refining the overall structure and subdomain orientation of ribosomal protein s4 Δ41 with dipolar...

Article No. jmbi.1999.3061 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 292, 375±387

Refining the Overall Structure and SubdomainOrientation of Ribosomal Protein S4 ��41 with DipolarCouplings Measured by NMR in Uniaxial LiquidCrystalline Phases

Michelle A. Markus1, Resi B. Gerstner2, David E. Draper2

and Dennis A. Torchia1*

1Molecular Structural BiologyUnit, National Institute ofDental and CraniofacialResearch, 30 Convent DriveRoom 106, BethesdaMD 20892-4310, USA2Department of ChemistryJohns Hopkins UniversityBaltimore, MD 21218, USA

E-mail address of the [email protected]

Abbreviations used: DHPC, dihexphosphocholine; DMPC, dimyristoyphosphocholine; HSQC, heteronuclecoherence; IPAP, in-phase, antiphasOverhauser effect; PMSF, phenyl m¯uoride; S4 �41, residues 1 (labeledthe Bacillus stearothermophilus sequeprotein S4; RMSD, root-mean-squar

0022-2836/99/370375±13 $30.00/0

Prokaryotic protein S4 initiates assembly of the small ribosomal subunitby binding to 16 S rRNA. Residues 43-200 of S4 from Bacillus stearother-mophilus (S4 �41) bind to both 16 S rRNA and to a mRNA pseudoknot.In order to obtain structure-based insights regarding RNA binding, wepreviously determined the solution structure of S4 �41 using NOE,hydrogen bond, and torsion angle restraints. S4 �41 is elongated, withtwo distinct subdomains, one all helical, the other including a b-sheet. Incontrast to the high resolution structures obtained for each individualsubdomain, their relative orientation was not precisely de®ned becauseonly 17 intersubdomain NOE restraints were determined. Compared tothe 1.7 AÊ crystal structure, when the sheet-containing subdomains aresuperimposed, the helical subdomain is twisted by almost 45 degreesabout the long axis of the molecule in the solution structure. Becausevariations in subdomain orientation may explain how the protein recog-nizes multiple RNA targets, our current goal is to determine the orien-tation of the subdomains in solution with high precision. To this end,NOE assignments were re-examined. NOESY experiments on a speci®-cally labeled sample revealed that one of the intersubdomain restraintshad been misassigned. However, the revised set of NOE restraints pro-duces solution structures that still have imprecisely de®ned subdomainorientations and that lie between the original NMR structure and thecrystal structure. In contrast, augmenting the NOE restraints with N-Hdipolar couplings, measured in uniaxial liquid crystalline phases, clearlyestablishes the relative orientation of the subdomains. Data obtainedfrom two independent liquid crystalline milieux, DMPC/DHPC bicellesand the ®lamentous bacteriophage Pf1, show that the relative orientationof the subdomains in solution is quite similar to the subdomain orien-tation in the crystal structure. The solution structure, re®ned with dipolardata, is presented and its implications for S4's RNA binding activity arediscussed.

# 1999 Academic Press

Keywords: ribosomal protein; atomic-resolution protein structure; NMRspectroscopy; dipolar couplings; RNA-protein interactions
*Corresponding author
ing author:

anoyl glycero-3-l glycero-3-ar single quantume; NOE, nuclearethyl sulfonyl

42) and 43-200 ofnce of the ribosomale deviation.

Introduction

Protein S4 initiates assembly of a set of proteinsin the small ribosomal subunit by binding to 16 SrRNA (Nowotny & Nierhaus, 1988). Like manyribosomal proteins, S4 also regulates its own trans-lation by binding to its own messenger RNA. Strik-ingly, the same region of the protein is required forbinding to both targets (Baker & Draper, 1995),

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376 Structure of S4 �41 Re®ned with Dipolar Couplings

even though the secondary structures and presum-ably the tertiary folds of the two RNA targets arequite different: the mRNA forms a pseudoknot(Deckman & Draper, 1987) while the rRNA siteincludes several hairpins (Sapag et al., 1990). Thestructure of the RNA binding domain of S4 fromBacillus stearothermophilus (S4 �41) has previouslybeen solved to provide insight into how the proteinrecognizes such disparate RNA structures (Davieset al., 1998; Markus et al., 1998). S4 �41 is orga-nized into two distinct subdomains. A positionknown to crosslink to 16 S rRNA in the context ofthe highly homologous Escherichia coli protein(Urlaub et al., 1997) corresponds to an exposedloop in the helical subdomain. A cluster of posi-tively charged side-chains lining the crevicebetween the subdomains suggests a likely site forinteraction with RNA.

The structures of S4 �41, solved independentlyby crystallography (Davies et al., 1998) and byNMR spectroscopy in solution (Markus et al.,1998), agree in the details of the secondary struc-ture and the overall fold, with the backbone heavyatoms for each subdomain superimposing towithin 1.36 AÊ . However, the solution and crystalstructures differ in the relative orientation of thesubdomains, by a twist of about 45 � of one subdo-main relative to the other about the long axis ofthe molecule (Figure 1). It has been noted that therelative orientation of the subdomains in solutionwas not well de®ned by the NOE, hydrogen bond,and dihedral angle restraints (Markus et al., 1998),largely because the intersubdomain NOEs accountfor less than 1 % of the NOE restraints used todetermine the structure. It is vital to establish theorientation of the subdomains in solution because(1) the orientation may provide insight into how

S4's structure enables it to recognize two appar-ently distinct RNA targets and (2) conformational¯exibility has been identi®ed as an important com-ponent of some protein-RNA interactions (forexample, Allain et al., 1996). Here, we use a deuter-ated sample labeled in speci®c amino acids toshow that one of the intersubdomain NOErestraints was previously misassigned. However,even after eliminating this NOE assignment andrechecking the NOE restraints, structure calcu-lations reveal that the revised set of NOE restraintsdoes not precisely de®ne the orientation of the sub-domains. The recalculated structures adopt a twistfor the subdomains somewhere between that of thecrystal structure and the original solution structure.To precisely de®ne the relative orientation of thesubdomains, we obtained additional informationfrom N-H dipolar couplings (Tjandra & Bax, 1997),measured in two different liquid crystalline phases.The N-H dipolar couplings determine the anglesbetween the N-H bond vectors and the alignmentcoordinate frame for the whole molecule. In bothliquid crystalline phases, the relative orientation ofthe S4 �41 subdomains in solution is approxi-mately the same as that in the crystal structure.The details of the structure determination and theimplications of these results for RNA recognitionare presented below.

Results

Comparison of the original NMR structurewith the crystal structure

Figure 1 shows the ribbon diagrams for a repre-sentative structure from the original NMR ensem-ble (a) and the crystal structure (b). The structures

Figure 1. Ribbon diagrams for(a) a representative structure fromthe original NMR ensemble ofS4 �41 and (b) the 1.7 AÊ crystalstructure. In (a) and (b) the sheet-containing subdomain is in thesame orientation, to emphasizeboth the similarity of the sheet-containing subdomains and thedifference in the relative orien-tations of the helical subdomains.Elements of secondary structureand the chain termini are labeled.This Figure was generated withMOLSCRIPT (Kraulis, 1991).

Structure of S4 �41 Re®ned with Dipolar Couplings 377

are very similar in that they both contain the sameelements of secondary structure in approximatelythe same place along the protein chain. In bothcases S4 �41 is organized into two distinct sub-domains, with helices a1, a2, a3, and a7 formingthe helical subdomain and strands b1 through b5forming an antiparallel b-sheet that combines withhelices a4, a5, and a6 to form the sheet-containingsubdomain. The ribbon diagrams in Figure 1(a)and (b) show the sheet-containing subdomains inthe same orientation; the pairwise RMSD for thebackbone residues in the sheet-containing sub-domain (residues 94 to 176) between the originalNMR ensemble and the crystal structure is only1.23(�0.07) AÊ . The pairwise RMSD for the helicalsubdomains (residues 47-92 and 192-199) is alsoquite small, at 1.36(�0.17) AÊ . The main differencebetween the structures is the relative orientation ofthe subdomains, re¯ected in the high pairwiseRMSD when both subdomains are considered sim-ultaneously (3.89(�0.31) AÊ for residues 47-199). Asseen in Figure 1, most of this difference can bedescribed as a twist of the helical subdomain aboutthe long axis of the molecule, by approximately45 �.

Examining the original NOE restraint list

Using the break between helix a3 and helix a4 asthe boundary between the subdomains, the helicalsubdomain can be de®ned as residues 42-92 and181-200, while the sheet-containing subdomain canbe de®ned as residues 94-180. (Residue 93, in thebreak between helices a3 and a4, is not assigned toeither subdomain.) With these boundaries, thereare only 17 intersubdomain NOE restraints in theoriginal list of 2196. For comparison, there are 19NOE restraints between helices a2 (residues 67-74)and a3 (residues 82-92). The NOE restraintsbetween the subdomains are expected to determinetheir relative orientation. However, all but one ofthese NOE restraints involve side-chain protonsthat require pseudoatom corrections, increasing theambiguity of the restraint. Of the 17 restraints, nineinvolve proton pairs separated by less than 4.0 AÊ

in the crystal structure, suggesting that theserestraints cannot establish the twist between thesubdomains. For the remaining eight intersub-domain restraints, in general, the correspondingcrosspeaks are relatively weak and involve at leastone degenerate chemical shift, calling for a re-examination of their assignments.

When the NOE restraints were checked for viola-tions against distances based on the crystal struc-ture, the M60 Hb to Y160 He intersubdomainrestraint gave the largest violation by far. (Thedetails are presented in Materials and Methods.)To unambiguously check this assignment, a speci®-cally labeled sample, with 13C only in the methion-ine side-chains and at the e-position of tyrosine,was produced by adding labeled amino acids tobacterial cultures before induction of proteinexpression. To further simplify the spectrum, the

bacteria were cultured in 2H2O to minimize thenumber of observable protons in the protein. Basedon the distances in the crystal structure, the mostlikely assignment for the crosspeak is Y160 He toP162 Hb, with interproton distances ranging from5.08 to 6.68 AÊ . To be absolutely sure that a cross-peak from P162 would not appear, proline deuter-ated at the b, g, and d positions was included inthe culture medium. Using this sample, a 2DNOESY spectrum was recorded, selecting for 13Cattached protons in both dimensions (Figure 2).Although crosspeaks between M60 Hb and Y61 He

and between M87 He and Y61 He are observed,consistent with both the original NOE restraint listand the distances in the crystal structure, no cross-peak is observed between M60 Hb to Y160 He.Therefore, we conclude that the original assign-ment of the crosspeak in the 3D 13C-separatedNOESY experiment was not correct; P162 Hb toY160 He, possibly enhanced by spin diffusionthrough P162 Ha, seems to be the best assignment.

Strikingly, removing the M60 Hb to Y160 He

restraint did not signi®cantly change the calculatedstructures. Furthermore, removing 14 of the inter-subdomain NOE restraints, including all that wereinconsistent with interproton distances the crystalstructure, resulted in a bundle of structures similarto the original solution structure (with a pairwiseRMSD of 1.65(�0.37) AÊ to the original solutionstructure and 3.74(�0.60) AÊ to the crystal structure,considering backbone atoms in residues 47-199).Apparently some of the remaining 2182 NOErestraints preserved a conformation resembling theoriginal twist despite the fact that none of theseNOEs correspond to intersubdomain restraints. Inan effort to remove any bias in the structure causedby the M60 to Y160 NOE assignment's in¯uenceon the other restraints, all NOE restraints werecompared against the interproton distances pre-dicted by the crystal structure. The 247 NOErestraints violated by more than 0.2 AÊ were deleted(11 % of the original restraint list). Not surprisingly,structures calculated with this restraint listresembled the crystal structure more than previousstructures, with a backbone RMSD of1.99(�0.35) AÊ to the crystal structure for residues47-199 compared with 3.24(�0.44) AÊ to the mini-mized average structure based on the originalrestraint list.

The energy landscape for thestructure calculations

What was surprising about the structure calcu-lations in X-PLOR (BruÈ nger, 1992) after 247restraints were removed is that the bundle ofcalculated and accepted structures no longerincluded the structure calculated from the originalrestraints, even though that structure had no viola-tions above 0.2 AÊ or 2 �, relatively low energy(225.8(�7.92) kcal/mole), and would still satisfythe remaining restraints. The new structures hadsomewhat lower energy (191.7(�3.8) kcal/mole)

Figure 2. 2D 13C-edited NOESYspectrum for (15N, 2H, YMP)S4 �41 in 2H2O. Only protonsattached to 13C are visible in bothdimensions of the spectrum. Thespectrum was recorded with 528scans per increment, 256 complexpoints in the indirect dimension,and a mixing time of 160 ms on aBruker DMX spectrometer operat-ing at a 1H frequency of 500 MHz.(a) Full spectrum. Notice the strongcrosspeaks near the diagonalaround 2.2 ppm for protons withinthe methionine residues. (b) Enlar-gement of the tyrosine ring tomethionine region of the spectrum.(c) Enlargement of the methionineto tyrosine ring region. Chemicalshift positions for the protons ofinterest are indicated with arrowsand labeled. Boxes surround cross-peaks between methionine andtyrosine protons. The empty boxindicates the expected position forthe crosspeak between M60 Hb andY160 He.


and a slightly higher RMSD to the averagestructure for the backbone in residues 47 to 199than before (1.17 AÊ versus 0.76 AÊ ). To test how theensemble of structures depends upon the re®ne-ment protocol, 26 structures based on the originalrestraint list were subjected to ten additionalrounds of simulated annealing re®nement in Carte-sian space, using re®ne.inp, with an initial tem-perature of 2000 K and 2000 steps for cooling.Over the course of ten additional rounds of re®ne-ment, the average energy of the structuresincreased slightly (to 252.9(�19.9) kcal/mole) butthe RMSD to the average for backbone atoms inresidues 47-199 went up dramatically, from 0.76 AÊ

to 1.78 AÊ for all 26 structures, or to 1.63 AÊ if onlythe ®ve structures with no restraint violations over0.2 AÊ and 2 � are considered. For the individualsubdomains, where the structure is better de®nedby the NOE restraints, the backbone RMSDs to theaverage structure went up much less (0.62 AÊ to

0.96 AÊ for the helical subdomain; 0.39 AÊ to 0.56 AÊ

for the sheet-containing subdomain, including all26 structures). For this set of restraints, theX-PLOR potential energy function apparently has abroad minimum with respect to the relative orien-tation of the subdomains, and the orientation thatresults from the calculations is somewhat sensitiveto the details of the calculation. Efforts to ®nd thebest energy minimum can lead to a tight bundle ofstructures which underestimates the conform-ational space allowed by the restraints.

In another comparison, structures based on theoriginal restraint list were calculated in DYANA(GuÈ ntert et al., 1997). The RMSDs to the averagestructure for the best 20 out of 30 calculatedstructures are similar to the values after repeatedrounds of simulated annealing in X-PLOR(1.29(�0.36) AÊ for the helical subdomain,0.64(�0.11) AÊ for the sheet-containing subdomain,and 1.73(�0.45) AÊ for residues 47-199). Surpris-

Figure 3. Ribbon diagram of S4 �41, showing thelocations of side-chains that lack proton assignments attwo or more side-chain carbon positions. The side-chains are shown as ball-and-stick models and labeledwith their amino acid type and sequence position. Thestructure is based on the restraint list that includes N-Hdipolar couplings, summarized in Table 1. ThisFigure was generated with MOLSCRIPT (Kraulis, 1991).


ingly, these structures do not have the samerelative orientation for the subdomains as thestructures calculated in X-PLOR; the pairwiseRMSD for residues 47-199 is 2.55(�0.60) AÊ to theX-PLOR structure and 3.35(�0.44) AÊ to the crystalstructure. This roughly corresponds to a twist ofabout 15 � away from the original NMR structuretoward the crystal structure, although there is alsoa small translational component to the change inthe relative orientation of the subdomains. Thedependence of the relative orientation of the sub-domains on the structure calculation program usedprovides further evidence that the orientation isunderdetermined by the experimental data.

Looking for additional NOE restraints

After the original NOE restraint list had been ®l-tered based on the distances determined from thecrystal structure, the resulting orientation of thesubdomains re¯ected a neutral consensus betweenthe solution and crystal structures, but it wasbased on very few intersubdomain NOE restraints.Therefore, an attempt was made to assign all theremaining unassigned peaks in all NOESY spectra,using a set of structures that included the originalsolution structure, the crystal structure, and thesolution structure calculated from the ®ltered NOElist for reference distances. In general, only weakpeaks remained unassigned. This process restored165 of the 247 NOE restraints previously removedbecause they were violated in the crystal structureby more than 0.2 AÊ . A total of 11 other NOErestraints were removed as questionable, and 67new NOE restraints were added. Of the resulting2170 NOE restraints, only 19 are intersubdomain,two of these are sequential and contribute mini-mally to establishing the orientation of the subdo-mains, and 12 of the 19 are also predicted by thecrystal structure. Structures calculated with thisrestraint list give results similar to the list with norestraints violated by more than 0.2 AÊ in the crys-tal structure, with a backbone RMSD for residues47-199 of 1.84(�0.21) AÊ to the crystal structure and3.59(�0.23) AÊ to the original minimized averagestructure.

In an effort to locate crosspeaks that unambigu-ously support the crystal structure in the NOESYspectra, all intersubdomain proton-proton dis-tances less than 4 AÊ in the crystal structure weretabulated. Many of the protons giving rise to shortintersubdomain distances are found in threearginine residues, located near the subdomaininterface. Unfortunately, these arginine residuesaccount for three of only four side-chains in theprotein with missing assignments at two or morepositions (Figure 3). R93 and R107 give weak sig-nals in spectra from the C(CO)NH and HC(CO)NHexperiments, which were key in assigning side-chain chemical shifts. R178 gives strong backbonesignals but lacks signals further along the side-chain. With 16 arginine residues in the molecule,these assignments were not readily available from

the HCCH-TOCSY spectrum, because side-chainchemical shifts for residues of the same type aretypically similar and therefore often overlap. Miss-ing chemical shift information at the subdomaininterface is a major reason why NOE restraintscannot, by themselves, establish the relative orien-tation of the subdomains.

No additional information about thesubdomain orientation from rotationaldiffusion anisotropy

Based on the original structure calculations,S4 �41 is elongated and approximately axiallysymmetric, with the principal components of itsinertia tensor in a ratio of 2.41 to 2.36 to 1.00(2.44:2.37:1.00 for the crystal structure). This shapepredicts that S4 �41 reorients in solution as anaxially symmetric rotor with signi®cant anisotropy(Dk/D? > 1). In agreement with this expectation, aplot of the 15N T2/T1 ratio versus sin2y, where y isthe angle between each N-H bond and the uniqueprincipal inertial axis, is linear, yielding a value ofapproximately 1.4 for Dk/D? (CopieÂ et al., 1998).Further ®tting of the 15N T1 and T2 data to a fully


asymmetric diffusion tensor (Tjandra et al., 1995)yields a ratio near one for the perpendicular com-ponents of the diffusion tensor (Dx/Dy � 1.03),con®rming the axial symmetry for tumbling pre-dicted from the inertia tensor. Unfortunatelybecause S4 �41 reorients as an axially symmetricrotor, the analysis of the relaxation data providesinformation only about the orientation of the N-Hbond vectors with respect to the long axis of themolecule and therefore will not distinguishbetween the original NMR structure and the crys-tal structure. The relative twist in the subdomainsis approximately about the long axis of themolecule and the angles between that axis and theN-H bond vectors are essentially the same for eachstructure.

Dipolar couplings in uniaxial liquid crystallinephases determine the relative orientation ofthe subdomains

Proteins dissolved in an aligned phase them-selves become slightly aligned so that their dipolarcouplings no longer average to zero, as they do inan isotropic phase (Tjandra & Bax, 1997; Hansenet al., 1998). The resulting dipolar couplings aregiven by equation (1):

1DNH�y;f� � S�m0=4p��gNgHh=4p2r3��Aa�3 cos2 yÿ 1�� 3

2 Ar sin2 y cos 2f��1�

where 1DNH is the one-bond dipolar couplingbetween the backbone amide nitrogen and proton,S is the generalized order parameter, r is the dis-tance between the coupled nuclei (for this work,N and H), Aa and Ar are the axial and rhombiccoef®cients of the molecular alignment tensor,respectively, and y and f are the spherical coordi-nates that specify the orientation of the N-H bondvector in the principal axis system of the molecularalignment tensor (Tjandra & Bax, 1997). Theremaining constants are m0, the magnetic per-meability of the vacuum, gN and gH, the gyromag-netic ratios for the N and H, and h, Planck'sconstant. If the rhombic term is zero, the angulardependence for the dipolar couplings is the sameas for the T2/T1 ratio in the axially symmetric case,and only the angle between the N-H vector andthe unique alignment axis (y) can be determined.However, if the rhombic term is non-zero, thedipolar couplings are sensitive to the twist aboutthe long molecular axis through f. Typically, therhombic term is signi®cant when the shape of theprotein lacks axial symmetry or when the proteininteracts weakly and asymmetrically (e.g. electro-statically) with the aligned phase.

Initial experiments with DMPC/DHPC bicellesand 15N-labeled S4 �41 were encouraging; themeasured dipolar couplings spanned a large range(ÿ38 to 40 Hz). The measured dipolar couplingswere ®t by Powell minimization to couplings cal-

culated with equation (1) and a reference structureto establish the orientation of the molecular align-ment tensor and the axial and rhombic coef®cients.Strikingly, ®ts using the crystal structure coordi-nates gave consistently lower values of the w2 errorfunction than ®ts using the NMR structure. A qual-ity factor, de®ned as:

Q � �p f�i�1;:::;N�DNHi�measured�

ÿDNHi�calculated��2g=N�=Drms �2�where Drms is an estimate of the overall size of thecouplings, taken as the root-mean-square value ofeither the calculated couplings or the measuredcouplings, can be used to assess the ®t (Ottiger &Bax, 1999). The ®t to the crystal structure has aquality factor of approximately 25 %, within therange of values observed when ®tting dipolarcouplings at pHs from 4 to 9 to a high resolutioncrystal structure for ubiquitin (16 % to 26 %, Ottiger& Bax, 1999). This quality factor is much betterthan the value obtained for the lowest energystructure from the original NMR ensemble (54 %)and suggests that the measured dipolar couplingsare consistent with the crystal structure. Further-more, ®tting the data one subdomain at a time tothe crystal structure produced similar quality fac-tors and axes for the alignment tensor that agree towithin 7 �, providing further support for that sub-domain orientation. Regardless of the coordinatesused for reference, ®ts revealed a large rhombicity(Ar/Aa � 0.39), suitable for resolving the twistbetween the subdomains.

However, the signal-to-noise ratio in the spec-trum acquired in DMPC/DHPC bicelles was low,due to the relatively low S4 �41 concentration(100 mM) and the high salt concentration (250 mMKCl before adding bicelles). S4 �41 aggregates athigh protein concentrations (1 mM) and low saltconcentrations (Markus et al., 1998), so very limitedimprovement could be expected from increasingprotein concentration or decreasing salt. Anotherfactor degrading the quality of the spectra was sig-ni®cant line broadening from proton-proton dipo-lar coupling in the aligned phase. In order toreduce the proton-proton dipolar couplings, thehighly deuterated protein, (15N, 2H, YMP) S4 �41,already produced for the ®ltered NOESY exper-iments, was used in subsequent alignment exper-iments. Unfortunately, the bicelle sample madewith (15N, 2H, YMP) S4 �41 was stable for only afew hours; the phases separated before data setscould be acquired, and the liquid crystal could notbe re-established by charging the bicelles withaddition of cetyl-trimethyl ammonium bromide(CTAB, cationic) or sodium dodecyl sulfate (SDS,anionic) (Losonczi & Prestegard, 1998). We hadattempted to recreate the conditions that gave avery stable system for 15N-labeled S4 �41 with anew bicelle stock solution and the (15N, 2H, YMP)labeled protein; it is unclear to us why the samplewas not more stable.

Figure 4. 2D IPAP 15N HSQC for (15N, 2H, YMP)S4 �41. The spectra were recorded on 210 mM (15N, 2H,YMP) S4 �41 with 2.5 mg/ml Pf1 in 300 mM KCl at750 MHz. Both the sum spectrum and the differencespectrum from the IPAP experiment have been superim-posed to demonstrate the splittings. Peaks are labeledwith their corresponding amino acid and sequenceposition. Peaks that form a pair are indicated withdouble-headed arrows. The arrow is labeled with thesplitting in hertz; note that this splitting corresponds tothe sum of the dipolar coupling and the 1JHN scalarcoupling (about 93 Hz).


For the second attempt to make a weaklyaligned sample of (15N, 2H, YMP) S4 �41, bac-teriophage Pf1 was added to 5 mg/ml in100 mM KCl. No protein signals were observedin an 15N HSQC experiment. This suggested thatthere was a strong electrostatic interaction of thepositively charged protein with the negativelycharged Pf1, so we increased the salt concen-tration to 300 mM KCl and reduced the Pf1 con-centration to 1 mg/ml. Under these conditions,backbone amide signals were observed in the15N HSQC spectrum. Further sample condition-ing established that at approximately 200 mMS4 �41 and 2.5 mg/ml Pf1, the salt concentrationcould be used to tune the dipolar interactions,which ranged from ÿ16 to 14 Hz in 400 mMKCl and increased to ÿ28 to 26 Hz in 300 mMKCl. Although the couplings scale up by a factorof about 1.8 from the higher to the lower saltconcentration, the orientation of the alignmenttensor is the same at both salt concentrations.Fitting either set of dipolar couplings to equation(1) gives a larger rhombicity (around 0.60) thancalculated for the bicelle sample. Note that thelow amount of Pf1 required for alignment of theprotein and the salt sensitivity of the degree ofalignment supports the hypothesis that S4 �41interacts with the Pf1 electrostatically rather thansimply colliding elastically with the ®lamentousphage to gain net alignment (see Hansen et al.,1998). This interaction, involving the axiallyasymmetric charge distribution on the proteinsurface, is the source of the highly rhombicalignment that proves crucial for determining therelative orientation of the subdomains. Represen-tative spectra of (15N, 2H, YMP) S4 �41 with2.5 mg/ml Pf1 in 300 mM KCl are overlaid inFigure 4.

Structure calculations including dipolarcoupling restraints

Dipolar coupling restraints based on the datacollected with Pf1 in 300 mM KCl were added tothe structure calculations through simulatedannealing in Cartesian space using X-PLOR 4.0; theexperimental restraints are summarized in Table 1.Only 35 couplings could be measured for the 71residues in the helical subdomain, due to overlap.A total of 65 couplings were determined for the 87residues in the sheet-containing subdomain, andthe coupling for R93, between the subdomains,was also determined. From a calculation of 50structures, the 16 structures with no restraint viola-tions above 0.4 AÊ (distance restraints), 3 � (dihedralangle restraints), nor 1.0 Hz (dipolar couplings) areshown in blue in Figure 5. The RMSDs from theinput values for bond lengths, bond angles, impro-per torsions, experimental distance restraints,experimental dihedral angles, and experimentaldipolar coupling restraints are relatively low, withvalues of 0.00329(�0.00011) AÊ , 0.424(�0.011) �,0.379(�0.16) �, 0.0259(�0.0016) AÊ , 0.186(�0.71) �,

and 0.088(�0.010) Hz, respectively, quoted as theaverage value over the set of 16 structures plus orminus the standard deviation. The stereochemicalquality of the structures is good, with 76.8 % of allresidues in all 16 structures in the most favoredregion of the Ramachandran plot, 19.2 % in theadditionally allowed region, 2.7 % in the gener-ously allowed regions, and only 1.2 % in the dis-allowed regions (based on PROCHECK,Laskowski et al., 1993).

The RMSD to the average structure for backboneatoms in residues 47-199 is 0.65 AÊ , slightly tighterthan the original structure (0.71 AÊ ). Unlike the cal-culations without dipolar restraints, the RMSD forthe ensemble is independent of the details of thesimulated annealing protocol when the dipolarrestraints are included. As before, the subdomainsare better de®ned than the whole structure, withbackbone RMSDs of 0.62 AÊ for residues 47-92 and192-199 (the helical subdomain) and 0.43 AÊ forresidues 94-176 (the sheet-containing subdomain).These structures closely resemble the crystalstructure (magenta in Figure 5), with pairwiseRMSDs of 1.29(�0.15) AÊ over all structuredresidues (residues 47-199), 1.16(�0.15) AÊ for thehelical subdomain, and 0.96(�0.06) AÊ for the sheet-containing subdomain. The individual subdomainsalso closely resemble the original solution struc-ture, with pairwise RMSDs of 1.09(�0.15) AÊ for thehelical subdomain and 1.05(�0.05) AÊ for the sheet-containing subdomain, but the relative orientationof the subdomains is different, so the pairwiseRMSD over all the structured residues is high(3.64(�0.21) AÊ for residues 47-199). Similar results

Figure 5. Stereoview of the sol-ution structure of S4 �41, based onthe NOE, hydrogen bond, dihedralangle, and N-H dipolar couplingrestraints summarized in Table 1.The best 16 structures out of a cal-culation of 50 are shown in blue.For comparison, the crystal struc-ture is shown in magenta and thesolution structure based on theoriginal restraint list, lacking dipo-lar couplings, is shown in black.All structures are aligned by thebackbone atoms in residues 94 to176 (the sheet-containing subdo-main). Some residues at the ends ofelements of secondary structureand the ends of the chain arelabeled for reference.


were obtained with the dipolar couplingsmeasured in Pf1 at 400 mM KCl or in DMPC/DHPC bicelles.

Discussion

The potential dif®culty in accurately establishinglong-range order from short-range constraints wasrecognized even as the methodology for proteinstructure determination by NMR spectroscopy insolution was developed (WuÈ thrich et al., 1982;Havel &WuÈ thrich, 1985). Due to the high protondensity in proteins, and the typical organization ofcompact, globular proteins around hydrophobiccores that bring many protons from remote partsof the amino acid sequence close together, there isusually enough information to determine long-range order. However, as NMR spectroscopy isapplied to larger, more elongated, and multi-

Table 1. Summary of experimental restraints used instructure calculations for S4 �41

Total constraints available 2471Total NOE restraints 2170

Intraresidue 700Sequential (ji ÿ jj � 1) 604Short range (1 < ji ÿ jj � 4) 353Long range (ji ÿ jj > 4) 513

Hydrogen bond constraints 86Total dihedral angle constraints 114

f 72w1 42

Stereospecific assignmentsb Methylene 28Valine g methyls 8 of 9Leucine d methyls 18 of 18

Residual dipolar couplingrestraints

HN-N 101

domain proteins, establishing the long-range struc-ture and the relative orientation of the domainshas proven more dif®cult (Tjandra et al., 1997a;CopieÂ et al., 1998; Spitzfaden et al., 1997; Wiles et al.,1997; BruÈ schweiler et al., 1995).

For E. coli S4, limited proteolysis establishes thatthe N-terminal 42 residues may be deleted and theremaining protein retains af®nity for RNA(Changchien & Craven, 1976). Based on chemicalcleavage, residues 46-124 from the E. coli sequence(corresponding to residues 42-120 in theB. stearothermophilus sequence) were described as aminimum ``domain'' required for RNA binding(Changchien & Craven, 1986). Thus cleavageexperiments gave no indication that S4 �41 isorganized into two distinct subdomains. (Furtherexperiments are necessary to determine if these``subdomains'' are capable of folding indepen-dently.) With a high degree of chemical shiftdegeneracy, especially in the helical subdomain,incomplete assignments near the subdomain inter-face, and less hydrophobic surface buried in theinterface than in a typical hydrophobic core, thereis not enough NOE information available to estab-lish the precise orientation of the subdomains. Thelack of information about the relative orientationsin the original restraints is clear from two resultsfrom the structure calculations: (1) the X-PLOR cal-culations identify conformations that accommodatethe original constraints, including the misassignedcrosspeak, with low energies and no outstandingviolations, and (2) starting from the same restraintlist, X-PLOR and DYANA yield structures inwhich the orientations of the subdomains differ byabout 15 �.

Techniques to measure dipolar couplings inpartially aligned protein samples (Tjandra & Bax,1997; Hansen et al., 1998; Clore et al., 1998) and to


include dipolar restraints in structure calculations(Tjandra et al., 1997b) provide essential informationabout long-range structure. The dipolar couplingsestablish the orientations of numerous internuclearvectors with respect to the common alignmenttensor axes ®xed in the molecule. This complemen-tary information is qualitatively different from theshort-range information available from NOE cross-peaks and dihedral angle restraints and thus is apowerful addition to the information base forstructure determination by NMR (Tolman et al.,1995). Here we have demonstrated that N-H dipo-lar couplings are suf®cient to establish long-rangeorder between two well-de®ned protein subdo-mains which are linked by very few NOErestraints.

Since ¯exibility, in both protein and RNA, hasbeen shown to be an important part of some pro-tein-RNA interactions (Markus et al., 1997; Allainet al., 1996), we have looked for evidence of ¯exi-bility in S4 �41 using relaxation experiments. 15Nrelaxation data show that local backbone motionson the nanosecond-to-picosecond time scale havesimilar amplitudes for the residues bridging thesubdomains as for the residues within elements ofsecondary structure; the residues bridging the sub-domains are not especially ¯exible. Preliminary T1rmeasurements fail to identify contiguous residueswith evidence of millisecond motions. Localmotions on time scales invisible to NMR are notruled out by these measurements. However, thegood ®t of the dipolar data to a single re®nedstructure suggests that any local conformational¯uctuations, if present, involve conformations withlow populations. The fact that a common orien-tation is observed in crystals, in solution with Pf1at various salt concentrations, and in solution withbicelles suggests that the structure shown inFigure 5 best represents the conformation of thefree protein under a wide range of conditions.

That S4 �41 presents one predominant confor-mation to interact with RNA in solution is consist-ent with its role in initiating ribosomal assembly.The current view of assembly of the small subunitis that S4 and S7 bind to separate regions of the16 S rRNA and facilitate binding of the other smallsubunit proteins either by affecting the RNA con-formation or providing an interaction surface,probably both (Nowonty & Nierhaus, 1988). If S4were to adopt multiple conformations while free insolution, complex formation would be less energe-tically favorable due to the decrease in con®gura-tional entropy. However, it would still beconsistent with S4's role in ribosomal assembly ifS4 were to change from one conformation toanother upon binding RNA. While the individualsubdomains have well-formed hydrophobic coresand are unlikely to rearrange upon binding RNA,the fact that so few intersubdomain NOEs areobserved suggests that the side-chains between thesubdomains are not tightly packed. Hence a shiftin subdomain orientation, driven by favorable con-tacts with the RNA, could accompany binding. We

suggest that S4 �41 maintains one major confor-mation both free in solution and when bound toRNA. RNA binding may or may not beaccompanied by rearrangement of the S4 subdo-main orientations as directed by interactions with aspeci®c RNA target.

Then how does the protein recognize two dis-tinct RNA molecules? Although a signi®cant com-ponent of the RNA binding site for both targetslies within S4 �41, the exact protein surfacesinvolved in binding are not known. A lysine thatcrosslinks to 16 S rRNA in the context of the E. coliprotein (Urlaub et al., 1997) is in the exposed loopbetween helices a2 and a3. The cluster of arginineand lysine side-chains along the cleft between thesubdomains seems like a very suitable place forbinding a polyanion like RNA. Limited proteolysisfor S4 bound to 16 S rRNA indicates that thesepositively charged residues are within the regionconsistently protected by RNA (Changchien &Craven, 1986). One reasonable model for RNAbinding is that the positively charged cleft interactsnon-speci®cally with the negatively charged RNAbackbone, positioning the RNA for speci®c recog-nition based on hydrogen bonds to bases andhydrophobic packing. Hence, even if S4 maintainsthe same overall conformation in the free andbound states, different side-chains could possiblyparticipate in the speci®c recognition of differentRNA targets. Furthermore, the amino-terminal resi-dues in the full-length S4 may contribute differ-ently to binding mRNA and rRNA (R.B.G. andD.E.D., unpublished results). To test this model,we need to establish which surfaces of S4 interactwith RNA through structural studies of the com-plex combined with extensive mutagenesis.

Materials and Methods

Protein samples

Ribosomal protein S4 from B. stearothermophilus withresidues 2-42 deleted (S4 �41) was overexpressed inE. coli and puri®ed by column chromatography as pre-viously described (Markus et al., 1998). The protein inthe bicelle sample was 15N-labeled by culturingBL21(DE3) cells expressing S4 �41 in M9 minimal med-ium with [15N]ammonium chloride (Cambridge IsotopeLaboratories) as the sole nitrogen source. The protein inthe Pf1 samples and in the ®ltered NOESY experimentswas 15N-labeled and approximately 85 % perdeuterated,with residue-speci®c labels including [1H, 13C, 15N]meth-ionine, [1H, e-13C]tyrosine, and [b,g,d-2H]proline, referredto as (15N, 2H, YMP) S4 �41. Cells were cultured in M9medium containing [15N]ammonium chloride as the solenitrogen source and 2H2O (99.9 % 2H, Cambridge IsotopeLaboratories) as the solvent but also containing proto-nated glucose, biotin, thiamine, and kanamycin, andtrace metals dissolved in very small volumes of H2O(®nal H2O estimated at 2 %). Therefore, the sample is notfully deuterated; methyl groups derived from theglucose contain notable levels of protonation. Speci®callylabeled amino acids were added 40 minutes beforeinduction at A600 � 0.600. Amide deuterons were readilyexchanged with protons, either by incubating the protein


in 20 mM KH2PO4, 250 mM KCl, with protease inhibitorPMSF in H2O at pH 7.00 at 55 �C for 22 hours or by dis-solving in 6.0 M urea, 20 mM KH2PO4, 250 mM KCl,0.5 mM dithiothreitol and 0.1 mM NaN3 in H2O atpH 5.5, incubating at room temperature for severalhours, and then removing the urea by dialysis.

Previous samples of S4 �41 were buffered with20 mM deuterated acetate at pH 5.4. Since both DMPC/DHPC bicelles and Pf1 are more stable and better charac-terized at more nearly neutral pH (Ottiger & Bax, 1998;Hansen et al., 1998), the buffer was changed to 10 mMphosphate at pH 6.5. Chemical shift positions changedonly slightly for most peaks with the change in pH;assignments were con®rmed with a 3D 15N-separatedNOESY spectrum at the new pH. The bicelle sample ofS4 �41 was made by adding DMPC:DHPC (ratio 3:1)(Avanti Polar Lipids) in 10 mM KH2PO4, 0.1 mM NaN3,7 % 2H2O at pH 6.5 to 15N-labeled protein in the originalNMR buffer (20 mM acetate, 250 mM KCl, pH 5.4). The®nal concentration of DMPC/DHPC was 5 % (w/v); the®nal protein concentration was about 100 mM. Thissample was stable for a few months. The Pf1 sample isapproximately 214 mM (15N, 2H, YMP) S4 �41, 2.5 mg/ml Pf1, 10 mM KH2PO4, 300 mM KCl, 0.2 mM NaN3,and 6 % 2H2O at pH 6.5.

The M60 Hbbb-Y160 Heee NOE restraint

As mentioned in Results, the NOE restraint from theoriginal set that gives the largest distance violation whencompared against the crystal structure is from M60 Hb toY160 He. The shortest M60 Hb -Y160 He distance in thecrystal structure is 14.88 AÊ , 9.39 AÊ larger than the mini-mum distance observed in minimized average structurefrom the NMR calculations. Although this restraint wasnot violated in the NMR calculations, the minimuminterproton distance in a representative NMR structure is5.49 AÊ , slightly greater than expected for an observableNOE crosspeak. The distance over 5 AÊ is tolerated in thestructure calculations because pseudoatom correctionswere added for the b-methylene, which lacks stereospeci-®c assignments, and for the chemically indistinguishablee positions across the tyrosine ring to give an upperdistance limit of 6.7 AÊ .

The peak assigned to M60 Hb -Y160 He was observedin a 3D 13C-separated NOESY spectrum acquired at 750MHz with a mixing time of 75 ms. Based on chemicalshifts alone (2.20 ppm 1H, 118.17 ppm 13C, 6.80 ppm13C-attached 1H), the 13C-attached 1H must be Y160 He,but there are seven possible assignments for the protonat 2.20 ppm. For those seven possible assignments,the tiers containing the attached 13C for the proton at2.20 ppm were searched for a crosspeak to 6.80 ppm, butno unambiguous peaks were found. However, the inten-sity for the aromatic ring protons was relatively weak inthis spectrum, the result of imperfect excitation at thearomatic carbon chemical shifts due to the carrierposition (64.0 ppm), so it was conceivable that the peak(at 2.20, 118.17, 6.80 ppm) was real despite the fact that acomplementary peak was not observed. Using distancesfrom preliminary NMR structures, the assignment toM60 Hb seemed reasonable.

13C filtered NOESY experiment

To identify NOE crosspeaks between protons in themethionine side-chains and protons in the e position ofthe tyrosine rings, a 2D 13C-edited NOESY spectrum was

recorded on 613 mM (15N, 2H, YMP) S4 �41 in 20 mMdeuterated acetate and 250 mM KCl in 2H2O, with anapparent pH of 5.36. The pulse sequence is essentiallythe same as the 4D NOESY previously recorded forS4 �41 (HMQC-NOESY-HSQC building blocks), exceptthat two 13C chemical shift evolution periods wereremoved to give a 2D experiment that selects for protonsattached to 13C in both dimensions. For (15N, 2H, YMP)S4 �41, 13C is present only at the e position of the tyro-sine rings and in the methionine side-chains, so the 2D1H-1H experiment provides suf®cient resolution. Thespectrum was recorded with a mixing time of 160 ms,256 complex points in the indirect dimension, and 528scans per increment on a Bruker DMX spectrometer at500 MHz.

Comparing the original hydrogen bond and torsionangle restraints to the crystal structure

Hydrogen bond restraints, two per hydrogen bond,had been added to the original structure calculations forbackbone amide protons that exchange slowly in regionsof regular secondary structure, based on the NOE cross-peaks and preliminary structure calculations. Of 45hydrogen bonds included, 43 were also found in thecrystal structure. For two hydrogen bonds, L89 O to R93HN, at the end of helix a3, and V98 O to G102 HN, atthe end of helix a4, the NOE data are consistent with thei to i � 4 hydrogen bonds, but the crystal structuresuggests i to i � 3 pairings, making turns of 310 helix atthe ends of otherwise a-helices. Since turns of 310 helixare often found at the ends of a-helices and since theavailable NMR data do not directly identify the oxygenpartner in the hydrogen bond, these restraints wereremoved.

Torsion angle restraints, based on measurements ofscalar coupling constants, had also been included in theoriginal structure calculations. Of 73 backbone f anglesrestrained, only one disagreed with the crystal structure.Angle f for residue R178 had been restrained toÿ65 � � 25 �, characteristic of helical conformation, basedon a measured 3JHNHA value of 5.16 Hz. This residue isin the extended chain running from the sheet-containingsubdomain back to the helical subdomain. In the crystalstructure, the f angle at R178 is ÿ169 �, which, althoughless common, also gives a small value for the 3JHNHA

coupling constant. This angle has been eliminated fromthe current restraint list. Four of 46 w1 restraints wereviolated in the crystal structure by 2 � or more (for resi-dues Q55, V126, T138, and D163). Although small differ-ences in side-chain conformations are possible fromcrystal to solution and there are no speci®c problemswith the coupling constants (3JNHb and 3JHaHb) used todetermine these restraints, they have been removed fromthe current calculations to eliminate any bias away fromthe crystal structure.

Measuring dipolar couplings

15N-1H dipolar couplings were measured in 2D exper-iments based on the 15N HSQC spectrum (Bodenhausen& Ruben, 1980). For a spectrum containing pairs ofpeaks for each residue, separated by the sum of the sca-lar coupling and the residual dipolar coupling, the 1Hdecoupling pulse during the 15N evolution period wasomitted. For pairs of spectra, each containing one of thepeaks for each residue and thus providing better resol-ution, the IPAP strategy was used (Ottiger et al., 1998).


Experiments were recorded on a DMX spectrometer(Bruker Instruments) at 750 MHz to maximize resolutionand sensitivity. The scalar couplings alone weremeasured for each residue using the IPAP 15N HSQCexperiment, recorded on 15N-labeled protein at 750 mMin 20 mM deuterated acetate, 250 mM KCl, 0.1 mMNaN3, 0.1 mg/ml PMSF, and 6 % 2H2O, at pH 5.4 at 500MHz.

Spectra were processed with nmrPipe (Delaglio et al.,1995). Spectra were processed two different ways, ®rst,to enhance resolution in the 15N dimension (using linearprediction and zero ®lling), second, to maximize sensi-tivity (using a Lorentz to Gauss transform parameterizedfor line broadening). Peak centers were determinedeither by peak detection in nmrDraw (Delaglio et al.,1995) or by mouse-directed picking in PIPP (Garrett et al.,1991). For strong well-resolved peaks, both peak pickingmethods and both processing strategies gave the sameresults within approximately 1.0 Hz. For weaker peaks,the spectra processed for sensitivity were deemed morereliable. For overlapped peaks, the spectra processed forresolution were used. Errors were estimated fromrepeated determination of the splittings using two differ-ent data sets, using differently processed spectra, andcomparing the result from the IPAP experiment to theresult using the difference spectrum of the IPAP 15NHSQC together with the decoupled 15N HSQC. The split-ting determined as the difference between the 15N chemi-cal shift position between the difference spectrum andthe sum spectrum in the IPAP experiment in principleequals twice the difference between the 15N chemicalshift position in the difference spectrum and thedecoupled 15N HSQC. Error estimates were made foreach residue.

Fitting the molecular alignment tensor

The principal axes and the axial and rhombic coef®-cients for the molecular alignment tensor were deter-mined by ®tting the measured couplings to calculatedvalues based on equation (1) and N-H vector orien-tations determined from a reference structure. Fittingwas performed by Powell minimization using the differ-ence between the calculated value and the measuredvalue, divided by the uncertainty in the measurement,then squared as the w2 error function (using a C programfrom Nico Tjandra). After initial ®tting, couplings thathad the largest disagreement with the calculated valueswere omitted and the ®tting was repeated until therewere no large disagreements. This procedure eliminatesoutlying data points, possibly caused by local errors inthe reference structure; typically, less than 10 % of thedata were omitted. Note that these data were onlyomitted from the ®tting step; they were included in thelist of couplings used for structure calculations. Forstructure calculations with the dipolar couplingsmeasured in Pf1 at 300 mM KCl, the axial and rhombiccoef®cients (see equation (1)) are required as input.Based on angles from the crystal structure, the axial coef-®cient and the rhombicity (Ar/Aa) are ÿ11.9 and 0.65,respectively. Based on the angles from the lowest energyNMR structure from the calculation with the originalNOE restraints and dihedral angles, the axial coef®cientand the rhombicity are ÿ11.6 and 0.53, respectively.Based on a preliminary structure calculated with dipolarrestraints in X-PLOR, the axial coef®cient and the rhom-bicity are ÿ12.5 and 0.63. All of the values are qualitat-ively similar and yield essentially the same calculatedstructures. Because the dipolar couplings scale with the

order parameter (equation (1)), dipolar couplings fromresidues with values over 90 ms for 15N T2 (averagevalue at 750 MHz: 68.3 ms) or values less than 0.70 forthe 1H-15N NOE (average value at 750 MHz: 0.83) werenot used in the structure calculations. Both criteria ident-ify the same set of seven residues to exclude (residues44, 45, and 46 from the amino terminus, 158, 159, and160 from an apparently ¯exible loop, and residue 200 atthe carboxyl terminus).

The same ®tting procedure was also applied to eachsubdomain separately. In general, the same data pointsgave the largest violations when the subdomains wereconsidered separately as when they were consideredtogether. Iterative ®tting, omitting the outlying datapoints at each step, produced values for the axial andrhombic coef®cients for each subdomain that were simi-lar in magnitude to the values for the full data set,regardless of the reference structure used. The molecularalignment tensors for the subdomains agreed to within7 � when the crystal structure was used as the referencestructure but showed a larger variation when the orig-inal NMR structure was used as a reference. Comparisonof the alignment tensors was complicated by the factthat the axial term and the rhombic term in equation (1)have similar magnitude; during ®tting, the axes some-times become interchanged.

Structure calculations

Structure calculations using only NOE, distance, andtorsion angle restraints were executed in X-PLOR(BruÈ nger, 1992), version 3.8, using the hybrid distancegeometry/simulated annealing (DGSA) protocol fol-lowed by two cycles of simulated annealing re®nement(based on dg sub embed.inp, dgsa.inp, and re®ne.inp).For calculations involving dipolar restraints, the DGSAapproach was used to calculate a starting structure usinga development version of X-PLOR 4.0 (maintained by G.Marius Clore). Then, dipolar restraints were added usinga square well energy term (implemented by Nico Tjan-dra) with the width of the well set to plus or minus theerror and structures were calculated using simulatedannealing with an initial temperature of 4000 K and60,000 cooling steps.

Structures were also calculated using torsion angledynamics as implemented in DYANA (GuÈ ntert et al.,1997). Structures based on only NOE, distance, andtorsion angle restraints could be readily calculated withthe default annealing schedule (invoked with calc all).Addition of orientational restraints for the dipolar datainterfered with convergence of the simulated annealing,so structures were calculated by simulated annealingwithout the orientational restraints. Then, the orienta-tional restraints were added gradually by specifyingweights of 0.005, 0.050, and 0.500 for the orientationalcontribution to the target function and minimizing thetarget function with vtfmin at each weighting. The low-est ®nal values for the target function with this protocolwere around 3.

PDB accession codes

The best 16 of 50 calculated structures have beendeposited in the Protein Data Bank with accession code1C06. The energy-minimized average structure has beendeposited as 1C05. The X-PLOR restraint tables havealso been deposited.


Acknowledgments

We thank Ad Bax for making the bicelle samples ofS4 �41 and for advising us on measurements in liquidcrystalline phases. John Marquardt and Marcel Ottigerhelped us with calculations and experiments using liquidcrystalline phases. Nico Tjandra provided ®tting pro-grams and advice on analyzing dipolar couplings. Wethank Art Pardi and Mark Hansen (University of Colora-do, Boulder) for Pf1 bacteriophage and advice aboutmaking the Pf1 samples. We thank Stephen W. Whiteand Chris Davies (St. Jude Children's Research Hospital)for the coordinates of the crystal structure of S4 �41 andfor discussions about the crystal packing. We also thankMary Starich, Frank Delaglio, and Dan Garrett forspectrometer and software support, and York Tomitaand Rieko Ishima for helpful discussions.

This work was supported in part by the AIDSTargeted Anti-Viral Program of the Of®ce of the Directorof the National Institutes of Health.

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Edited by P. E. Wright

(Received 21 May 1999; received in revised form 16 July 1999; accepted 19 July 1999)

refining the overall structure and subdomain orientation of ribosomal protein s4 Δ41 with dipolar...

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