The Structure and Thermal Motion of the B800–850 LH2 ... · The structure at 100 K of integral...

The Structure and Thermal Motion of the B800–850LH2 Complex from Rps. acidophila at 2.0 A Resolutionand 100 K: New Structural Features and FunctionallyRelevant Motions

Miroslav Z. Papiz1*, Steve M. Prince2, Tina Howard3

Richard J. Cogdell3 and Neil W. Isaacs4

1Department of SynchrotronRadiation, CCLRC DaresburyLaboratory, Keckwick LaneWarrington, CheshireWA4 4AD, UK

2Department of BiomolecularSciences, UMISTManchester M60 1QD, UK

3Division of Biochemistryand Molecular BiologyDavidson BuildingUniversity of GlasgowGlasgow G12 8QQ, UK

4Department of ChemistryJoseph Black BuildingUniversity of GlasgowGlasgow G12 8QQ, UK

The structure at 100 K of integral membrane light-harvesting complex II(LH2) from Rhodopseudomonas acidophila strain 10050 has been refined to2.0 A resolution. The electron density has been significantly improved,compared to the 2.5 A resolution map, by high resolution data, cryo-cooling and translation, libration, screw (TLS) refinement. The electrondensity reveals a second carotenoid molecule, the last five C-terminalresidues of the a-chain and a carboxy modified a-Met1 which forms theligand of the B800 bacteriochlorophyll. TLS refinement has enabled thecharacterisation of displacements between molecules in the complex.B850 bacteriochlorophyll molecules are arranged in a ring of 18 pigmentscomposed of nine approximate dimers. These pigments are stronglycoupled and at their equilibrium positions the excited state dipoleinteraction energies, within and between dimers, are ,370 cm21 and280 cm21, respectively. This difference in coupling energy is similar inmagnitude to changes in interaction energies arising from the pigmentdisplacements described by TLS tensors. The displacements appear tobe non-random in nature and appear to be designed to optimise themodulation of pigment energy interactions. This is the first time thatLH2 pigment displacements have been quantified experimentally. Thecalculated energy changes indicate that there may be significant contri-butions to inter-pigment energy interactions from molecular displace-ments and these may be of importance to photosynthetic energy transfer.

q 2003 Elsevier Science Ltd. All rights reserved

Keywords: light-harvesting; TLS refinement; X-ray structure; carotenoid;energy transfer*Corresponding author

Introduction

The structures of a number of macromoleculesinvolved in the first stages of photosynthesis havebeen determined in recent years. These revealhighly organised membrane-associated complexesof peptides and pigments that are well adaptedto trapping and transporting solar energy. In non-sulphur purple bacteria this energy is trapped bythe peripheral light-harvesting complexes (LH2)

and core complexes composed of light-harvesting1 and reaction centre (LH1/RC). The propertiesand times scales of energy transfer arise from therelative pigment interaction energies and pigmentsite energy disorder. These in turn are controlledby factors such as inter-pigment geometries andtheir interactions with protein and membraneenvironments. Some events, between the mostclosely spaced bacteriochlorophyll pigments (BChla), are completed in 100 fs, whilst the final step ofenergy transfer to the RC occurs in ,35 ps.1 – 7

Important to understanding the nature of energytransfer in these systems has been the 2.5 A X-raystructures of B850-800 LH2 complexes fromRps. acidophila8 and Rs. molischianum.9 These havebeen shown to be cyclic structures of Cn symmetrycomposed of low molecular weight peptides, BChl

0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved

E-mail address of the corresponding author:[email protected]

Abbreviations used: LH, light-harvesting; TLS, trans-lation, libration, screw; BChl, bacteriochlorophyll; ADP,anisotropic displacement parameter; RG, rhodopinglucoside; APE, accumulated photon echo.

doi:10.1016/S0022-2836(03)00024-X J. Mol. Biol. (2003) 326, 1523–1538

a and carotenoid pigments. For LH2 of Rps. acidophilathe oligomer symmetry is n ¼ 9 whilst forRs. molischianum it is n ¼ 8.

B800-850 LH2 of Rps. acidophila is organised ina ring of nine radially arranged inner a and outerb-peptides. In between the b-peptides and close tothe cytoplasmic surface are nine well-separatedBChl a molecules absorbing at 800 nm. Near theperiplasmic surface, and between a and b- pep-tides, are a ring of 18 BChl a molecules that absorbat 857 nm at room temperature. The BChl a Qytransition dipole moments are responsible for thenear infrared absorption by Bchl a molecules atgeneric wavelengths 800 nm and 850 nm, and forthis reason these pigments are called B800 andB850. Carotenoid pigments are also present andabsorb in the visible part of the spectrum andperform the additional role of protection againstphoto-induced oxidation by quenching BChl atriplet states before the formation of singlet-excitedstates of oxygen radicals. The a and b-peptidesform trans-membrane a-helices with short surface-lying helices and extended structures. The B850pigments ligate a-His31 and b-His30 while B800 isbound to a-Met1. The structure of Rs. molischianumis similar but has C8 symmetry and B800 is ligatedto the carboxylate of a-Asp6. In addition the B800is also tilted and rotated in the plain of the mem-brane in comparison to B800 of Rps. acidophila.Both structures have been described with asingle carotenoid molecule in the repeating unit:rhodopin glucoside in LH2 of Rps. acidophila8 andlycopene in Rs. molischianum.9

Many spectroscopic experiments have beeninterpreted in the light of these structures. Impor-tant to understanding how these complexes trapenergy is the degree of delocalisation of thelowest energy states between 850 nm and 870 nm.Whilst it is clear, from CD calculations and singlemolecule spectroscopy, that the lowest energystates are delocalised over most of the B850pigments,10 – 12 as the energy states evolve withtime they become localised on two to fourpigments.13 – 16 The degree of delocalisation is stilla subject of debate and estimates of 14–16 pig-ments have been reported elsewhere.17,18 Delocali-sation of excited energy states depends on thetime scale of experiments, temperature, the energyof coupling between pigments and energy disorderof the system. Modelling of the spectroscopicdata requires assumptions about the nature of thedifferent kinds of energy disorder. In calculationsboth diagonal (site) and off-diagonal (interaction)energy disorder have been considered but withthe simplifying assumption that disorder is essen-tially random. Although this may be a reasonableassumption it is known that motions in proteinsare dominated by a few low frequency/largeamplitude principal modes, which have non-random trajectories.19,20 As the process of energyrelaxation through the energy states dependson static energy disorder and dynamic phononscattering it maybe useful to characterise, in

greater detail, the thermal motions in the crystalstructure. Here we describe the high-resolutionB800-850 LH2 structure of Rps. acidophila measuredat 100 K and refined at 2.0 A. At this resolution it isusual to refine isotropic thermal parameters andonly at 1.2A, or better, is it possible to refine aniso-tropic displacement parameters (ADPs). However,many crystals do not diffract better than 1.8 A,and integral membrane proteins less so. Fortu-nately X-ray thermal parameters are dominatedby a few low frequency modes correlated overmany atoms so that refinement of ADPs for eachatom is, in most cases, an over parameterisation ofthe problem. TLS group refinement comprisingtranslation, libration and screw tensors is wellknown for small molecule refinement21 but itsuse in macromolecular refinement is only nowbecoming common.22 – 25 Recently, TLS refinementhas been implemented in REFMAC5,26 which hasenabled thermal anisotropy to be modelled withlimited data in the range of 2.5–1.5 A. AlthoughTLS tensors do not describe normal modes ofvibration they approximate the effects of a collec-tion of these modes. Normal modes analysis,which describes collective atomic motions, hasbeen used to constrain ADP refinement19 and thismay be a more appropriate way to model thermalmotion. This method of refinement has shown thatonly the ten lowest frequency modes are neededto account for the majority of ADPs. However,this method requires prior knowledge of normalmodes and is more difficult to implement thanTLS refinement.

Here we show that data anisotropy can beaccounted for using a TLS thermal model refine-ment. The much-improved electron density revealsfeatures that would otherwise be near the noiselevel of the map. We show that the additionalbenefits of TLS refinement is an insight into pos-sible dynamic modes and relative disorder invarious parts of the structure. The TLS modelprovides an alternative to the stochastic models ofthermal motion employed in the interpretation ofspectroscopic experiments and gives some infor-mation about the amplitudes and directions ofcollective modes of motion involving specificatomic groups. In this way X-ray structuralinformation can give complementary informationthat maybe useful in interpreting energy transferexperiments.

Results

X-ray structure refinement was greatly improvedby TLS modelling of thermal parameters, loweringthe crystallographic R-factor from 0.205 to 0.173and improving electron density in regions wheredisordered was present in the original 2.5 A map.Features, such as the second carotenoid and thelast five residues of the a-peptide were revealed(Figure 1(c)). It was possible to reassign the ligandof B800, which was previously identified as a

1524 The Structure and Thermal Motion of B800–850 LH2

formyl-a-Met1, to carboxyl-a-Met1 (Figure 2).Examples of atomic structures that have a modifiedcarboxyl-Met1 group are a number of thymidylatesynthases,27 engineered in Escherichia coli, anda non-engineered urease from Bacillus pasteurii,which has a modification at the N terminus of theg-chain.28 Most of the LH2 structure is as reportedelsewhere8,29,30 and here we concentrate on newstructural details including more accurate pigmentgeometries, hydrogen bonding distances and adescription of correlated thermal motion/disorderarising from the TLS refinement. It should benoted that much of the spectroscopic data havebeen measured at cryogenic temperatures and thestructural parameters described here may be morerelevant to those experiments. Preliminary TLSrefinement results, using the program RESTRAIN,have been reported31,32 and are mostly consistentwith the results given here using a more robust

implementation in the program REFMAC5.26,33

These results offer the first opportunity for reliableanalysis of the TLS tensors of an LH2 complex.

Peptides

The a and b-proteins form inner and outer ringsof peptides and cross the membrane once. Resi-dues 11–36 in both a and b-peptides form trans-membrane a-helical domains. In comparison tothe 2.5 A structure8,29 the main differences occur atthe N and C termini of the a-peptides. A formyl-a-Met1 was originally postulated with the formyloxygen atom (O1) ligating Mg2þ of B800. In thehigh resolution structure electron density wasobserved at the H position of the formyl groupand refined better as a carboxyl rather than anacetyl group. The surrounding residues donateseveral hydrogen bonds to the O2 oxygen atom of

Figure 1. A schematic representation of the B800-850 LH2 complex: (a) and (b) viewed into the membrane surface;(c) and (d) view along the membrane surface. The a-chains are drawn as light-green and b-chain as purple ribbons.Bacteriochlorophylls are a-B850 (red), b-B850 (green), B800 (blue) and rhodopin glucoside (orange). The cytoplasmicsurface (N terminus) is at the bottom of (c) and (d) where glucoside head groups of the first carotenoid (RG1)are located while at the periplasmic surface (C terminus, top ((a) and (d)) are located the glucosides of the secondcarotenoid (RG2). The newly found C terminus residues can be seen extending upwards (light-green) at the top of c.

The Structure and Thermal Motion of B800–850 LH2 1525

the COO-aMet1 (Figure 2). The donating atomsare: N-aAsn2 3.08 A, N-aGln3 2.95 A and NE2-b-His12 2.95 A. The presence of these hydrogenbonds strengthens the case for assigning a carboxylrather than an acetyl group. The structural role ofb-His12 can be explained with this assignment.Residues equivalent to b-His12 are conserved inmost LH1 and LH2 complexes, suggesting that thepresence of COO-a-Met1 may be a determiningfactor in the formation of B800-850 LH2, rather

than LH1 complexes which contain only the onetype of BChl. The exception to this is B800-850LH2 of Rs. molischianum where the equivalent Hisresidue is too far for H-bonding to a-Asp6 whichligates B800.9 However, this LH2 is atypical as it isan octamer and has a very different sequence atthe N-terminal of the a-chain compared to otherLH2 a-peptides. Single site mutation in LH1 hasestablished that this residue is important forcomplex assembly,34 although in the present struc-ture there is no evidence for this apart from theinteraction with COO-a-Met1. As this residue isnot present in LH1 and LH2 of Rs. molischianumit appears that the reasons why the His residueis conserved may be different. However, LH2 ofRps. acidophila shares strong sequence homology,at the N terminus of the a-chain, with most of theLH2 complexes from non-sulphur purple bacteriaand it is likely that they all utilise COO-a-Met1 asa ligand to B800. It would be of interest to estab-lish, by other methods, if these LH2 complexeshave carboxyl modified a-Met1.

At the C terminus of the a-chain the 2.5 A struc-ture stops at a-Gly48 but at high resolution, andwith TLS refinement, the density map showsunbroken electron density which can be fittedwith the remaining residues a-Val49-Lys50-Lys51-Ala52-Ala53. Val and Lys side-chains are clearlyvisible but the last two Ala residues occupy largevolumes, indicating that there may be a number ofconformational states. This part of the structure isin an extended confirmation that passes betweenthe b-chains of the neighbouring complex relatedby 2-fold crystallographic symmetry. There areno close contacts with the neighbouring complexhence the large atomic thermal parameters of,55 A2.

The hydrogen bonds between peptides appear tohave different functions at the N and C termini.Whereas at the N terminus contacts are made onlywithin a repeating ab-heterodimer, at the C termi-nus hydrogen bonds are made only betweenneighbouring repeating units (Table 1). At the Cterminus each a-chain forms hydrogen bondswith either an a or b-chain advanced or retardedby one repeating unit. So for example, a-Thr39H-bonds with (þ )a-Gln46 in an advanced a-chainwhile a-Tyr44 bonds to (2 )b-Trp39 retarded byone subunit. Interestingly the complete a-chainreveals that a-Lys50 makes an inter-subunit contactwith (2 )b-His41 which may further stabilisecomplex formation.

Bacteriochlorophylls

B850 pigments are arranged in a circle ofdiameter ,25 A composed of nine approximateB850 dimers. The dimers are approximate becausethey are formed through association with a pair ofradially associated chemically distinct a and b-pro-teins and the pigments are not related by exact 1808rotations. The Mg-a-B850 to Mg-b-B850 distanceis 9.5 A within a dimer and 8.8 A between dimers.

Figure 2. Hydrogen bonding network surroundingB800. The COO-modification of a-Met1 ligates Mg2þ onB800. Inset is the final refined electron density in theregion of COO-a-Met1 and B800 with superimposedatomic model. B800 interacts with O1 of the carboxylforming a 2.04 A link to Mg2þ, while O2 hydrogenbonds to a-Asn2-N (3.08 A) and a-Gln3-N (2.90 A) andb-His12-NE2 (2.95 A). The C30 acetyl OBB of B800 formsH-bonds with b-Arg20-NE 2.79 A and NH2 2.97 A.There is a possible weak H-bond 3.34 A between the SDatom of COO-a-Met1 and NH2 of b-Arg20 providingadditional stability to the ligating amino acid.

Table 1. Inter-chain peptide-peptide hydrogen bonds

Residue Atom Residue Atom Distance (A)

COO-Met a1 O1 His b12 NE2 2.96COO-Met a1 SD Arg b20 NH2 3.31Gly a4 O Ser b8 OG 2.65Gly a4 O Ser b8 O 3.30Trp a7 N Ser b8 OG 3.07Trp a7 NE1 His b12 ND1 3.01Trp a7 O Leu b3 N 2.96Asn a11 OD1 Ala b1 N 2.67Thr a39 N Gln (þ )a46 OE1 2.85Thr a39 OG1 Gln (þ )a46 NE2 2.83Trp a40 NE1 Trp (þ )a45 O 2.89Tyr a44 OH Trp (2)b39 NE1 3.31Trp a45 O Trp (2)a40 NE1 2.87Gln a46 OE1 Thr (2)a39 N 2.90Gln a46 NE2 Thr (2)a39 OG1 2.88Lys a50 NZ His (2)b41 ND1 2.86

þ refers to a residue in an adjacent peptide in the forwarddirection; 2 to adjacent peptides in the reverse direction relatedby one repeat of the nonamer.


In the 2.5 A structure,8,30 measured at room tem-perature, these distances are 9.7 A and 8.7 A,respectively. These changes are probably not sig-nificant given the limiting resolution of the earlierstructure and the present co-ordinate error of0.1 A (Table 2). The planes of the macro-cyclerings are normal to the membrane plane. The B850Qy transition dipole moments have the followingorientation: for a-B850 they are 81.28 from themembrane normal and, projected onto the mem-brane plane, they are 1618 from the tangent of thecircle passing through the Mg atoms. For b-B850these angles are 81.28 and 288, respectively.Hydrogen bonds are formed between a-B850-OBBand a-Trp45-NE1 2.89 A, and b-B850-OBB anda-Tyr44-OH 2.68 A. The phytyl of a-B850 formsextensive contacts with the residues of twoa-chains before passing the inner edge of B800making contacts with O1A, O1D and CBD. Theb-B850 phytyl chain makes contact with a numberof b-chain residues then passes over the uppersurface of B800 on the same repeating unit formingextensive contacts with many atoms of its macro-cycle ring system. Most of the phytyl contacts arewith peptide residues rather than other phytylgroups and explains why the peptide sequence ishighly conserved in this region.

The B800 macro-cycle lies in the membraneplane and Qy makes a 1508 angle to the tangent ofthe circle passing through the B800 pigments.Again OBB forms H-bonds with b-Arg20-NE,2.79 A and NH2, 2.97 A. Unlike B850, B800 is wellseparated from other BChl molecules with nearestdistances of 18.4 A to a-B850, 24.0 A to b-B850 and21.3 A to a neighbouring B800. Apart from theH-bonding to b-Arg20 and ligation to COO-a-Met1, B800 is further stabilised by van der Waalscontacts with b-Val15-Ile16, b-Thr19 and b-His30.

Carotenoids

The major carotenoid of B800-850 LH2 fromRps. acidophila strain 10050 is rhodopin glucosidewith a measured BChl/carotenoid ratio of 2:1.35,36

Whilst the 2.5 A resolution structure showed awell- defined carotenoid (RG1) the second caro-tenoid could not be identified unambiguously dueto poor electron density in the relevant region.This was tentatively modelled as detergent or athird portion of a carotenoid molecule. Chemicalanalysis of LH2 from Rs. molischianum37 produceda pigment ratio of 2:1; however, the X-raystructure9 gave no indication of a second caro-tenoid. Measurements on related LH2 complexesform Rb. sphaeroides38,39 and Rb. capsulatus40 havealso revealed values close to 2:1. More recent esti-mates, using spectral reconstruction methods,41

have produced ratios of 3:1 for LH2 of Rps. acidophilaand Rs. molischianum, although a carotenoid under-estimation of 15% is possible. The carotenoidcomposition is cell culture condition dependant,and has been measured to be 81.6% rhodopinglucoside, 13.9% rhodopin and 4.5% lycopenewithin this LH2 complex (S. Takaichi, personalcommunication). This heterogeneous compositionmay be a contributing factor to some of the dis-order observed in the earlier electron density.However, the 2.0 A electron density map is sub-stantially improved and reveals clearer evidencefor a possible second rhodopin glucoside (RG2)molecule. The putative RG2 lies on the outside ofthe complex between b-peptides with an averagepeak height about half that of the RG1. Theelectron density is weak, but unbroken from theglucoside head group to two-thirds along the iso-prene chain (Figure 3). Some of the isoprenemethyl groups are visible but the rest, due to

Table 2. Parameters and refinement

Data resolution 2.0 ALow resolution cut off 8.0 ASpace group R32Cell dimensions a ¼ 117.05 A; c ¼ 298.44 ANumber of reflections (Free) 48,978 (2431)Rmerge (intensities) 0.05Data completeness 99.3%Rcryst 0.173Rfree 0.194Correlation coefficient on F 0.96Number of atoms 3439Number of water molecules 242DPI Cruickshank co-ordinate error 0.110 ADPI estimated co-ordinate error on Rfree 0.105 ARMS bond deviation from ideal 0.019 ARMS angle deviation from ideal 1.628Number of NCS constraints 7Number of TLS groups 30

Rmerge ¼P

hkl

Pj lIj 2 kIll=

PkIl is the intensity R-factor between symmetry-related reflections of index hkl and intensity Ij relative to

their mean intensity kIl: Rcryst ¼P

ðkFobsl2 lFcalckÞ=P

lFobsl is the crystallographic R-factor, where Fobs and Fcalc are the observed andcalculated structure factor amplitude, respectively. Rfree is the crystallographic R-factor calculated from a 5% subset of randomlyselected reflections not used in phasing. Co-ordinate errors are estimated according to the Cruickshank60 dispersion precision indi-cator (DPI) for all the data and the subset of reflections used to calculate Rfree:


large thermal parameters, are difficult to locate.However, at the periplasmic surface the glucosidemoiety can easily be identified and so defines thestart of the molecule. In RG2 the glucoside headgroups are more recognisable than in RG1, asthese are partially disordered even though theirisoprene parts are well defined. The detergentused in LH2 complex preparation is b-octylgluco-side, but it can be discounted as a candidate forthis electron density as it is too short and lacks themethylation apparent in the electron density. RG2makes a severe turn towards the detergent micelleforming an acute bend of ,508. To accommodatethe severe turn in RG2 two cis bonds are required.The cis bonds are cautiously located at C12 andC15 in the current model. The last third of thechain could not be located and it is assumed thata number of conformations exist. For this reasonthe last third was set to zero occupancy in therefinement. RG2 is orientated in the opposite direc-tion to RG1 with the glucoside moiety located onthe periplasmic surface rather than the cytoplasmic(Figure 1(c) and (d)). The glucoside moiety of RG2lies in a pocket created by a-Trp40,Ala43-Tyr44and b-Leu40-His41 with H-bonds to glucosidehydroxyl groups through a network of eight watermolecules. In contrast RG1 H-bonds directly toamino acid residues a-Lys5 and b-Glu10: with O6

to NZ of 2.33 A and O2 to OE1 of 2.77 A. Theisoprene chain of RG2 passes over the outersurfaces of B850 while RG1 terminates at its innersurface. Both carotenoids make van der Waalscontacts with the three BChl a pigments. However,for RG1 these contacts are distributed equallybetween all types of BChl a molecules, whilst RG2makes contacts mostly with the outer macro-cyclesurface of a-B850. Peptide contacts with RG2 areb-Ala29, Leu32, Ala33, Ala36, Thr37 and a-Trp 40and Tyr44. A question remains as to whether itis native confirmation or whether changes haveoccurred during sample preparation. Positioned asit is on the outside of the complex it is very vulner-able to detergent attack. This is consistent withhigh thermal parameters and a measured BChl/carotenoid ratio that does not constitute an integernumber of carotenoids. The latter indicates thatcarotenoid loss may have occurred early duringdetergent solubilisation. If this is the case weshould ask the question, what is the conformationin vivo?

RG2 could be in a cis conformation as foundin some reaction centres, which have 15,150-cisspheroidenone42 and 15,150-cis 1,2-dihydroneuro-sporene.43 Alternatively it may have undergone anoxidative degradation to an epoxide derivative bythermal cis–trans isomerization.44 Unfortunatelythe electron density is not sufficiently well definedto determine this. In vivo this present conformationwould extend into the lipid bilayer and perhapshinder inter-complex contacts. For this reason, andbecause of its obviously disordered state, a morelikely native conformation is as in RG1, whichwould place it along the groove between b-chains.RG2 covers the outer surface of a-B850 and makesmore extensive contacts with the B850 macro-cyclering than does RG1, and it may be that RG2 hasmore efficient Car-B850 energy transfer than RG1.Also, if in its native state it is in an extended transconformation, it too would make close contactswith B800 and be a possible channel for caro-tenoid-B800 energy transfer. In the present dis-ordered state, RG2 is likely to have little effect onCar-B800 transfers but may have measurableeffects on Car-B850 energy transfers. Finally,the presence of RG2 means that B850 pigmentsare surrounded by carotenoids and this wouldoffer greater protection against photo-oxidativedamage.

TLS tensors

The combined effect of thermal translation,libration and screw rotations can be visualisedwith an ellipsoidal representation45 of the atomicanisotropic Uij parameters derived from thedecomposition of TLS tensors using the programTLSANL.46 TLS displacements are best thoughtof as correlated atomic group motions ratherthan independent atomic motions. They can beimagined as arising from the collective effects ofa number of low frequency normal modes that

Figure 3. 2Fo 2 Fc electron density maps of the secondrhodopin glucoside, RG2. The red electron density iscalculated with phases and FCs obtained after isotropicthermal parameter refinement while the lilac electrondensity is after TLS refinement: both are contoured at2s. The refinements were done before the additionalfive C terminus amino acid residues and RG2 carotenoidwere found. The superimposed atomic structure is of thefitted RG2 but prior to its refinement.


dominate the TLS group of atoms. The modes thatwill contribute the most will have large amplitudesand vibrational phases that constructively combinemode amplitudes. In the present implementationof REFMAC525 the TLS components are refinedfollowed by the atomic co-ordinates and B-factors(Biso). In this second stage of refinement Bisorepresents the residual thermal contributionswhich can not be modelled by TLS tensors. Dueto correlation between thermal parameters thedivision between Biso and the TLS derived aniso-tropic Uij parameters is to some extent arbitraryand it is difficult to say whether Biso representslocal atomic deviations or is part of the TLS tensor.An indication that Biso could be a global para-meter, that should be subtracted from the TLS

tensors, can be seen in Figure 4(a) where apartfrom the partially disordered a-chain C-terminal,the Bisos remain remarkably constant in bothchains with the TLS components providing themain variation in the thermal parameters. Afurther complication is the possibility that some ofthe deviations originate from static disorder ratherthan dynamic thermal motion. This issue couldbe resolved by X-ray diffraction measurements ata number of temperatures down to 20 K, providinga significant number of trapped conformationalsub-states do not exist. Of importance to this workis an estimate of global or lattice motion and dis-order, if the underlying internal displacements areto be modelled. Global effects can be estimatedif we compare the results of one or three with 30TLS tensor refinement (Figure 9). For an increaseof one to three TLS tensors the fall in R-free is 45%of that for 1–30 TLS tensors, also the translationaldisplacement, for one TLS, is one-third and libra-tion one-quarter of that from 30 TLS tensors. Thispoints to around 25–45% contribution from latticedisplacements with the rest represented by internaldisplacements between the various TLS groups.The following results have been analysed for TLSgroup anisotropy and direction of displacementby monitoring two parameters, Pmax and E; whichcan be calculated from Uij parameters. A moredetailed explanation can be found in Methodsbut in brief, Uij parameters describe an ellipsoidalsurface of atomic displacements. This surface isdefined by three principal axes of the ellipsoid.Pmax is the largest and Pmin is the smallest ellipsoidaxis and the ratio E ¼ lPmaxl=lPminl defines thedegree of anisotropy or distortion from a sphere.It follows that vector Pmax provides informationon the relative amplitudes and direction of thedominant TLS group displacement.

Peptide thermal parameters

In the region of B850 pigments the a-chains havesmaller thermal amplitudes and less anisotropythan the b-chains (Figure 4(b)). The N and Ctermini are thermally hotter and the b-chain Nterminus has large librational motion of ,45 deg2,with an axis of rotation in the plane of the mem-brane and along the direction of the chain. Onaverage the b-chains are hotter than a-chains andPmax; calculated from Ca atoms around the B850pigments, lies almost in the membrane plane(Figure 5(d)). The largest component of librationfor the trans-membrane helices is along their helicalaxes and is ,12 deg2 for both chains. For trans-membrane helices the components of libration inthe membrane plane are ,2 deg2 but act on longerlever arms so that at the ends of the helices thedisplacements resulting from these small rotationsare comparable to the libration along the helicalaxes. The point of intersection of the axes of libra-tion are between the B850 and B800 pigments:5 A and 13 A below the Mgþ2 atoms of B850 forthe a and b-chains, respectively. The total thermal

Figure 4. (a) Isotropic thermal parameters for Ca atomsof a (squares) and b (triangles) chains. The lower curves(dotted) are atomic residual B-factors converted tokUisol

2¼ Biso=8p2: The upper curves (continuous) are

the equivalent isotropic thermal factors kUeql2¼ Tr{U};

where Tr{U} is the trace of atomic anisotropic thermaltensor Uij: kUisol

2 is included in kUeql2 to give the full

thermal parameter. For each peptide an average wastaken from the NCS repeating unit. The standard devia-tions of the three copies were in the range 0.01–0.02.The abcissa gives the residue number of each chain.(b) Pmax and Pmin are the largest and smallest principalaxes of the thermal anisotropic ellipsoid derived fromthe eignavalues of tensor Uij with kUisol

2 included. Thelower curves (thick lines) are Pmax and the upper curves(thin lines) are E ¼ Pmax=Pmin: E is the degree of aniso-tropy and E ¼ 1 corresponds to isotropic thermal para-meters while increasing E corresponds to larger atomicanisotropy. These values were calculated from the meanof three NCS copies with a corresponding standarddeviation of 0.02 for Pmax and 0.1 for E:


contribution TLS þ ISO, expressed as the equiva-lent isotropic thermal parameter Ueq; in the regionof B850 pigments is ,0.4 A2 and 0.6 A2 for a andb-chains, respectively (Figure 4(a)). The observedoscillations in Ueq; along the b-chain, are greateston the outer surface, suggesting more freedom forthese atoms. In contrast the a-chains exhibit nooscillations, are less anisotropic, are thermallycooler and have a minimum displacement in theregion of the phytyl chains. The biggest values ofPmax coincide with the outer surface of the b-chainsand are directed tangentially to the nonameric ring.For example the Ca atom of b-Gly24 on the firstb-peptide lies exactly on the co-ordinate x-axiswith Pmax making an angle of 1008 to the x-axis atthe same time the angle of the line joining thisatom to the same atom on the next b-chain is,1108 relative to the co-ordinate x-axis. This isalso observed for a-chains but the direction ofPmax is less well defined as the atoms tend to bemore isotropic (Figure 4(b)). The out-of-membraneplane angle of Pmax is around 30–408 along mostof the length of the trans-membrane helices, butfor b-chains it becomes progressively morein-plane reaching an almost in-plane orientationat b-His30. For a-chains it is almost in-plane atthe end of the trans-membrane helix and at thebeginning of the surface-lying segment betweenresidues 38 and 48.

B850 thermal parameters

The largest B850 librations are about axes normalto the membrane and are ,6 deg2 for a-B850 and12 deg2 for b-B850. The in-membrane librationsare small apart for b-B850 which has a significantlibration of 7 deg2 along Qy. At the extreme pointsof the macro-cycle ring the atomic displacementscaused by the large librations are ,0.5 A2 and1.0 A2, for a and b-B850, respectively, and can bevisualised as a rocking motion of the B850 pig-ments about the membrane normal. The macro-cycle as a whole experiences average librationaldisplacements of 0.2–0.4 A2 while the largest trans-lational motions act along the line joining pigmentsand are about 0.3 A2. These values arise from theTLS components alone and when the individualisotropic parts are added result in Pmax of around0.5 A2. Anisotropy factors E; obtained from TLS þISO, are 1.5 for B850 pigments but when LS tensorsalone are used to calculate Uij no change in E isobserved for b-B850 but a decrease is seen for a-B850 to 1.25. This may indicate that relative contri-butions from T and LS tensors are different for thetwo pigments. In particular a-B850 is more con-strained and is less able to perform librationalmotion. Assuming equipartition of Biso betweenthe T and LS tensors the contribution to ADPsfrom libration is about half that of translation

Figure 5. The angles made, by that the largest principal axis of the thermal ellipsoid ðPmaxÞ; with the membraneplane (u) and with the a crystallographic axis (c) when projected onto the a; b plane. Only the Ca atoms were chosenfor each amino acid residue. (a) and (b) the a-chains and (c) and (d) b-chains. Each NCS repeating peptide is shown,in the order from a to b axis (as a S, A and K). The accuracy of these curves depends on the amount of anisotropy ineach atom. For example, in the case of an isotropic atom these angles are completely undefined and have no meaning.


(Figure 6(b)). The direction of mean Pmax for themacro-cycle atoms is, on average, 258 out of themembrane plane but is larger if only LS com-ponents are used in the calculation. This appearsto indicate that contributions to TLS tensors fromtranslational motions alone are along membraneplane (Figure 6(a)). Pmax is around 10–308 awayfrom the Qy transition dipole moments and is atthe larger end of this range for b-B850 pigments.On looking into the membrane plane the anglebetween Pmax of B850 pigments in a dimer is,1208 (Figure 7(a)) whilst between dimers theyare almost in line. This could be interpreted asshowing a more correlated motion between dimersthan within dimers. The angle made by Pmax rela-tive to the co-ordinate x-axis is dominated bythe nonameric geometry of the complex and asexpected increases linearly. Comparing linesobtained with LS tensors alone is instructive as itgives information about the librational motion.

The deviation from a straight line for a-B850 pig-ments 1 and 3 is not significant as it is due to theisotropic nature of these pigments (E of 1.25),which means that the principal axes of the ellipsoidare not well defined. However, the b-B850 pig-ments at positions 2, 4 and 6, with relatively largeranisotropies, fall on a straight line and give avalue close to 908 for the direction of motionrelative to Qy.

In summary, there are two types of motion: atranslational movement along a line between thecentres of B850 pigments of different dimers and adisplacement at right angles to this imparted bylibration or rocking motion about an axis normalto the membrane plane (Figure 7). There are othermotions which, although significant, vary the interpigment angles and displacements by smalleramounts and in directions that have lesser effectson pigment interaction energies. TLS refinementcannot reveal the relative phases of the motionsthat are important in understanding the magnitudeof energy changes arising from molecular displace-ments. If the relative motions of nearest pigmentsare positively correlated then their relative dis-placements will be unchanged. However, ifmotions are anti-correlated then the relative dis-placement is the sum of their individual displace-ments. In fact the situation is more complex thanthis because within a TLS tensor group all atomscan move in a positively correlated way and thisis indistinguishable from the situation of half the

Figure 7. B850 pigments with the directions of Pmax

marked with copper bars. Pigments from left to rightare alternatively a-B850, b-B850 with a dimer of onerepeating unit coloured green. (a) Looking into theplane of the membrane; (b) looking along the membrane.The largest libration is marked with dark blue arcs andis a rotation about an axis normal to the membrane.The amplitudes and approximate directions of Pmax asmarked with yellow arrows.

Figure 6. (a) The angles made by the largest principalaxis of the thermal ellipsoid ðPmaxÞ for each pigment,with the membrane plane (dotted) and with the a crystal-lographic axis (continuous) when projected onto themembrane plane. Pmax is the mean vector calculatedfrom BChl a macrocycle atoms or all the isoprene atomsfor the carotenoids. Curves marked with circles werecalculated with full TLS tensors and those with squareswere calculated with only the libration and screw tensorsLS. The unmarked line is the angle that Qy transitiondipole moment makes with the a axis. a-B850 pigmentsare 1, 3 and 5; b-B850 is 2, 4 and 6; B800 is 7, 8 and 9and the carotenoids RG1 are S, T and U. (b) Pmax is indotted lines and anisotropy E in continuous lines. Themarkings are as for (a).


group moving in one direction and the other halfin the opposite direction at half this displacement.For this scenario an anti-correlated motion will bethe sum of half displacements. Even so, for thissecond case the inter B850 amplitude displace-ments (i.e

pPmax) will be relatively high at 0.7 A.

The likelihood is that B850 neighbours are anti-correlated as extensive normal mode analysis ofseveral proteins20 show that nearest-neighbourresidues with bonded contacts have positivelycorrelated motions while non-bonded neighbourstend towards anti-correlated motions. Becausea-B850 and b-B850 have similar environments andlocations within the complex then it maybeexpected that they should have similar thermalproperties. Although for translational motionthere is only a very slight difference betweena-B850 and b-B850 the difference is more pro-nounced for librational motion, which is greaterfor b-B850. In addition the translation is morealigned in a direction that is between dimers.The source of thermal asymmetry between a andb-B850 may originate from the b-chain which hasgreater thermal motion and freedom of movementand may impart some of its motion to b-B850. Itmay be noticed that Pmax; for a-B850, is in themacrocycle plane but for b-B850 (Figure 7) it is

aligned more in the direction of the b-chain. It isthis difference in b-B850 motion which causes thedisplacement to be more in a direction that isbetween dimers and may implicate b-chain move-ment in driving b-B850 motion.

B800 thermal parameters

The equivalent mean isotropic thermal para-meter, Ueq; for B800 is around 0.6 A2 and is largerthan for B850 which is 0.4 A2. Anisotropy is alsolarger, 1.7 rather than 1.5 and Pmax is alignedalong the Qy transition dipole vector which is 1508relative to the tangent of the nonamer circle. Thelargest libration is a compound motion of twoorthogonal angles of rotation in the plane of themacro-cycle. The combined effect is 12 deg2

rotation about an axis parallel to the radial vector.There is a smaller libration of 4 deg2 about an axisnormal to the membrane and the centre of actionof the TLS tensors is 4.5 A above the Mg2þ atomstowards the B850 pigments. The contribution toADPs from librations are similar to B850 but withlarger translational components of around 0.4 A2.The largest anisotropy is in the B800 phytyl chainwhich reaches a maximum of 6 deg2 at the termi-nus with a Pmax value of 1.79 A2. Interestinglyb-phytyl-B850 shows the same behaviour while a-phytyl-B850 is thermally cooler and more isotropicalong its length (Figure 8). The phytyl groups ofB800 and b-B850 pass over each other’s macrocyclerings, while a-phytyl-B850 passes between B800and an a-chain (Figure 1(c)). As both B800 andb-B850 have librations of 12 deg2 in their macro-cycle rings, which are also aligned with the dis-placements of the phytyl chains, this may indicatesome coupled motion. It may also account forsome of the differences observed in the motions ofa and b-B850 mentioned in the previous section.

Carotenoid thermal parameters

Only the first carotenoid (RG1) has been TLSrefined as RG2 is less well defined. The mean ani-sotropy E of RG1 is 2.0, mean Pmax amplitude0.62 A2 and Ueq 0.45 A2. The direction of Pmax is atright angles to the local direction of the isoprenechain. The largest anisotropy of 3.8 is near theglucoside head group with a Pmax value of 1.1 A2

while at the other end, near the B850 pigments, Efalls to 1.6 and Pmax to 0.5 A2. Here thermalmotions matches that of B850, both in amplitudeand anisotropy and Pmax is directed towardsb-B850. Again there is a directional pigment-pigment displacement with b-B850 that does notexist for a-B850.

Discussion

Atomic displacements within the crystal canarise from a number of sources: dynamic vibra-tions and static disorder within a macromolecule

Figure 8. Anisotropic thermal ellipsoids of bacterio-chlorophyll pigments. Only one asymmetric unit isshown. (a) B850 and (b) B800 pigments. Both are viewedalong the membrane surface and have been drawn asseparate diagrams for clarity using Ortep-345 and ren-dered with POV-ray.† The largest thermal motion isseen at the ends of the phytyl chains of b-B850 and B800.


and lattice disorder or dynamic motions betweenmacromolecules. Limited data resolution can alsomimic thermal motion. For example a refinementof LH2 at 2.5 A inflates thermal parameters by12%. In principal the relative contributions ofdynamic vibration and static disorder can bequantified with data measured at several tempera-tures. Temperature dependence of isotropicthermal parameters in ribonuclease a47 haverevealed a biphasic reduction in thermal para-meters with a fast decline from room temperatureto 200 K followed by a slower rate at lower tem-peratures. The increase in thermal parameters,in the range 98–200 K, was ascribed to harmonicvibrations and anharmonic above 200 K. Atomicnormal mode analysis20 and X-ray structure refine-ment using normal mode constraints19 haveshown that the ten lowest modes, in the range of3–10 cm21, can account for ,70% of the observedthermal parameters. The dominance of these lowfrequency modes suggests that data well below100 K must be measured if dynamic vibrationamplitudes are to be significantly reduced toreveal the temperature-independent static disorder.

At 98 K ribonuclease a thermal parameters are halfthose observed for the LH2 complex and it maybe supposed that LH2 has significant contributionsfrom lattice disorder. This difference in thermalparameter could be, in part, a resolution effect asthe ribonuclease data were collected at 1.5 A withthermal behaviour typical of a protein diffractingto atomic resolution. However, a small compactprotein composed of a single peptide with disul-phide bridges may be expected to have a verydifferent thermal response compared to LH2which is composed of 63 separate small peptidesand pigments. It is not obvious whether the dif-ferences are due only to lattice disorder. LH2diffraction patterns display pronounced acousticthermal diffuse scattering, which is characteristicof disorder on the scale of sizeable domains, butthis can also originate from dynamic fluctuations.There is also evidence that special thermal proper-ties may exist in long stretches of a-helices.The TLS refinement of Ca2þ bound calmodulin, at1.0 A resolution and at 100 K,48 revealed an averageT tensor eigenvalue of 0.25 A2 while for the six-turn a-helix it was 0.52 A2. The authors concludedthat there exists a small number of conformationalsub-states indicating flexibility in this long helix.These results suggest that large thermal para-meters may be an intrinsic property of some longhelices and may explain the refinement of LH2 forwhich several long trans-membrane helices exist.

Up to 45% of the TLS tensor amplitudes may beattributable to lattice displacements or displace-ments between nonameric repeating units, asjudged by the drop in the R-free value for 3 and30 TLS tensors (Figure 9). However, one TLS tensorrefinement has components one-quarter to one-third of 30 TLS tensors which may indicate a smal-ler contribution from inter-complex displacements.Such displacements may account for ,0.25 A2 ofthe total and is comparable to the mean residualisotropic B-factor. With this contribution removedthe B850 root-mean-squared displacements become^0.5 A rather than ^0.7 A. If the motions are anti-correlated between pigments then the change ininter-pigment separation r is dr , 1:0 �A: For aninter-pigment separation of r , 9 �A this modulatesthe pigment-pigment Coulomb interaction energiesV by the factor dV=V ¼ 3dr=r ¼ ^0:33: The rota-tional motion will add to this depending on therelative phases of the modes and may contributedV=V ¼ 0:1 and 0.07 for the 12 deg2 and 6 deg2

librations separately, or 0.12 if they are also anti-correlated. Several estimates of V have been pro-posed ranging from 200 cm21 to 500 cm21; how-ever, all agree that the intra aB850-bB850 V valueis larger than inter dimer (2 )bB850-aB850 Vvalue. The most detailed calculations, using the abinitio transition density cubic method,49 give367 cm21 and 284 cm21 for the intra and inter-dimer B850 interaction energies. The displacementsoutlined above indicate that these energies maychange by ^100 cm21. At the extremes of pigmentdisplacement anti-correlated motion will have the

Figure 9. TLS refinement R-factor (diamond) and Rfree

(square) statistics as a function of number of TLS tensors.REFMAC5 was used for refinement.26 Rfree was calcu-lated from a randomly selected set of reflections whichcomprised 5% of the total and which were excludedfrom refinement. For 0 TLS tensor an isotropic B-factorwas refined only; one TLS was a single tensor for thewhole asymmetric unit; three, one for each NCS repeat-ing unit each comprising two peptides, three BChl aand one carotenoid molecules; 18, one for each molecule;30, as 18, but each peptide partitioned into surface/trans-membrane/surface helices and 48 TLS tensors where thetrans-membrane helices were divided into four equalsegments.


effect of reversing the relative strengths of theinteractions; however, even for random uncorre-lated motion this will make them about equal.During this part of the vibrational cycle thelikelihood that excited energy will concentratebetween dimers rather than within dimers may beincreased. If this is a dynamic time-dependantchange then it could be a mechanism for drivingenergy transfer from pigment to pigment. Silbeyand co-workers50 have proposed models for B850excited state diagonal (pigment site energy) andoff-diagonal ðdVÞ disorder which could explain anumber of experimental parameters concerningthe positions and separations of the lowest energyk ¼ ð0 ^ 1Þ exciton states that have been measuredby low temperature hole burning and singlemolecule spectroscopy.51,52 They concluded thatinhomogeneous broadening may originate fromapproximately equal amounts of diagonal and off-diagonal disorder. A random off-diagonal disorderof standard deviation s ¼ 0:35V or a disorder ofs ¼ 0:19V that is anti-correlated with pigment siteenergy can well explain the observations andgives an alternative model to one of ellipticaldeformation proposed to explain the singlemolecule spectroscopy experiments.52 In fact theelliptical deformation (,7%) used to model thesingle molecule spectra52 introduced a correlateddisplacement of pigments around the ellipse andthis has some similarity with the models proposedby Silbey and co-workers.50 The main difference isthat elliptical deformation produces a systematicdisplacement while the models described by Silbeyare stochastic and uncorrelated displacementsaround the ring of pigments. Nevertheless bothmethods take into account the observed spectro-scopic data through the lowering of C9 symmetryof the complex by varying inter-pigment sepa-rations and angles. The off-diagonal energydisorder proposed, from the modelling of spectro-scopic data, is similar in magnitude to that whichwe derived from thermal parameters. Motionsmay be correlated over several pigments for somelow frequency modes and it not clear whether thecombined mode displacements reduce the systemto C1 symmetry or whether, on average, someintermediate symmetry between C1 and C9 exists.These motions will of course be more complexdue to anharmonic interactions, which can causediscontinuous jumps across energy barriers intodifferent conformational substates. Such inter-actions have been described for bovine pancreatictrypsin inhibitor by principal component analysisbased on molecular dynamics (MD) calculation.53,54

It was shown that anharmonic interactions canarise at frequencies below 80 cm21 and theseincrease mean square displacements by a factor of1.5 compared to harmonic modes. The principalmodes with the largest amplitudes were found tobe composed of normal modes across a broadrange of frequencies and these shared similartrajectories. For LH2 complexes, which arecomposed of many molecules, the number of

anharmonic modes may be larger compared to thesmall proteins used in MD simulation.53,54 Never-theless, complicated though these motions maybe, a degree of correlation in trajectories of normalmodes that form principal modes may be approxi-mated by large harmonic displacements53 such asthose defined by TLS tensors. This may be a wayof using X-ray derived TLS displacements in themodelling of spectroscopic data and should bepreferable to the models currently employedwhich are based on plausible assumptions ratherthan experimental observations.

The displacements we see in all X-ray structuresare dominated by a small number of modes in the3–10 cm21 range19,20 and, if we assume harmonicmotion, these will have time periods of 3–7 ps.Half periods describe the extremes of spatial dis-placement so that any energy transfer processestaking place within time scales ! 1 ps will see dis-placed, but essentially static, atomic distributionsand hence static V values; whereas for energytransfers at times $1 ps a time dependant vari-ation of V values will be observed. With thissimple harmonic model it can be stated that for66% of time pigments will be displaced in theextreme half and at ! 1 ps times there will be sig-nificant displacements from equilibrium. It wouldbe of interest to know if low frequency modes dochange their character from apparent static dis-placements, below 1 ps, to dynamic displacementsover several picoseconds or whether thesedynamic motions take place, by discontinuousanharmonic jumps, at times longer than those ofphotosynthetic transfer processes. Displacementsmay play a different role in the two time domains,especially if there is a degree of non-stochasticinteraction between a number of pigments, so thatthe coupling energy change, dV; retains a degreeof correlation with time and pigment position.

Normal mode analysis20 shows that each indi-vidual low frequency mode defines large seg-mental motion correlated over the whole complex.Even without knowledge of mode phases it ispossible to say that as these modes combine themotion will become less correlated globally butmay remain strongly correlated over a smallerpart of the structure. This must be the case other-wise we would not be able to observe measurableTLS displacements. Obviously if a large numberof modes contribute the motion will be almostrandom and uncorrelated. However, as the lowest,10 modes have the largest amplitudes and domi-nate displacements it is likely that such a smallsample of modes will achieve only partial ran-domisation and significant correlated or anti-corre-lated motion may remain spanning a large partof the structure. The majority of modes are highfrequency small amplitude modes involving smal-ler structural segments and this would be expectedto contribute a background noise of superimposedrandom displacement. For LH2, significant corre-lated motion exists: TLS refinement reveals rela-tively large segmental motions that appear to be


well reproduced between NCS molecular copies.Recently accumulated photon echo (APE) andpump-probe measurements have revealed thatB850 pigments have a relatively short dephasingtime, T2

p , 130 ps at 4.2 K, which points to strongpigment–protein interactions. TLS refinementshows that these interactions may be more exten-sive than the immediate neighbourhood of thepigments. For example the in membrane planemotion of b-chains culminates at b-B850 butinvolves the whole trans-membrane helix. Alsothe large librations of B850 pigments, about themembrane normal, is also mirrored in the peptidechains which suggests that these motions arecoupled in some way. Interestingly there is only asmall B800 libration normal to the membraneand the biggest libration is about an axis in themembrane plane. The different behaviour of B800compared B850 and peptides may indicate a B800motion that is less coupled to protein motion. Thisappears to be indicated by APE measurementswhich show a dephasing time of 300 ps for B800,55

which is significantly longer than that for B850.56

These motions could have important biologicalrelevance and may have arisen by the naturalselection of physical and dynamic properties tooptimise energy transfer.

Methods

The LH2 complex was purified and crystallised asdescribed.57,8 X-ray diffraction data were collected at100 K from crystals that were stabilised and preparedfor cryo-freezing by equilibrating in K2HPO4, 350 mMNaCl, 0.5% (v/v) b-octyl glucoside (BOG) by vapourdiffusion against 2.3 M ammonium sulphate at pH 9.3for two days then placed in Hampton Research dialysisbuttons covered with 3 kDa membrane and dialysedagainst a 1:1 mixture of 3 M K2HPO4/Na (pH 10.5)700 mM NaCl 1% BOG: saturated sucrose in waterfor 8–16 hours. Data were collected with an ADSCQuantum 4 CCD area detector on the SRS wiggler beamline 9.6 at Daresbury Laboratory. Data were visiblyanisotropic showing weaker diffraction in the ab planefor which the limit of resolution was 2.0 A. Cell dimen-sions were significantly different from the crystals usedto determine the room temperature structure at 2.5 Aresolution (1KZU) with a shortening in the ab directions117.05 A (ca. 120.3 A) and lengthening in the c dimension298.44 A (ca. 296.2 A). The crystal structure was refinedusing REFMAC533 initially using isotropic thermal para-meters then TLS thermal refinement25 (Table 2). SevenNCS constraints were imposed between three repeatingunits of a, b-peptides, a-B850, b-B850, B800 andcarotenoid molecules. Within the hole, enclosed by thenonamer, are several segments of weak electron densitywhich were fitted with a number of b-octylglucosideand benzamidine molecules. TLS tensors groups wereassigned to molecules or well defined segments such astrans-membrane helices and surface lying helices. Thenumber of groups was chosen by monitoring changes inR-free as the number of groups was increased (Figure9). The groups were increased from 1 to 48 as follows:(a) one tensor for the whole asymmetric unit; (b) threetensors, one for each NCS repeating unit of molecules;

(c) 18 tensors one for each peptide and pigment; (d) 30tensors which were as in (c) but with peptides brokendown into surface-membrane-surface domains; and(e) with trans-membrane domains broken down intofour additional trans-membrane regions. These calcu-lations were made with all the water molecules in placebut before the last five a-peptide residues and secondcarotenoid were located. A significant drop in R-factorand Rfree was observed for one TLS tensor but only aslight improvement to three tensors then a large drop at18 tensors with a small change at 30 and none at 48.Although a small change was observed on going from18 to 30 tensors, 30 was finally chosen as relativelylarge changes in TLS tensors elements were seen for theadditional surface segments and these were consistentlyrepeated for the three NCS repeated groups. MediumNCS constraints were applied to co-ordinates (0.5 A)and isotropic B-factors (2 A2) while TLS parameterswere unconstrained. Usually refinement proceeded inrounds of six cycles of TLS refinement, with atomicresidual isotropic B-factors (Biso) set at 20 A2, followedby 15 cylces of co-ordinate and residual Biso refinement.Overall scale and thermal parameters were refined andapplied to Fobs before TLS refinement, hence TLS para-meters define the anisotropy after the isotropic part isremoved. The program TLSANL58 was used to analysethe TLS parameters and to output coordinates and ortho-gonalised anisotropic Uij from the decomposition of theTLS tensors. The atomic Biso residual was included inthe Uij values so that the equivalent isotropic U is Ueq ¼Tr{U}; where Tr{U} is the trace of the atomic Uij ADPs.It was found to be more useful to analyse the Uij valuesfor anisotropic trends in the following way: the eigen-values and eigenvectors were calculated for each Uij

tensor giving the amplitude and directions of the prin-cipal axis of the atomic thermal ellipsoids. The followingparameters were calculated: Pmax; the magnitude of thelargest principal axis of ellipsoid, the angle (u) it makesabove the membrane plane, the angle (c) it makes withthe a crystallographic axis when projected onto thea=b plane and an anisotropy factor E defined aslPmaxl=lPminl; where Pmin is the smallest principal axis.Electron density was fitted with an atomic model usingthe graphics program O59 and the electron density andatomic structure were rendered with the ray tracingprogram POV-Ray.†

Data Bank accession number

Atomic co-ordinates and structure factors have beendeposited under the RCSB PDB ID code 1NKZ.

Acknowledgements

The authors thank the BBSRC and CCLRC forsupporting the research programme over manyyears.

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† http://www.povray.org


http://www.povray.org

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Edited by R. Huber

(Received 25 September 2002; accepted 19 December 2002)


The Structure and Thermal Motion of the B800–850 LH2 ... · The structure at 100 K of integral...

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