 (See pictograms in Fig. 2). In the d-layers, the Cα–Cβ bonds of the side chains are parallel to the Cα–Cα bonds in the holes on the adjacent helix (clockwise; viewed down the helix from the N-terminus)10 (Fig. 2). Classical coiled coils have a left-handed supercoil in order to keep the two hydrophobic interfaces in register12. Insertion of skip residues into the heptad register may alter the supercoiling characteristics12,13. Such skip residues have been identified in the structures of the trimeric influenza virus hemagglutinin at neutral and low pH14,15 and the Ebola virus GP2 at low pH16, in which the Cα–Cβ bonds of the three side chains point to the three-fold axis rather than to the Cα–Cα bond in a neighboring hole. Layers with this geometry have been named x-layers14 and occur after the d positions (a-d-x-a-d). Lupas et al.13 have suggested another type of skip geometry occurring after the a positions (d-a-da-d-a). In these layers, the side chains of the d residues interact with the side chains of the e residues of the adjacent helix rather than packing in a hole (Fig. 2). Because of distortion of the coiled coil geometry, a da-layer is often followed by an x-layer14. The coiled coil of the SeV P protein may be divided into an N-terminal region (Tyr 364–Ser 382), a middle region (Ala 383–Val 400) and a C-terminal region (Glu 401–His 429) (Fig. 3a,b). The N-terminal region contains a mixture of canonical a-layers and d-layers mixed with x-layers and da-layers. The a-layers and d-layers are composed of nonpolar residues whereas the x-layers and da-layers contain polar or even charged residues. The middle region contains only regular a-layers and d-layers composed of apolar side chains. The C-terminal region has a succession of da-layers and x-layers that contain large polar or charged residues; the x-layers and da-layers do not serve as skip residues in a hydrophobic heptad repeat but form a new type of coiled coil structure. One example each of packing in a- and d-layers, and all cases of packing in the x- and da-layers are shown in Fig. 2. Depending on the side chain characteristics, the d residue in da-layers can pack more towards the outside of the coil (Fig. 2, top four frames below the pictogram for the da-layer) or more towards the center of the coil (Fig. 2, bottom frame below the pictogram for the da-layer). The N-terminal region is virtually not supercoiled and has a radius, calculated using the local center of mass of the helix, of 9–7.2 Å. The middle region has an average nature structural biology • volume 7 number 9 • september 2000
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letters Fig. 3 Sequence and geometry of the oligomerization domain. a, Sequence of the oligomerization domain. The amino acids involved in α-helices (A–D) are boxed in gray, the residues involved in the packing of the core, and the type of layer, are specified above the sequence. b, Helical net diagram for the long helix of the oligomerization domain of P. The residues forming the central core are marked by boxes. The sequence should be read from right to left and from top to bottom. Residue number and layer types are specified next to the sequence (*Tyr 407 is discussed in the text). c, Representation of the side chain packing angles. This angle is defined between the projection onto a plane perpendicular to the four-fold symmetry axis of the Cα–Cβ vector and the vector defined between the local center of mass of the helix and the four-fold symmetry axis when viewed from the N-terminal end of the helix. The local center of mass of the helix is calculated by averaging the positions of the atoms of the main chain (N-Cα-C) of seven consecutive residues. The value for the middle residue of these seven consecutive residues was used. Angles observed for the P protein are represented by solid lines and the angles observed for GCN4-pLI by dotted lines. The colored sectors for each kind of layer are defined by the angles for the P protein. Asterisks indicate two of the four layers previously defined as d for GCN4-pLI that we classify as x-layers.
a
mENTSSMKEMATLLTSLGVIQSAQEFESSRDASYVFARRALKSAN 320 . . . .
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heli D da x da x a x RFSEYQKEQNSLLMSNLSTLHIITD 409. . . 433
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Helical net: E N
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nature structural biology • volume 7 number 9 • september 2000
helix C
x da d a da x a d a d a da * YAEMTFNVCGLILSAEKSSARKVDENKQLLKQIQESVESFRDIYK 364 . . . .
S
radius of 7.1 Å (range 6.9–7.5 Å) and a supercoiling pitch of 235 Å. The C-terminal region has an average radius of 8.0 Å (range 7.5–8.5 Å) and a pitch of 278 Å. For comparison, the GCN4-pLI tetramer has an average diameter of 7.6 Å and a smaller supercoil pitch of 205 Å (ref. 10). Brown and colleagues17 have suggested that a description of heptad layers by a, d, x or da geometries is too limited and that a more continuous and graded description would be required. Therefore, we have determined the angles between the vectors for each Cα–Cβ bond and the vector from the local center of mass of the α-helix to the center of the tetramer (Fig. 3c). It appears that each type of layer occupies its own angular range and, as such, the four geometries seem to be quite distinct. The angle for Tyr 407 lies between the ranges for x-layers and a-layers. We interpret this residue as an outlier from an x-layer due to the bulky side chain rather than a part of an a-layer. For comparison, we also included the angles for the a-layers and d-layers of the GCN4-pLI tetramer10 (Fig. 3c; dotted lines). Although all GCN4 a-layers are within the same wedge of angles as the a-layers of the SeV P protein tetramer, the angles of two of the GCN4 d-layers seem to be closer to those of the x-layer wedge. These exceptional geometries, plus that of Tyr 407, seem to fill the gaps between the d and x, and between the x and a geometries, confirming the suggestion of Brown et al17. Unlike the GCN4-pLI structure, water molecules could be found within or very close to the da-layers in the structure presented here. The internal channel of the coiled coil is almost contiguous but variable in size (Fig. 4a). There are 23 ordered water molecules inside this channel, of which seven, as well as one calcium ion, are located on the four-fold axis. The calcium ion is coordinated by four Asn 389 residues (Fig. 1a) (OD1–Ca2+ distance is 3.47 Å) and its B-factor is similar to those of its ligands. Calcium was present in the crystallization media and the density for the atom disappeared from crystals that had been soaked in 10 mM EDTA. Because the center of the coiled coil is open to water and other ions, the percentage of total water excluded surface area is lower than the 61% calculated for the GCN4 tetramer. For the N-terminal region, 28% of the surface area of each monomer is excluded by the coiled coil partner helices. However, this part of the coiled coil is in contact with the small helical bundle, which leads to 64% buried surface area between the monomers. For the middle and C-terminal regions, 50% and 43% of the surface of the
helix B
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Y364 M367-T368 V371 I375 A378-E379 S382 V386 N389 L393 I396 V400 F403-R404 Y407 F410-S411 Q414 Q417-N418 L421 L425 L428
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monomer is excluded, respectively, giving a total excluded area for the whole structure of 50%. However, since the tetramer of P is much longer than the GCN4 tetramer, the total buried surface area of P is larger than that of GCN4 (2,570 Å2 compared to 1,640 Å2 per monomer). There is only one interhelix salt bridge in the tetramer of P. Stability and function of the P protein tetramer Parallel coiled coils have been described with two, three, four or even five helices10,18–20. We present here the structure of a new four-stranded parallel coiled coil, which is longer than that of the previously described GCN4 mutant p-LI but much less regular. Nevertheless, functional studies with deletion mutants of P indicate that the P tetramer is a sturdy structure. Deletion of amino acids 344–373 (helix C and the first 10 residues of the long helix D), of amino acids 374–411 (37 residues in the coiled coil) or of amino acids 412–479 (the last 779
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letters a
Fig. 4 Implications of the structure for the interactions with other proteins. a, The central channel. Cross section of the surface along the coil axis shows the central channel and the charge distribution. This figure was generated with GRASP31. b, CPK representation of the tetramer colored according to temperature factors (from blue to red for 20–60 Å2). The charged amino acids, which were mutated in ref. 22 and affect the interaction between P and the L protein, are indicated.
b
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K408A R409A E412A K415A E416A
20 residues of the coiled coil plus some of the following residues) did not affect oligomerization8. Only the deletion of amino acids 344–411 led to the loss of oligomerization8. Furthermore, mutations known to destabilize classic coiled coils, such as V400P, a residue at an a-layer, or insertion of AlaPro or Ala-Ala after Val 400 that should lead to the proposed dephasing of the residues involved in coiled coil interactions, did not affect the activity of P for either viral RNA transcription or genome amplification8.
The SeV polymerase progresses over the template RNA in steps of one nucleotide whereas the P protein can only be displaced on the N-RNA in steps of one N monomer, that is, per six nucleotides. It has been proposed that P cartwheels over the N-RNA template21, in which case the interaction between the tetrameric P and the monomeric L proteins would have to be continuously broken and remade. The residues on P that have been identified as binding to the L protein22 are shown in Fig. 4b. Both points mentioned above imply that there is a need for mobility in the interface between these two proteins. This is probably provided by the plasticity of the noncanonical coiled coil due to its increased radius and the mobility of the side chains at the interface, reflected by their high B-factors (Fig. 4b). In general, the C-terminal, noncanonical region of the coiled coil has much higher temperature factors than the middle, classic coiled coil (average value of 43.7 versus 30.8 Å2). We suggest that the region of P that binds the L protein acts as a molecular axle using the plasticity of the noncanonical L-binding portion of the coiled coil to allow a rotation of P while bound to the L protein.
Table 1 Statistics on data collection1 and refinement and the composition of the model Data set
λ (Å)
Resolution (Å)
Native Native 1 0.94 2.6 EMTS derivatives 1 0.94 2.2 2 0.94 2.42 3 0.94 2.53 4 0.94 1.9 EMTS plus selenomethionine derivatives M1 (peak Se) 0.9793 2.3 M2 (inflection Se) 0.9794 2.3 M3 (peak Hg) 1.0064 2.42 M4 (remote) 0.9310 2.7 M5 (inflection Hg) 1.0093 3.0 Phasing quality Figure of merit (centric / acentric; 34–2.2 Å) 0.49 / 0.45 Overall FOM (after solvent flattening; 34–2.2 Å) 0.79 Refinement statistics2–5 Rcryst (15–1.9 Å) 0.259 Rfree (15–1.9 Å) 0.288 R.m.s. deviations Bonds (Å) 0.006 Angles (°) 0.97
Unique reflections
Multiplicity Completeness (%)
Rsym (%)
4173
11.2/8.9
98.9/100
10.1/26.5
6537 5142 4376 9175
6.4 / 6.1 7.2 / 7.0 7.2 / 7.3 3.7 / 3.8
98 / 101 98.7 / 99.3 99.4 / 100.5 92.1 / 94.5
6.2 / 40.7 6.1 / 27.8 4.3 / 32.2 8.9 / 38.6
5894 5901 5062 3593 2296
7.4 / 7.8 7.4 / 7.7 7.4 / 7.7 5.7 / 3.8 5.2 / 3.4
99.2 / 99.9 99.2 / 99.9 99.3 / 102 95.8 / 80.5 83.7 / 82.5
7.0 / 26.2 6.5 / 38.3 7.7 / 46.0 8.2 / 39.6 7.7 / 14.6
Model composition6 Number of atoms Number of residues Number of water molecules Number of calcium atoms Number of EMC (Hg-CH2-CH3) molecules Number of glycerol molecules Number of residues with alternative conformations
967 112 60 1 1 1 1
Values for the highest resolution shell (1.97-1.90 Å) are given after a slash. All reflections between 15 and 1.9 Å (excluding the 10 % in the test set) were used in the refinement. 3A bulk solvent correction was applied. Restrained individual temperature factors have been refined. 4The loop Glu 344–Arg 347 is involved in a crystal contact and has poor electron density and high B-factor values. 5One residue (Ser 346) falls outside the energetically favored regions of the Ramachandran plot. 6Cys 372 takes an alternate conformation when it binds ethylmercury of which the occupancy refined to a value of 0.39. 1 2
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letters Methods
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Protein purification and crystallization. The oligomerization domain (12.5 kDa), containing residues 320–433, was subcloned from a previous construct9 by PCR mutagenesis and overexpressed in Escherichia coli. Selenomethionine labeled protein was obtained by overexpression in a minimal medium as described23. After cell lysis, the protein was bound to a Q-Sepharose fast flow column (Pharmacia) and eluted with a 50–500 mM NaCl gradient. The fractions of interest were precipitated in 50% saturated ammonium sulfate at 4 °C. The pellet was resuspended in 10 mM potassium phosphate, pH 6.8, and loaded onto a FPLC hydroxyapatite column (CHT2-I, Bio-rad). The last purification step was done on a FPLC Superdex 75 (Pharmacia) as described9. For the selenomethionine protein all the buffers were complemented with 10 mM dithiothreitol. The protein was crystallized at 20° C at a concentration of 8 mg ml-1 in 20 mM HEPES, pH 7.0, 50 mM NaCl using the hanging drop method. The reservoir contained 100 mM Tris-HCl pH 9.0, 200 mM NaCl, 10 mM CaCl2, 15–17% (w/v) PEG 3000. The best crystals were obtained by cocrystallization with 5 mM ethylmercurythiosalicylic acid (EMTS). The selenomethionine crystals were obtained under the same conditions. Data collection and structure determination. The crystals were transferred to a solution of 100 mM Tris-HCl pH 9.0, 200 mM NaCl, 10 mM CaCl2, 13% (w/v) PEG 3000, containing 30% (v/v) glycerol in three steps of 10, 20 and 30% glycerol and flash frozen in a nitrogen gas stream (Oxford Cryosystem) at 100 K. Data were collected at ESRF beamline ID14-3 equipped with a MAR CCD detector (Mar Research) for collection of the native and mercury derivative data and at beamline ID14-4 with a Quantum-4 CCD detector (ADSC) for the MAD experiment at the selenium and mercury edges. All data were processed using MOSFLM24 and the CCP4 program suite25 except derivative 4, which was processed using the DENZO/SCALEPACK package26. Table 1 gives the data collection and refinement statistics. The mercury position was located on anomalous difference and difference Patterson maps and refined using MLPHARE25. The selenium sites were located in an anomalous difference Fourier synthesis. The data from the native set, derivatives 1–3 and the five wavelengths of the MAD experiment were used together with the heavy atom positions in SHARP27 to calculate the best phases. The map obtained after modification using SOLOMON27 was then used for model building using O28 and allowed 85% of the model to be built. The refinement was carried out using CNS_SOLVE 0.9 (ref. 29) against derivative 4.
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Coordinates. Coordinates have been deposited in the Protein Data Bank (accession code 1EZJ).
Acknowledgments We thank J.-B. Marq (CMU, Geneva) for technical assistance, D. Kolakofsky (CMU, Geneva) for discussions and support and W. Weissenhorn (EMBL, Grenoble) for critical comments on the text.
Correspondence should be addressed to W.P.B. email:
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