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Mapping proteins of the 50S subunit from Escherichia coli ribosomes
Biochimica et Biophysica Acta 1520 (2001) 7^20
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Mapping proteins of the 50S subunit from Escherichia coli ribosomes Regine Willumeit 1;a; *, Gundo Diedrich 1;2;b , Stefan Forthmann a , Jo«rn Beckmann a , Roland P. May c , Heinrich B. Stuhrmann 3;a , Knud H. Nierhaus b a
GKSS Forschungszentrum Geesthacht GmbH, Institut fu«r Werksto¡forschung, WFS, Max-Planck-StraMe, 21502 Geesthacht, Germany b Max-Planck-Institut fu«r Molekulare Genetik, AG Ribosomen, IhnestraMe 73, 14195 Berlin, Germany c Institute Laue Langevin, F-38042 Grenoble Cedex 9, France Received 8 November 2000; received in revised form 20 March 2001 ; accepted 16 May 2001
Abstract Mapping of protein positions in the ribosomal subunits was first achieved for the 30S subunit by means of neutron scattering about 15 years ago. Since the 50S subunit is almost twice as large as the 30S subunit and consists of more proteins, it was difficult to apply classical contrast variation techniques for the localisation of the proteins. Polarisation dependent neutron scattering (spin-contrast variation) helped to overcome this restriction. Here a map of 14 proteins within the 50S subunit from Escherichia coli ribosomes is presented including the proteins L17 and L20 that are not present in archeal ribosomes. The results are compared with the recent crystallographic map of the 50S subunit from the archea Haloarcula marismortui. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Neutron scattering; Ribosomal protein; Spin-contrast variation
1. Introduction The ribosome as the site of protein synthesis in every living cell is one of the most intensively investigated macromolecular structures. A wide set of di¡erent biological and physical methods are applied to determine structure and function of the ribosome. Beside cross-linking and footprinting, methods which reveal the interaction of constituents of the ribosome [1^4], scattering techniques such as neutron and X-ray scattering, immunoelectron microscopy (IEM) and cryoelectron microscopy (cryo-EM) have added to the picture of what the ribosome looks like [4^ 11]. The remarkable achievements by X-ray crystallography now give the possibility to become familiar with the
Abbreviations : IEM, immunoelectron microscopy; CP, central protuberance; TP50, total proteins of the 50S ribosomal subunit; SANS, small angle neutron scattering; EHBA, sodium bis(2-ethyl-2-hydroxybutyrato)oxo-chromate(V) monohydrate {Na[Cr(C6 H10 O3 )2 ]WH2 O}; DNP, dynamic nuclear polarisation * Corresponding author. Fax: +49-4152-87-13-56. E-mail address : [email protected] (Regine Willumeit). 1 These authors contributed equally to the paper. 2 Present address: Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. 3 Present address: Institut de Biologie Structurale, IBS-LCM, 41 Avenue des Martyrs, F-38027 Grenoble Cedex 1, France.
structure of the subunits on an atomic level [12^14]. The 50S data were obtained from the ribosomes of Haloarcula marismortui which with respect to the three fundamental categories of the living systems, viz. the bacteria, archea and eucarya [15], belongs to the archea. In this work neutron scattering results for the localisation of ¢ve proteins of the large subunit of the bacterium Escherichia coli are presented. They are combined with previously published results [16^19] giving the positions of 14 proteins in the 50S subunit. These positions are reviewed taking into account the now available high resolution structures. The method used was spin-contrast variation, which has been successfully applied to position the protein L2 within 50S subunits and 70S ribosomes, and that of tRNAs and the mRNA within elongating ribosomes [19^23]. 2. Materials and methods Deuterated cells of the E. coli strain MRE600rif were purchased from CDN (Tallinn, Estonia), L-[U-14 C]Phe from Amersham-Buchler (Braunschweig, Germany), chemicals for gel preparation from Bio-Rad (Richmond, CA, USA), poly(U) mRNA from Boehringer (Mannheim, Germany), HEPES from Calbiochem (Frankfurt, Germany) and all other chemicals including 2 H2 O and d8 -
0167-4781 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 1 ) 0 0 2 4 5 - 7
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glycerol were purchased from Merck (Darmstadt, Germany).
described in [25]. A summary of the quality checks of the various particles is given in Fig. 1.
2.1. Isolation of ribosomal components
2.5. Spin-contrast variation
Protonated E. coli MRE600rif cells were grown in a 100 l batch fermentation according to Rheinberger et al. [24] at the MPI fu«r Molekulare Genetik, Berlin. The isolation of deuterated and protonated crude 70S ribosomes and ribosomal subunits followed [25], that of TP50 and total rRNA from 70S ribosomes [26].
The measurements made use of the variation of the contrast by isotopic substitution (H [ = proton, 1 H]3D [ = deuteron, 2 H] exchange) and di¡erent polarisation dependent scattering lengths. A detailed description of the experimental conditions and the techniques can be found in [30] or [20]. The di¡erent samples consist of a mixture of 2 H2 O, d8 -glycerol (¢nal concentration 54% v/w), an EHBAchromium(V) complex (¢nal concentration 0.85% v/w as paramagnetic impurities for DNP (dynamic nuclear polarisation), EHBA-chromium(V) (sodium bis(2-ethyl-2hydroxybutyrato)oxo-chromate(V) monohydrate {Na[Cr(C6 H10 O3 )2 ]WH2 O}) and the speci¢cally labelled ribosomal particles. The composition of the samples can be seen in Table 2. The components were mixed together, stirred for 20 s and poured into a 3U17U17 mm3 copper mould cooled by liquid nitrogen. A dark red glassy sample plate was obtained. The measurements for all samples were performed in a î with vV/V = similar way using a wavelength V of 8.5 A 10%. The sample detector distances were 0.7, 1.8 and 4.5 m respectively resulting in a q-range from 0.01 to 0.25 î 31 . All samples were measured in the unpolarised state A (PH = PD = 0, 1^2 days) and as proton spin targets (PH g0, PD = 0, 3^4 days) with di¡erent relative orientations of neutron and hydrogen spins: parallel or antiparallel to each other. The measuring time for a single measurement was 1000 s resulting in about 320 measurements per sample during the whole 5 day period of measurements. The two-dimensional data sets were summarised with respect to the sample detector distance and the sample polarisation. From the two-dimensional data sets one-dimensional intensity curves were calculated. All data sets were corrected for the in£uence of the wavelength distribution and collimation. After subtraction of the solvent scattering and incoherent scattering the three basic scattering functions MU(q)M2 , Re[U(q)V(q)] and MV(q)M2 were calculated [31]. These functions described the macroscopic scattering intensity of polarisation dependent scattering:
2.2. Puri¢cation of the proteins The isolation procedure for ribosomal proteins followed the methods described in [27]. The actual isolation strategy for the various proteins is overviewed in Table 1. 2.3. Preparation of selectively protonated and deuterated 50S subunits The reconstitution procedure for the large scale preparation of deuterated particles containing one protonated protein (D 50S[H Lx]) followed that described in [19]. In brief, H Lx and deuterated 70S rRNA were mixed in the ionic milieu of the Rec4 bu¡er (20 mM HEPES^KOH, pH 7.6 at 0³C, 4 mM Mg2 and 400 mM NH4 Cl) and incubated at 44³C for 1 h. Then the magnesium level was raised to 20 mM and a second incubation step of 4 h at 50³C followed. The puri¢cation of the reconstituted particles was identical to that described before [19]. The particles were ¢nally dissolved in a 2 H2 O bu¡er containing 100 mM imidazole/2 HCl (p2 H 7.6), 20 mM MgCl2 and 400 mM KCl and extensively dialysed against the same bu¡er to remove exchangeable protons from the sample. The imidazole, MgCl2 and KCl in the deuterated bu¡er were dissolved in 2 H2 O and lyophilised three times before they were used in the bu¡er to get rid of exchangeable protons or crystal water respectively. 2.4. Analysis of the rRNA, proteins, subunits and the 70S ribosomes All preparation steps were monitored with di¡erent analytical methods to prove the quality of the preparation. The particle homogeneity of the isolated subunits obtained from the cells in the ¢rst step was tested with an analytical sucrose gradient and the biological activity was checked with an in vitro protein synthesis assay (described in [25]). The protein content of the particles was examined by means of two-dimensional polyacrylamide gel electrophoresis [28]. The RNA was tested by polyacrylamide gel electrophoresis following the protocol of [29]. The biological activity of the samples was determined by means of a poly(U) dependent poly([14 C]Phe) synthesis, as
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I q MU qM2 2Pn ReU qV q MV qM2 while Pn is the neutron spin polarisation and q = (4Z/V) sin(a) with V = wavelength and a = scattering angle. From these basic scattering functions the in situ structure of the labelled proteins is determined in a model calî electron culation procedure. For the 50S subunit a 40 A microscopy structure according to Frank et al. [32] and information about the protein distribution according to Sto«¥er-Meilicke and Sto«¥er [4] were taken into account. Even though higher resolution ribosomal structures are
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Table 1 Preparation of speci¢cally protonated ribosomal 50S subunits Protein L1 L3 L4 L5 L14 L18 L24 L25
Salt wash
Mono-S pH 6.0
1
2
1 1
2 2
1
1 2
Mono-S pH 7.0
Mono-S pH 8.0
Mono-Q pH 9.5
1
Sephadex 75 2
1
2
1
For each protein one or two of the following methods were used: salt washes of the 50S subunit with 3.3 mM LiCl, cation-exchange chromatography at the pH indicated on a Pharmacia Mono-S column, anion-exchange chromatography on a Pharmacia Mono-Q column or gel ¢ltration on a Pharmacia Sephadex 75 column. The numbers (1 or 2) indicate which step had to be taken ¢rst. For details see [27].
î resolution structure [33], a available, for example a 15 A î structure did calculation of scattering curves with the 15 A not give a better representation of our measured ribosome data. The unknown position of the labelled protein is added to the theoretical scattering contribution of the ribosome and varied throughout the volume of the ribosome. Additional ¢t parameters are the contrast values due to the partly unknown degree of deuteration of the ribosome (95^98%). The position of the protein is found, when the theoretical scattering intensity Icalculated ¢ts best with the measured Imeasured by means of a least square ¢t minimising the R-value: ( )1=2 N 1X I qmeasured;i 3I qcalculated;i 2 minimum R N i1 ci for N = 40 intervals of q in 0.01 6 q 6 0.2 for all basic scattering functions. In the next step also the shape of the particle (spherical or elongated) is taken into account. To test the uniqueness of the found position for a minimal R-value a so-called minimum map is calculated (Figs. 2B,D, 3B,D and 4B). In this map the R-values for all distances d starting from the centre of mass of the riboî , vd = 5 A î ) and polar angles a some (0 9 d 9 150 A (0 9 a 9 Z, va = Z/19) and P (0 9 P 9 2 Z, vP = 2Z /19) are
represented. If the found position is unique only one smallest R-value should show up, if it is a deep global minimum only neighboured positions should have R-values slightly larger than the minimal one. 3. Results 3.1. Preparation of speci¢cally labelled subunits and ribosomes Spin-contrast variation requires selectively deuterated ribosomal samples. For this purpose 50S subunits were totally reconstituted starting from the following three ribosomal fractions: (I) a protonated puri¢ed protein (H Lx), (II) a deuterated total protein fraction of the 50S subunit lacking the protein Lx (D TP50-Lx) and (III) a deuterated rRNA fraction containing 23S and 5S rRNA. An e¡ective separation of the TP50 proteins is possible by a combination of di¡erent methods such as ion exchange chromatography, gel ¢ltration and salt washing. For each protein puri¢cation a two-step combination of these techniques exists [27] (see Section 2 and Table 1). The deuterated total rRNA fraction was isolated from D 70 ribosomes since it is known that the presence of 16S rRNA does not in£uence or impair the reconstitution of 50S subunits [34]. The use of total rRNA from 70S ribo-
Table 2 Chemical composition of the measured samples for the nuclear spin-contrast variation Sample
D
Concentration (A260 ) Biological activitya (%) Incorporation of the protein (%) Percentage of 2 H label Ribosomes (mg) 2 H2 O (mg) H2 O (mg) D-Glycerol (mg) H-Glycerol (mg) Cr(V) (mg) Imidazole, MgCl2 , KCl (total) (mg)
469 76 100 15 11.34 617.98 1.24 828.49 10.91 12.98 17.97
a
50S[H L5]
D
50S[H L14]
531 61 100 5 12.74 611.57 1.23 822.76 10.84 13.34 17.83
D
50S[H L18]
462 68 100 0 11.35 603.76 1.21 850.60 11.20 12.84 18.26
The biological activity was determined with respect to native deuterated 50S subunits.
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50S[H L24]
447 43 100 0 11.10 631.19 1.26 829.77 10.93 12.90 18.45
D
50S[H L25]
571 68 75 0 13.68 593.07 1.19 827.50 10.90 11.65 17.81
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Fig. 1. Quality parameters of the particles prepared for this work. For details see text.
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activity measured with the reconstituted particles is unusually high [26]. The protein analysis was performed from the deuterated TP50-Lx that indicated the residual Lx content as given in the header of the protein parameter pro¢les (Fig. 1). The residual amount ranged from 0% (L18, L24 and L25) over 5% (L14) up to 15% (L5). Furthermore, after adding the protonated protein Lx to the deuterated TP50-Lx, another gel control was made in order to verify the incorporation of the protein Lx under observation. Finally, the protein content of the reconstituted and puri¢ed particle 50S[Lx] is shown in Fig. 1, left panel at the bottom. 3.2. In situ structure determination of ribosomal proteins
Fig. 1 (Continued).
somes for the reconstitution of 50S subunits has two advantages. (a) There is no need to isolate 50S subunits, and (b) the 23S rRNA is completely intact in contrast to isolated 50S subunits that can contain up to 30% degraded 23S rRNA under careful isolation conditions. Fig. 1A^E contains the parameter pro¢les of reconstituted D 50S particles containing one protonated protein, viz. H L5, H L14, H L18, H L24 and H L25. Controls of the rRNA of the reconstituted particles demonstrated that the 23S rRNA was intact to 60^80%, the activity in poly(Phe) synthesis after complementing with protonated 30S subunits was between 43% and 76% compared to native deuterated 50S subunits. The fact that we did not have a 100% homogeneity of active particles does not compromise a structural analysis. The particles moved as a single, slim peak in the zonal centrifuge and thus are well suited for a position analysis of the protonated protein. In fact, the
The chemical composition of the samples used for analysis by nuclear spin-contrast variation is compiled in Table 2. The scattering data were measured at the SANS1 (SANS = small angle neutron scattering) instrument at the GKSS research centre. Spectra were taken for the unpolarised target and with either parallel or antiparallel aligned proton spins relative to the spins of the incoming neutrons. The collected data were processed according to standard corrections and deconvoluted into the three basic scattering functions MU(q)M2 , Re[U(q)V(q)] and MV(q)M2 . Theoretical basic scattering functions derived from an electron microscopy structure of the 50S subunit [32] containing the protonated protein under observation at all possible positions were ¢tted to the experimental scattering functions. After the determination of the radius of gyration RG and the coordinates of the protein centre of mass, in a second step of calculation also a possible elongated shape of the protein was taken into account. Unless a protein was extremely elongated no statement about the orientation could be made. The ¢nal results from the ¢tting procedure are found in Table 3. 3.3. In situ structure determination of L5 in the 50S subunit Following the procedure described above we localised protein L5 in the rear part of the central protuberance of the 50S subunit. IEM proposed a position towards the intersubunit interface [4]. The results of the ¢tting procedure can be seen in Fig. 2A. The data analysis gave an RG î . The theoretical radius of gyration assuming a of 17 þ 1 A
Table 3 Position of proteins L5, L14, L18, L24 and L25 in the 50S subunit determined by spin-contrast variation î) RG (A î) Distance (A î) Dimensions (A î ) centre of mass Coordinates (A î ) for ellipsoid Coordinates (A
L5
L14
17 þ 1 92 þ 7 22 (4)U22 (4)U51 (9) 37 (6)/90 (7)/312 (8) 316/98/328 6/84/316
15 þ 3 15 þ 1 12 þ 3 95 þ 4 100 þ 3 33 þ 6 25 (5)U25 (5)U38 (6) 80 (6)/40 (5)/33 (9) 328 (4)/95 (3)/37 (15) 35 (7)/315 (4)/9 (11) 79/43/33 85/33/28
L18
L24
L25 13 þ 1 93 þ 9 20 (5)U20 (5)U35 (6) 16 (8)/91 (8)/0 (7) 10/91/32 8/75/33
The coordinates of the proteins are calculated with respect to the ribosome structure from Frank et al. [32]. All distances and coordinates (X/Y/Z) refer to the centre of mass of the ribosome structure. Numbers in parentheses are standard deviations.
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Fig. 2. Results of the ¢tting procedure of proteins L5 (A,B) and L14 (C,D). The measured and the modelled (calculated) basic scattering curves for the best ¢t are shown for protein L5 in A and for protein L14 in C. In B (protein L5) and D (protein L14) the respective minimum maps are calculated. The R-values are presented for three di¡erent distances around the minimum R-value which gives the position of the labelled protein. To illustrate the uniqueness of the position found for each protein, R-values up to 10% higher than the best solution are taken into account. The minimum R-values for proteins L5 and L14 were 3.507 and 2.400, respectively.
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Fig. 3. Results of the ¢tting procedure of proteins L18 (A,B) and L24 (C,D). In A the best ¢t for the measured and the modelled (calculated) basic scattering curves are shown for protein L18 while for protein L24 they can be found in C. In B (protein L18) and D (protein L24) the respective minimum maps are calculated. To illustrate the uniqueness of the position found for each protein, R-values up to 5% (protein L18) and 10% (protein L24) higher than the best solution are taken into account. The minimum R-values for proteins L18 and L24 were 3.310 and 1.379, respectively.
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Fig. 4. Results of the ¢tting procedure of protein L25 (A,B) and the map of the 14 localised proteins in the 50S subunit (C). The measured and the modelled (calculated) basic scattering curves for the best ¢t of the data of protein L25 in the 50S subunit are shown in A. In B the respective minimum map is calculated. The R-values are presented for three di¡erent distances around the minimum R-value of 3.465, which gives the position of the labelled protein. To illustrate the uniqueness of the found position, R-values up to 5% higher than the best solution are taken into account. It can be seen that, in contrary to the other minimum maps, the position of L25 is not unequivocal. In C the 14 protein positions so far localised in the 50S subunit are presented. The red labelled proteins were localised by classical contrast variation [16] and the black labelled ones are localised by spin-contrast variation (L1 [17], L2 [19], L3, L4 [18]). The ribosome structure is by courtesy of J. Frank. Figures were generated with the programme LOCALITE Molecule Navigator.
î . It follows that L5 is spherical shape of L5 would be 14 A slightly elongated, and further data analysis revealed an î, ellipsoidal shape with the three main axes of 22 þ 4 A î î 22 þ 4 A and 51 þ 9 A. This elongated shape was also
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seen in the recently published high resolution structure of the 50S subunit [12]. The radius of gyration from this î which is in L5 structure can be calculated to be 17 A excellent agreement with our measurements.
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Fig. 5. Visualisation of additional positions (centre of mass) according to the minimum maps displayed in Figs. 2^4. For simplicity similar positions are represented only by one small orange sphere. Standard deviations are not represented here. (Left) Crown view; (right) ribosomal subunit turned by 90³, view towards L1. (A) Positions of L5, up to 10% higher R-value. (B) Positions of L14, up to 10% higher R-value. (C) Positions of L18, up to 5% higher R-value. (D) Positions of L24, up to 10% higher R-value. (E) Positions of L25, up to 5% higher R-value. Red sphere, equally good R-value compared to the medium-sized light green sphere (only for L25). Large spheres or ellipsoids in yellow, in situ protein structures; medium light green spheres, best R-value of the minimum map. In B this sphere is located inside the larger yellow in situ structure. If the position of the in situ structure is a global minimum the yellow coloured sphere or ellipsoid should be at the same position as the light green sphere. Small orange spheres, other possible positions if a higher R-value is taken into account.
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The position was found between L25 and L18 (see Fig. 4C). The distance of the centre of mass of the protein with respect to the centre of mass of the 50S subunit was deî . The determination of the angles a termined to be 92 þ 7 A and P was possible with a precision of 5 and 4%, respectively (a = 1.70 þ 0.08, P = 1.65 þ 0.07 [rad]). As can be seen from the minimum map in Fig. 2B the position is unequivocal up to 5% deviation of the R-values. The visualisation of additional positions for R-values higher than 5% can be found in Fig. 5A. 3.4. In situ structure determination of L14 in the 50S subunit The measured and modelled basic scattering curves are displayed in Fig. 2C. The position of the protein does not coincide with the position obtained from immunoelectron microscopy [4] but is in quite good agreement with a ¢t into the crystallographic map of the 50S subunit [35]: instead of being localised in the centre of the ribosome, L14 is found near the L7/L12 stalk. The distance from the centre of mass of the subunit was determined to be î , the angles were a = 1.20 þ 0.20, P = 0.50 þ 0.10 95 þ 4 A [rad]. For protein L14 additional information is available from crystallography. The structure of the isolated L14 is known [36] and a calculation using the programme î. CRYSOL by D. Svergun gave a theoretical RG of 14 A The calculation of RG from the scattering curves showed that the radius of gyration corresponds markedly well with î . The spin-contrast variation this prediction : RG = 15 þ 3 A measurements suggested that L14 is not spherical but that it can be described rather by an ellipsoid with main axes of î , 25 þ 5 A î and 38 þ 6 A î . This value corresponds 25 þ 5 A well with the dimension of the protein obtained from the crystal structure if the non-structured loops of the protein î U22 are omitted (estimated to have the dimensions 22 A î î AU38 A). The uniqueness of the position can be seen in Fig. 2D: no signi¢cantly di¡erent positions are found for this protein within up to 5% higher R-values. Additional positions showing up for R-values up to 10% can be seen in Fig. 5B. 3.5. In situ structure determination of L18 in the 50S ribosome Protein L18 with its mass of 12.770 kDa is a small protein attached to the 5S rRNA. Its theoretical radius î . The measured radius was of gyration would be 12 A î . Even though this is slightly larger: RG equals 15 þ 1 A an indication that the protein is not spherical, there was no signi¢cant improvement in the ¢tting procedure when an elongated L18 model was used. A possible explanation could be a shape of L18 neither spherical nor elongated. It is interesting to note that the radius of gyration determined from the L18 structure in Ban et al. [12] can be
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î , even larger than that decalculated to be approx. 18 A termined here. L18 is displayed as a sphere in Fig. 4C despite knowing that this is not the correct description of the shape. The distance between L18 and the centre of mass of the 50S î . The angles of the determined radius subunit is 100 þ 3 A vector are a = 1.72 þ 0.17 and P = 1.78 þ 0.07 [rad], which localises L18 in the central protuberance. The basic scattering function U(q)2 is a representative of the scattering of the ribosomal matrix, and thus it should be almost the same for every sample. We note that the shape of U(q)2 from this particle is a bit di¡erent from the other measurements (Fig. 3A). The basic scattering curve V(q)2 shows that the shape of the labelled protein is not su¤ciently described by a sphere. The minimum map shows only one deep minimum (Rvalue 3.310) up to 5% higher R-values (Figs. 3B and 5C). For higher R-values multiple positions show up. 3.6. In situ structure determination of L24 in the 50S ribosome The determination of the position of L24, which was so far not localised by other methods in E. coli ribosomes, turned out to give a markedly stable result even though the protein belongs to the smaller proteins investigated (molecular mass 13.541 kDa). The radius of gyration î which agrees well with the small was found to be 12 þ 3 A mass of L24. From crystallographic data [12] the RG (L24) î. was calculated to be approx. 15 A The protein is localised in a central position, the distance to the centre of mass of the 50S subunit is only î . The determination of the angles gave 33 þ 6 A a = 1.40 þ 0.34 and P = 5.87 þ 0.17 [rad]. As can be seen in Fig. 3C, the basic scattering curves are very well modelled. The minimum map indicates an unequivocal localisation of L24 (Figs. 3D and 5D; minimum R-value 1.379). 3.7. In situ structure determination of L25 in the 50S ribosome Protein L25 is so far the smallest protein whose in situ structure is determined by spin-contrast variation. Its molecular mass of 10.693 kDa corresponds to a theoretical î . From NMR and crysradius of gyration of approx. 11 A tal structures of L25 [37,38] it can be seen that L25 is slightly elongated while it interacts with the 5S rRNA. The dimensions of the protein were estimated from the î U25 A î U45 A î . With the procrystal structure to be 25 A î was calgramme CRYSOL a radius of gyration of 13 A culated. Our spin-contrast measurements of the in situ î. radius of gyration of L25 gave the value RG = 13 þ 1 A Model calculation with an elongated shape of L25 led to î U(20 þ 5) slightly smaller dimensions of (20 þ 5) A î î AU(35 þ 6) A. The protein was localised at a distance of
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î in the central protuberance of the 50S subunit. 93 þ 9 A The angles a and P were determined to be a = 1.55 þ 0.09 and P = 1.41 þ 0.07 [rad]. Fig. 4A shows that for almost the whole q-range of the basic scattering functions a good agreement between measured and modelled data was found. More surprising was the ¢nding that a second equally possible position of L25 shows up as displayed in the minimum map (Figs. 4B and 5E). This position is below the L7/l12 stalk and hardly touches the ribosome. Furthermore the minimum of the R-value is not very pronounced (smallest R-value 3.465). Other possible positions, for example near L2, were detected. A possible reason for this ambiguity is that with this small protein the limits of the technique are reached because even with the increase of the contrast by polarisation dependent scattering the scattering contribution of labels smaller than 0.5% of the total mass is almost not distinguishable from the matrix and solvent scattering. 4. Discussion Relative positions of proteins can be determined by cross-linking and IEM methods. These methods resulted in the ¢rst map of the 50S proteins [39]. An alternative to the structure determination by biochemical means is neutron scattering which was used to develop a protein map of the 30S subunit [11]. Both techniques together with high resolution X-ray crystallography of the protein and 30S subunit crystals produced a well resolved view of the rRNA and protein distribution in the 30S subunit [13]. The recent crystallographic map of the large subunit of î localised 27 proteins H. marismortui ribosomes at 2.4 A
17
and revealed their in situ structure [12]. Here we present a 50S map with the positions of 14 out of the 33 proteins of the E. coli large subunit of ribosomes (Fig. 4C, Table 5). The map was determined independently by spin-contrast variation and contained seven more proteins than our previous map [16]. A striking feature is that most of the proteins investigated by spin-contrast variation are elongated, only L1 and L24 are largely spherical. In the following paragraphs we will discuss our ¢ndings in comparison with the results of IEM and crystallography. 4.1. Resolution of the methods The IEM positions are determined with a precision of î , in some cases even better. It is obvious that approx. 40 A in the crystallographic map we see the position with a 2.4 î resolution, the best precision reached at the moment. A However, protein L1 and the L7/L12 stalk were not seen in the crystal map but could be localised in the electron microscope. For the neutron scattering analysis we used a î resolution. cryoelectron microscopy structure with 40 A The standard deviations were calculated taking into account systematic and statistical errors. 4.2. Species di¡erences of the ribosomes For IEM and spin-contrast variation ribosomes from E. coli were investigated, and the crystal structure was obtained from the archea H. marismortui ribosomes. These ribosomes di¡er in their composition: the archeal ribosome does not contain proteins L9, L17, L20 and L25. Furthermore the sequence identities between the ortho-
Table 4 Comparison of the protein positions and radii of gyration determined by spin-contrast variation and crystallography [12] î ) SANS î ) cryst. [12] î ) towards IEM î ) towards cryst. Protein RG (A RG (A Distances (A Distances (A L1 L2 L3 L4 L5 L13 L14 L17 L18 L20 L22 L23 L24 L25
24 þ 1 22 þ 1 23 þ 2b 17 þ 2b 17 þ 1 not measured 15 þ 3 24 þ 2 15 þ 1 18 þ 2 21 þ 2 23 þ 2 12 þ 3 13 þ 1
18.8a 22.2 23.6 26.4d 17.0 14.9 13.8c no data available 18.4 no data available 17.1 11.5 15.5 12.8a
15 45 30 15 53 53 94 38 59 60 51 10 not detected 38
not 51 75 28 36 38 56 not 10 not 53 49 93 not
detected
detected detected
detected
The distance between the positions was determined as follows : the neutron scattering coordinate system was rotated by 45³ around the z-axis, followed by a 90³ rotation around the y-axis. Then the coordinates of the centre of mass of the proteins were translated into the coordinate system of the crystallographic structure. The distance was then calculated from the centre of mass of the crystallographic protein positions with respect to the translated neutron scattering positions. The coordinate systems of immunoelectron microscopy and neutron scattering were identical. a Structure of L1: PDB entry 1AD2, structure of L25 : PDB entry 1DFU. b î and RG (L4) = 19 þ 2 A î (private communications by R.P. May). RG (L3) = 35 þ 2 A c î using PDB entry 1WHI. Corresponding to RG = 13.9 A d î. Structure of isolated L4: PDB entry 1DMG gives RG (L4) = 17 A
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Table 5 Comparison of the coordinates of protein positions determined by crystallography (H. marismortui) [12] and spin-contrast variation (E. coli) Protein
H. marismortui X
L1 L2 L3 L4 L5 L13 L14 L17 L18 L20 L22 L23 L24 L25
E. coli Y
Z
120.4 43.8 22.9 91.6 22.8 86.6
134.6 158.4 111.9 24.6 123.0 149.4
61.4 139.9 37.6 108.1 134.2 143.3
63.4
17.4
83.0
29.4 68.4 20.1
161.5 181.7 152.9
87.1 38.7 29.8
X
Y
Z
109.4 110.4 67.4 42.4 60.4 50.4 82.4 45.4 62.4 0.4 59.4 29.4 67.4 79.4
61.5 109.5 166.5 101.5 30.5 99.5 103.5 171.5 19.5 101.5 121.5 156.5 140.5 42.5
37.1 17.9 68.9 19.9 90.9 123.9 174.9 87.9 72.9 71.9 70.9 22.9 108.9 95.9
The coordinates refer to the center of mass of the proteins with respect to the center of mass and the coordinate system of the PDB structure 1FFK [12].
logue proteins of E. coli and H. marismortui are in the range of 24^37% (L1 (29%), L2 (36%), L3 (25%), L4 (24%), L5 (34%), L13 (26%), L14 (37%), L18 (25%), L23 (35%) (BLAST search)). Other proteins correspond to eucaryotic ribosomal proteins but have no counterpart in procaryotic ribosomes. All these di¡erences could shift the relative positions of those proteins of the H. marismortui ribosome that could be compared with E. coli proteins, partially explaining the di¡erences of the corresponding positions described below. Proteins L5, L18 and L25 are the so-called 5S rRNA binding proteins that are located in the central protuberance of the 50S subunit together with the 5S rRNA. IEM investigations had positioned these proteins near to the intersubunit interface, whereas our analysis places these proteins more towards the back of the central protuber-
ance (CP). This coincides well with the distribution of rRNA as presented by Mu«ller et al. [40], and is also in agreement with the relative positioning of the CP proteins that were also found mainly in the cytoplasmic region of the central protuberance as proposed by Svergun and Nierhaus [6]. A comparison of the positions of the three proteins proposed by IEM with those determined by neutron scattering shows that the deviation between the corî for responding positions of each method is approx. 53 A î protein L5, approx. 59 A for protein L18 and approx. 38 î in the case of L25 (Table 4). The corresponding values A for the distances between the neutron scattering and the corresponding crystal positions [12] agree better : L5 was î away from the crystallographic posifound approx. 36 A î (see also Tables 4 and 5 and Fig. 6). tion, L18 about 10 A All three CP proteins are next to each other, probably
Fig. 6. Comparison of the protein positions obtained by neutron scattering and crystallography [12]. The spheres symbolise the centre of mass of the protein positions determined by spin-contrast variation. The crystallographic structures of the respective proteins are highlighted in the same colour. Front view (A) is left (interface side towards the 30S subunit), rear view (C) right (cytoplasmic side with the exit of the polypeptide channel). The picture in the middle (B) shows the subunit from the L1 side (90³ rotation).
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close in contact. L25 was found on the `left' side of the strand identi¢ed as part of the 5S rRNA in the central protuberance which is contrary to the results published previously [40,41]. L5 was found on the back side of the CP next to L18. This position agrees well with the correî structure [12]. The quality sponding position in the 2.4 A of the calculated solutions for the positions of the three CP proteins is rather di¡erent. In the case of L5 a deep minimum was obtained (Fig. 2B). For R-values of up to 10% higher than the best ¢t further possible positions show up in a similar distance to the centre of mass of the ribosome, mainly near the L7/L12 stalk or in a symmetric location (Fig. 5A). In the case of proteins L18 and L25 the minimum is not as deep as for protein L5 (Figs. 3B and 4B). The distance to the centre of mass was determined precisely, but the determination of the angles a and P turned out to be more complicated (L18, Fig. 5C). With L25 the limits of the SANS procedure applied here seems to be reached, since a second position on the L7/L12 stalk side of the ribosome was calculated with an equally good R-value. Other positions can be found in symmetric locations (Fig. 5E). Protein L14 was found near the L7/L12 stalk which does not correspond with the position found by IEM [4] î ). In the 2.4 A î structure L14 is 56 A î (distance approx. 94 A apart from our position [12] (Fig. 6). Again the determination of the distance to the centre of mass was unique. The angles a and P gave several other positions in the CP, near L1 and on the back side of the ribosome (Fig. 5B). Since all these positions have R-values about 10% higher than the minimal value, they are less likely. L24 is located near the intersubunit interface, but is not exposed at the interface. The representation of additional possible positions for higher R-values is shown in Fig. 5D. A symmetric location near the centre of mass of the ribosome was also detected. The distance between L24 from î H. marismortui and our position was calculated to be 93 A (Fig. 6). An overall comparison of the protein positions obtained from neutron scattering with recently published high resolution structures shows that in some respects the similarities are markedly consistent, while for other aspects striking discrepancies are seen. For example, the radii of gyration as determined by neutron scattering are very similar to the corresponding values derived from the crystal structure or from isolated proteins (exception : proteins L1, L4 and L23, see Table 4). The protein positions of L4 and L18 coincide within the limits of the experimental and statistical error with the crystallographic structure, and there is a quite good agreement for the positions of L2, L5, L13, L14, L22 and L23 (Table 4). The relatively large deviations from the crystal positions concerning L2 and L14 might be partially due to the fact that L1, L9 (important for L2) and the L7/L12 stalk (important for L14) are absent in the crystal structure.
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For L17 and L20 no comparison with the crystal map can be made since these proteins do not exist in that structure. Our data compare very well with the corresponding data from the older IEM map except for proteins L14, L18 and L20 (Table 4). Proteins L3 and L24 are found at rather di¡erent locations inside the ribosome. Note that with classical contrast variation methods L3 was also found on the right hand side of the subunit (crown view [16]). It has to be seen whether or not the di¡erences are `real', i.e. at least partially due to domain di¡erences of the respective organisms E. coli and H. marismortui, respectively, or, alternatively, are caused by drawbacks of the low resolution method of neutron scattering.
References [1] R. Brimacombe, J. Atmadja, W. Stiege, D. Schuler, J. Mol. Biol. 199 (1988) 115^136. [2] A. Malhotra, S.C. Harvey, J. Mol. Biol. 240 (1994) 308^340. [3] S. Stern, B. Weiser, H.F. Noller, J. Mol. Biol. 204 (1988) 447^481. [4] M. Sto«¥er-Meilicke, G. Sto«¥er, in: W.E. Hill, A. Dahlberg, R.A. Garrett, P.B. Moore, D. Schlessinger, J.R. Warner (Eds.), The Ribosome. Structure, Function and Evolution, American Society for Microbiology, Washington, DC, 1990, pp. 123^133. [5] R.R. Crichton, D.M. Engelman, J. Haas, M.H.J. Koch, P.B. Koch, R. Parfait, H.B. Stuhrmann, Proc. Natl. Acad. Sci. USA 74 (1977) 5547^5550. [6] D.I. Svergun, K.H. Nierhaus, J. Biol. Chem. 275 (2000) 14432^14439. [7] D.I. Svergun, Biophys. J. 76 (1999) 2879^2886. [8] J. Frank, J. Zhu, P. Penczek, Y.H. Li, S. Srivastava, A. Verschoor, M. Radermacher, R. Grassucci, R.K. Lata, R.K. Agrawal, Nature 376 (1995) 441^444. [9] J. Frank, A. Verschoor, Y.H. Li, J. Zhu, R.K. Lata, M. Radermacher, P. Penczek, R. Grassucci, R.K. Agrawal, S. Srivastava, Biochem. Cell Biol. 73 (1995) 757^765. [10] H. Stark, F. Mueller, E.V. Orlova, M. Schatz, P. Dube, T. Erdemir, F. Zemlin, R. Brimacombe, M.v. Heel, Structure 3 (1995) 815^ 821. [11] M.S. Capel, D.M. Engelman, B.R. Freeborn, M. Kjeldgaard, J.A. Langer, V. Ramakrishnan, D.G. Schindler, D.K. Schneider, B.P. Schoenborn, I.-Y. Sillers, S. Yabuki, P.B. Moore, Science 238 (1987) 1403^1406. [12] N. Ban, P. Nissen, J. Hansen, P.B. Moore, T.A. Steitz, Science 289 (2000) 905^920. [13] W.M.J. Clemons, J.L. May, B.T. Wimberly, J.P. McCutcheon, M.S. Capel, V. Ramakrishnan, Nature 400 (1999) 833^840. [14] F. Schlu«nzen, A. Tocilj, R. Zarivach, J. Harms, M. Glu«hmann, D. Janell, A. Bashan, H. Bartels, I. Agmon, F. Franceschi, A. Yonath, Cell 102 (2000) 615^623. [15] C.R. Woese, O. Kandler, M.L. Wheelis, Proc. Natl. Acad. Sci. USA 87 (1990) 4576^4579. [16] R.P. May, V. Nowotny, P. Nowotny, H. Voss, K.H. Nierhaus, EMBO J. 11 (1992) 373^378. [17] R. Willumeit, N. Burkhardt, G. Diedrich, J. Zhao, K.H. Nierhaus, H.B. Stuhrmann, J. Mol. Struct. 383 (1996) 201^211. [18] J. Zhao, H.B. Stuhrmann, J. Phys. Coll. C8 3 (1993) 233^236. [19] R. Willumeit, S. Forthmann, J. Beckmann, G. Diedrich, R. Ratering, H.B. Stuhrmann, K.H. Nierhaus, J. Mol. Biol. 305 (2000) 167^177. [20] R. Willumeit, N. Burkhardt, J. Wadzack, K.H. Nierhaus, H.B. Stuhrmann, J. Appl. Crystallogr. 30 (1997) 1125^1131. [21] J. Wadzack, N. Burkhardt, R. Ju«nemann, G. Diedrich, K.H. Nier-
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[26]
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R. Willumeit et al. / Biochimica et Biophysica Acta 1520 (2001) 7^20 haus, J. Frank, P. Penczek, W. Meerwinck, M. Schmitt, R. Willumeit, H.B. Stuhrmann, J. Mol. Biol. 266 (1997) 343^356. K.H. Nierhaus, J. Wadzack, N. Burkhardt, R. Ju«nemann, W. Meerwinck, R. Willumeit, H.B. Stuhrmann, Proc. Natl. Acad. Sci. USA 95 (1998) 945^950. R. Ju«nemann, N. Burkhardt, J. Wadzack, M. Schmitt, R. Willumeit, H.B. Stuhrmann, K.H. Nierhaus, Biol. Chem. 379 (1998) 807^818. H.-J. Rheinberger, U. Geigenmu«ller, M. Wedde, K.H. Nierhaus, Methods Enzymol. 164 (1988) 658^670. U. Bommer, N. Burkhardt, R. Ju«nemann, C.M.T. Spahn, F.J. Triana-Alonso, K.H. Nierhaus, in: J. Graham, D. Rickwoods (Eds.), Subcellular Fractionation. A Practical Approach, IRL Press at Oxford University Press, Oxford, 1996, pp. 271^301. K. Nierhaus, in: G. Spedding (Ed.), Ribosomes and Protein Synthesis. A Practical Approach, IRL Press at Oxford University Press, Oxford, 1990, pp. 161^189. G. Diedrich, N. Burkhardt, K.H. Nierhaus, Protein Expr. Purif. 10 (1997) 42^50. D. Geyl, A. Bo«ck, K. Isono, Mol. Gen. Genet. 181 (1981) 309^ 312. H. Ceri, P.Y. Maeba, Biochim. Biophys. Acta 312 (1973) 337^348. J. Zhao, W. Meerwinck, T. Niinikoski, A. Rijllart, M. Schmitt, R. Willumeit, H.B. Stuhrmann, Nucl. Inst. Methods Phys. Res. A 356 (1995) 133^137.
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[31] A. Abragam, M. Goldman, Nuclear Magnetism, Order and Disorder, Clarendon Press, Oxford, 1992. [32] J. Frank, P. Penczek, R. Grassucci, S. Srivastava, J. Cell Biol. 115 (1991) 597^605. [33] A. Malhotra, P. Penczek, R.K. Agrawal, I.S. Gabashvili, R.A. Grassucci, R. Ju«nemann, N. Burkhardt, K.H. Nierhaus, J. Frank, J. Mol. Biol. 280 (1998) 103^116. [34] R. Lietzke, K.H. Nierhaus, Methods Enzymol. 164 (1988) 278^ 283. [35] N. Ban, P. Nissen, J. Hansen, M. Capel, P.B. Moore, T.A. Steitz, Nature 400 (1999) 841^847. [36] C. Davies, S.W. White, V. Ramakrishnan, Structure 4 (1996) 55^ 66. [37] M. Stoldt, J. Wohnert, O. Ohlenschlager, M. Gorlach, L.R. Brown, EMBO J. 18 (1999) 6508^6521. [38] M. Lu, T.A. Steitz, Proc. Natl. Acad. Sci. USA 97 (2000) 2023^2028. [39] J. Walleczek, R. Albrecht-Ehrlich, G. Sto«¥er, M. Sto«¥er-Meilicke, J. Biol. Chem. 265 (1990) 1338^1344. [40] F. Mu«ller, I. Sommer, P. Baranov, R. Matadeen, M. Stoldt, J. Wo«hnert, M. Go«rlach, M. van Heel, R. Brimacombe, J. Mol. Biol. 298 (2000) 35^59. [41] R. Matadeen, A. Patwardhan, B. Gowen, E.V. Orlova, T. Pape, M. Cu¡, F. Mueller, R. Brimacombe, M. van Heel, Structure 7 (1999) 1575^1583.
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Mapping proteins of the 50S subunit from Escherichia coli ribosomes Regine Willumeit 1;a; *, Gundo Diedrich 1;2;b , Stefan Forthmann a , Jo«rn Beckmann a , Roland P. May c , Heinrich B. Stuhrmann 3;a , Knud H. Nierhaus b a
GKSS Forschungszentrum Geesthacht GmbH, Institut fu«r Werksto¡forschung, WFS, Max-Planck-StraMe, 21502 Geesthacht, Germany b Max-Planck-Institut fu«r Molekulare Genetik, AG Ribosomen, IhnestraMe 73, 14195 Berlin, Germany c Institute Laue Langevin, F-38042 Grenoble Cedex 9, France Received 8 November 2000; received in revised form 20 March 2001 ; accepted 16 May 2001
Abstract Mapping of protein positions in the ribosomal subunits was first achieved for the 30S subunit by means of neutron scattering about 15 years ago. Since the 50S subunit is almost twice as large as the 30S subunit and consists of more proteins, it was difficult to apply classical contrast variation techniques for the localisation of the proteins. Polarisation dependent neutron scattering (spin-contrast variation) helped to overcome this restriction. Here a map of 14 proteins within the 50S subunit from Escherichia coli ribosomes is presented including the proteins L17 and L20 that are not present in archeal ribosomes. The results are compared with the recent crystallographic map of the 50S subunit from the archea Haloarcula marismortui. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : Neutron scattering; Ribosomal protein; Spin-contrast variation
1. Introduction The ribosome as the site of protein synthesis in every living cell is one of the most intensively investigated macromolecular structures. A wide set of di¡erent biological and physical methods are applied to determine structure and function of the ribosome. Beside cross-linking and footprinting, methods which reveal the interaction of constituents of the ribosome [1^4], scattering techniques such as neutron and X-ray scattering, immunoelectron microscopy (IEM) and cryoelectron microscopy (cryo-EM) have added to the picture of what the ribosome looks like [4^ 11]. The remarkable achievements by X-ray crystallography now give the possibility to become familiar with the
Abbreviations : IEM, immunoelectron microscopy; CP, central protuberance; TP50, total proteins of the 50S ribosomal subunit; SANS, small angle neutron scattering; EHBA, sodium bis(2-ethyl-2-hydroxybutyrato)oxo-chromate(V) monohydrate {Na[Cr(C6 H10 O3 )2 ]WH2 O}; DNP, dynamic nuclear polarisation * Corresponding author. Fax: +49-4152-87-13-56. E-mail address : [email protected] (Regine Willumeit). 1 These authors contributed equally to the paper. 2 Present address: Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. 3 Present address: Institut de Biologie Structurale, IBS-LCM, 41 Avenue des Martyrs, F-38027 Grenoble Cedex 1, France.
structure of the subunits on an atomic level [12^14]. The 50S data were obtained from the ribosomes of Haloarcula marismortui which with respect to the three fundamental categories of the living systems, viz. the bacteria, archea and eucarya [15], belongs to the archea. In this work neutron scattering results for the localisation of ¢ve proteins of the large subunit of the bacterium Escherichia coli are presented. They are combined with previously published results [16^19] giving the positions of 14 proteins in the 50S subunit. These positions are reviewed taking into account the now available high resolution structures. The method used was spin-contrast variation, which has been successfully applied to position the protein L2 within 50S subunits and 70S ribosomes, and that of tRNAs and the mRNA within elongating ribosomes [19^23]. 2. Materials and methods Deuterated cells of the E. coli strain MRE600rif were purchased from CDN (Tallinn, Estonia), L-[U-14 C]Phe from Amersham-Buchler (Braunschweig, Germany), chemicals for gel preparation from Bio-Rad (Richmond, CA, USA), poly(U) mRNA from Boehringer (Mannheim, Germany), HEPES from Calbiochem (Frankfurt, Germany) and all other chemicals including 2 H2 O and d8 -
0167-4781 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 1 ) 0 0 2 4 5 - 7
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glycerol were purchased from Merck (Darmstadt, Germany).
described in [25]. A summary of the quality checks of the various particles is given in Fig. 1.
2.1. Isolation of ribosomal components
2.5. Spin-contrast variation
Protonated E. coli MRE600rif cells were grown in a 100 l batch fermentation according to Rheinberger et al. [24] at the MPI fu«r Molekulare Genetik, Berlin. The isolation of deuterated and protonated crude 70S ribosomes and ribosomal subunits followed [25], that of TP50 and total rRNA from 70S ribosomes [26].
The measurements made use of the variation of the contrast by isotopic substitution (H [ = proton, 1 H]3D [ = deuteron, 2 H] exchange) and di¡erent polarisation dependent scattering lengths. A detailed description of the experimental conditions and the techniques can be found in [30] or [20]. The di¡erent samples consist of a mixture of 2 H2 O, d8 -glycerol (¢nal concentration 54% v/w), an EHBAchromium(V) complex (¢nal concentration 0.85% v/w as paramagnetic impurities for DNP (dynamic nuclear polarisation), EHBA-chromium(V) (sodium bis(2-ethyl-2hydroxybutyrato)oxo-chromate(V) monohydrate {Na[Cr(C6 H10 O3 )2 ]WH2 O}) and the speci¢cally labelled ribosomal particles. The composition of the samples can be seen in Table 2. The components were mixed together, stirred for 20 s and poured into a 3U17U17 mm3 copper mould cooled by liquid nitrogen. A dark red glassy sample plate was obtained. The measurements for all samples were performed in a î with vV/V = similar way using a wavelength V of 8.5 A 10%. The sample detector distances were 0.7, 1.8 and 4.5 m respectively resulting in a q-range from 0.01 to 0.25 î 31 . All samples were measured in the unpolarised state A (PH = PD = 0, 1^2 days) and as proton spin targets (PH g0, PD = 0, 3^4 days) with di¡erent relative orientations of neutron and hydrogen spins: parallel or antiparallel to each other. The measuring time for a single measurement was 1000 s resulting in about 320 measurements per sample during the whole 5 day period of measurements. The two-dimensional data sets were summarised with respect to the sample detector distance and the sample polarisation. From the two-dimensional data sets one-dimensional intensity curves were calculated. All data sets were corrected for the in£uence of the wavelength distribution and collimation. After subtraction of the solvent scattering and incoherent scattering the three basic scattering functions MU(q)M2 , Re[U(q)V(q)] and MV(q)M2 were calculated [31]. These functions described the macroscopic scattering intensity of polarisation dependent scattering:
2.2. Puri¢cation of the proteins The isolation procedure for ribosomal proteins followed the methods described in [27]. The actual isolation strategy for the various proteins is overviewed in Table 1. 2.3. Preparation of selectively protonated and deuterated 50S subunits The reconstitution procedure for the large scale preparation of deuterated particles containing one protonated protein (D 50S[H Lx]) followed that described in [19]. In brief, H Lx and deuterated 70S rRNA were mixed in the ionic milieu of the Rec4 bu¡er (20 mM HEPES^KOH, pH 7.6 at 0³C, 4 mM Mg2 and 400 mM NH4 Cl) and incubated at 44³C for 1 h. Then the magnesium level was raised to 20 mM and a second incubation step of 4 h at 50³C followed. The puri¢cation of the reconstituted particles was identical to that described before [19]. The particles were ¢nally dissolved in a 2 H2 O bu¡er containing 100 mM imidazole/2 HCl (p2 H 7.6), 20 mM MgCl2 and 400 mM KCl and extensively dialysed against the same bu¡er to remove exchangeable protons from the sample. The imidazole, MgCl2 and KCl in the deuterated bu¡er were dissolved in 2 H2 O and lyophilised three times before they were used in the bu¡er to get rid of exchangeable protons or crystal water respectively. 2.4. Analysis of the rRNA, proteins, subunits and the 70S ribosomes All preparation steps were monitored with di¡erent analytical methods to prove the quality of the preparation. The particle homogeneity of the isolated subunits obtained from the cells in the ¢rst step was tested with an analytical sucrose gradient and the biological activity was checked with an in vitro protein synthesis assay (described in [25]). The protein content of the particles was examined by means of two-dimensional polyacrylamide gel electrophoresis [28]. The RNA was tested by polyacrylamide gel electrophoresis following the protocol of [29]. The biological activity of the samples was determined by means of a poly(U) dependent poly([14 C]Phe) synthesis, as
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I q MU qM2 2Pn ReU qV q MV qM2 while Pn is the neutron spin polarisation and q = (4Z/V) sin(a) with V = wavelength and a = scattering angle. From these basic scattering functions the in situ structure of the labelled proteins is determined in a model calî electron culation procedure. For the 50S subunit a 40 A microscopy structure according to Frank et al. [32] and information about the protein distribution according to Sto«¥er-Meilicke and Sto«¥er [4] were taken into account. Even though higher resolution ribosomal structures are
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Table 1 Preparation of speci¢cally protonated ribosomal 50S subunits Protein L1 L3 L4 L5 L14 L18 L24 L25
Salt wash
Mono-S pH 6.0
1
2
1 1
2 2
1
1 2
Mono-S pH 7.0
Mono-S pH 8.0
Mono-Q pH 9.5
1
Sephadex 75 2
1
2
1
For each protein one or two of the following methods were used: salt washes of the 50S subunit with 3.3 mM LiCl, cation-exchange chromatography at the pH indicated on a Pharmacia Mono-S column, anion-exchange chromatography on a Pharmacia Mono-Q column or gel ¢ltration on a Pharmacia Sephadex 75 column. The numbers (1 or 2) indicate which step had to be taken ¢rst. For details see [27].
î resolution structure [33], a available, for example a 15 A î structure did calculation of scattering curves with the 15 A not give a better representation of our measured ribosome data. The unknown position of the labelled protein is added to the theoretical scattering contribution of the ribosome and varied throughout the volume of the ribosome. Additional ¢t parameters are the contrast values due to the partly unknown degree of deuteration of the ribosome (95^98%). The position of the protein is found, when the theoretical scattering intensity Icalculated ¢ts best with the measured Imeasured by means of a least square ¢t minimising the R-value: ( )1=2 N 1X I qmeasured;i 3I qcalculated;i 2 minimum R N i1 ci for N = 40 intervals of q in 0.01 6 q 6 0.2 for all basic scattering functions. In the next step also the shape of the particle (spherical or elongated) is taken into account. To test the uniqueness of the found position for a minimal R-value a so-called minimum map is calculated (Figs. 2B,D, 3B,D and 4B). In this map the R-values for all distances d starting from the centre of mass of the riboî , vd = 5 A î ) and polar angles a some (0 9 d 9 150 A (0 9 a 9 Z, va = Z/19) and P (0 9 P 9 2 Z, vP = 2Z /19) are
represented. If the found position is unique only one smallest R-value should show up, if it is a deep global minimum only neighboured positions should have R-values slightly larger than the minimal one. 3. Results 3.1. Preparation of speci¢cally labelled subunits and ribosomes Spin-contrast variation requires selectively deuterated ribosomal samples. For this purpose 50S subunits were totally reconstituted starting from the following three ribosomal fractions: (I) a protonated puri¢ed protein (H Lx), (II) a deuterated total protein fraction of the 50S subunit lacking the protein Lx (D TP50-Lx) and (III) a deuterated rRNA fraction containing 23S and 5S rRNA. An e¡ective separation of the TP50 proteins is possible by a combination of di¡erent methods such as ion exchange chromatography, gel ¢ltration and salt washing. For each protein puri¢cation a two-step combination of these techniques exists [27] (see Section 2 and Table 1). The deuterated total rRNA fraction was isolated from D 70 ribosomes since it is known that the presence of 16S rRNA does not in£uence or impair the reconstitution of 50S subunits [34]. The use of total rRNA from 70S ribo-
Table 2 Chemical composition of the measured samples for the nuclear spin-contrast variation Sample
D
Concentration (A260 ) Biological activitya (%) Incorporation of the protein (%) Percentage of 2 H label Ribosomes (mg) 2 H2 O (mg) H2 O (mg) D-Glycerol (mg) H-Glycerol (mg) Cr(V) (mg) Imidazole, MgCl2 , KCl (total) (mg)
469 76 100 15 11.34 617.98 1.24 828.49 10.91 12.98 17.97
a
50S[H L5]
D
50S[H L14]
531 61 100 5 12.74 611.57 1.23 822.76 10.84 13.34 17.83
D
50S[H L18]
462 68 100 0 11.35 603.76 1.21 850.60 11.20 12.84 18.26
The biological activity was determined with respect to native deuterated 50S subunits.
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D
50S[H L24]
447 43 100 0 11.10 631.19 1.26 829.77 10.93 12.90 18.45
D
50S[H L25]
571 68 75 0 13.68 593.07 1.19 827.50 10.90 11.65 17.81
10
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Fig. 1. Quality parameters of the particles prepared for this work. For details see text.
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11
activity measured with the reconstituted particles is unusually high [26]. The protein analysis was performed from the deuterated TP50-Lx that indicated the residual Lx content as given in the header of the protein parameter pro¢les (Fig. 1). The residual amount ranged from 0% (L18, L24 and L25) over 5% (L14) up to 15% (L5). Furthermore, after adding the protonated protein Lx to the deuterated TP50-Lx, another gel control was made in order to verify the incorporation of the protein Lx under observation. Finally, the protein content of the reconstituted and puri¢ed particle 50S[Lx] is shown in Fig. 1, left panel at the bottom. 3.2. In situ structure determination of ribosomal proteins
Fig. 1 (Continued).
somes for the reconstitution of 50S subunits has two advantages. (a) There is no need to isolate 50S subunits, and (b) the 23S rRNA is completely intact in contrast to isolated 50S subunits that can contain up to 30% degraded 23S rRNA under careful isolation conditions. Fig. 1A^E contains the parameter pro¢les of reconstituted D 50S particles containing one protonated protein, viz. H L5, H L14, H L18, H L24 and H L25. Controls of the rRNA of the reconstituted particles demonstrated that the 23S rRNA was intact to 60^80%, the activity in poly(Phe) synthesis after complementing with protonated 30S subunits was between 43% and 76% compared to native deuterated 50S subunits. The fact that we did not have a 100% homogeneity of active particles does not compromise a structural analysis. The particles moved as a single, slim peak in the zonal centrifuge and thus are well suited for a position analysis of the protonated protein. In fact, the
The chemical composition of the samples used for analysis by nuclear spin-contrast variation is compiled in Table 2. The scattering data were measured at the SANS1 (SANS = small angle neutron scattering) instrument at the GKSS research centre. Spectra were taken for the unpolarised target and with either parallel or antiparallel aligned proton spins relative to the spins of the incoming neutrons. The collected data were processed according to standard corrections and deconvoluted into the three basic scattering functions MU(q)M2 , Re[U(q)V(q)] and MV(q)M2 . Theoretical basic scattering functions derived from an electron microscopy structure of the 50S subunit [32] containing the protonated protein under observation at all possible positions were ¢tted to the experimental scattering functions. After the determination of the radius of gyration RG and the coordinates of the protein centre of mass, in a second step of calculation also a possible elongated shape of the protein was taken into account. Unless a protein was extremely elongated no statement about the orientation could be made. The ¢nal results from the ¢tting procedure are found in Table 3. 3.3. In situ structure determination of L5 in the 50S subunit Following the procedure described above we localised protein L5 in the rear part of the central protuberance of the 50S subunit. IEM proposed a position towards the intersubunit interface [4]. The results of the ¢tting procedure can be seen in Fig. 2A. The data analysis gave an RG î . The theoretical radius of gyration assuming a of 17 þ 1 A
Table 3 Position of proteins L5, L14, L18, L24 and L25 in the 50S subunit determined by spin-contrast variation î) RG (A î) Distance (A î) Dimensions (A î ) centre of mass Coordinates (A î ) for ellipsoid Coordinates (A
L5
L14
17 þ 1 92 þ 7 22 (4)U22 (4)U51 (9) 37 (6)/90 (7)/312 (8) 316/98/328 6/84/316
15 þ 3 15 þ 1 12 þ 3 95 þ 4 100 þ 3 33 þ 6 25 (5)U25 (5)U38 (6) 80 (6)/40 (5)/33 (9) 328 (4)/95 (3)/37 (15) 35 (7)/315 (4)/9 (11) 79/43/33 85/33/28
L18
L24
L25 13 þ 1 93 þ 9 20 (5)U20 (5)U35 (6) 16 (8)/91 (8)/0 (7) 10/91/32 8/75/33
The coordinates of the proteins are calculated with respect to the ribosome structure from Frank et al. [32]. All distances and coordinates (X/Y/Z) refer to the centre of mass of the ribosome structure. Numbers in parentheses are standard deviations.
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Fig. 2. Results of the ¢tting procedure of proteins L5 (A,B) and L14 (C,D). The measured and the modelled (calculated) basic scattering curves for the best ¢t are shown for protein L5 in A and for protein L14 in C. In B (protein L5) and D (protein L14) the respective minimum maps are calculated. The R-values are presented for three di¡erent distances around the minimum R-value which gives the position of the labelled protein. To illustrate the uniqueness of the position found for each protein, R-values up to 10% higher than the best solution are taken into account. The minimum R-values for proteins L5 and L14 were 3.507 and 2.400, respectively.
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Fig. 3. Results of the ¢tting procedure of proteins L18 (A,B) and L24 (C,D). In A the best ¢t for the measured and the modelled (calculated) basic scattering curves are shown for protein L18 while for protein L24 they can be found in C. In B (protein L18) and D (protein L24) the respective minimum maps are calculated. To illustrate the uniqueness of the position found for each protein, R-values up to 5% (protein L18) and 10% (protein L24) higher than the best solution are taken into account. The minimum R-values for proteins L18 and L24 were 3.310 and 1.379, respectively.
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Fig. 4. Results of the ¢tting procedure of protein L25 (A,B) and the map of the 14 localised proteins in the 50S subunit (C). The measured and the modelled (calculated) basic scattering curves for the best ¢t of the data of protein L25 in the 50S subunit are shown in A. In B the respective minimum map is calculated. The R-values are presented for three di¡erent distances around the minimum R-value of 3.465, which gives the position of the labelled protein. To illustrate the uniqueness of the found position, R-values up to 5% higher than the best solution are taken into account. It can be seen that, in contrary to the other minimum maps, the position of L25 is not unequivocal. In C the 14 protein positions so far localised in the 50S subunit are presented. The red labelled proteins were localised by classical contrast variation [16] and the black labelled ones are localised by spin-contrast variation (L1 [17], L2 [19], L3, L4 [18]). The ribosome structure is by courtesy of J. Frank. Figures were generated with the programme LOCALITE Molecule Navigator.
î . It follows that L5 is spherical shape of L5 would be 14 A slightly elongated, and further data analysis revealed an î, ellipsoidal shape with the three main axes of 22 þ 4 A î î 22 þ 4 A and 51 þ 9 A. This elongated shape was also
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seen in the recently published high resolution structure of the 50S subunit [12]. The radius of gyration from this î which is in L5 structure can be calculated to be 17 A excellent agreement with our measurements.
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15
Fig. 5. Visualisation of additional positions (centre of mass) according to the minimum maps displayed in Figs. 2^4. For simplicity similar positions are represented only by one small orange sphere. Standard deviations are not represented here. (Left) Crown view; (right) ribosomal subunit turned by 90³, view towards L1. (A) Positions of L5, up to 10% higher R-value. (B) Positions of L14, up to 10% higher R-value. (C) Positions of L18, up to 5% higher R-value. (D) Positions of L24, up to 10% higher R-value. (E) Positions of L25, up to 5% higher R-value. Red sphere, equally good R-value compared to the medium-sized light green sphere (only for L25). Large spheres or ellipsoids in yellow, in situ protein structures; medium light green spheres, best R-value of the minimum map. In B this sphere is located inside the larger yellow in situ structure. If the position of the in situ structure is a global minimum the yellow coloured sphere or ellipsoid should be at the same position as the light green sphere. Small orange spheres, other possible positions if a higher R-value is taken into account.
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The position was found between L25 and L18 (see Fig. 4C). The distance of the centre of mass of the protein with respect to the centre of mass of the 50S subunit was deî . The determination of the angles a termined to be 92 þ 7 A and P was possible with a precision of 5 and 4%, respectively (a = 1.70 þ 0.08, P = 1.65 þ 0.07 [rad]). As can be seen from the minimum map in Fig. 2B the position is unequivocal up to 5% deviation of the R-values. The visualisation of additional positions for R-values higher than 5% can be found in Fig. 5A. 3.4. In situ structure determination of L14 in the 50S subunit The measured and modelled basic scattering curves are displayed in Fig. 2C. The position of the protein does not coincide with the position obtained from immunoelectron microscopy [4] but is in quite good agreement with a ¢t into the crystallographic map of the 50S subunit [35]: instead of being localised in the centre of the ribosome, L14 is found near the L7/L12 stalk. The distance from the centre of mass of the subunit was determined to be î , the angles were a = 1.20 þ 0.20, P = 0.50 þ 0.10 95 þ 4 A [rad]. For protein L14 additional information is available from crystallography. The structure of the isolated L14 is known [36] and a calculation using the programme î. CRYSOL by D. Svergun gave a theoretical RG of 14 A The calculation of RG from the scattering curves showed that the radius of gyration corresponds markedly well with î . The spin-contrast variation this prediction : RG = 15 þ 3 A measurements suggested that L14 is not spherical but that it can be described rather by an ellipsoid with main axes of î , 25 þ 5 A î and 38 þ 6 A î . This value corresponds 25 þ 5 A well with the dimension of the protein obtained from the crystal structure if the non-structured loops of the protein î U22 are omitted (estimated to have the dimensions 22 A î î AU38 A). The uniqueness of the position can be seen in Fig. 2D: no signi¢cantly di¡erent positions are found for this protein within up to 5% higher R-values. Additional positions showing up for R-values up to 10% can be seen in Fig. 5B. 3.5. In situ structure determination of L18 in the 50S ribosome Protein L18 with its mass of 12.770 kDa is a small protein attached to the 5S rRNA. Its theoretical radius î . The measured radius was of gyration would be 12 A î . Even though this is slightly larger: RG equals 15 þ 1 A an indication that the protein is not spherical, there was no signi¢cant improvement in the ¢tting procedure when an elongated L18 model was used. A possible explanation could be a shape of L18 neither spherical nor elongated. It is interesting to note that the radius of gyration determined from the L18 structure in Ban et al. [12] can be
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î , even larger than that decalculated to be approx. 18 A termined here. L18 is displayed as a sphere in Fig. 4C despite knowing that this is not the correct description of the shape. The distance between L18 and the centre of mass of the 50S î . The angles of the determined radius subunit is 100 þ 3 A vector are a = 1.72 þ 0.17 and P = 1.78 þ 0.07 [rad], which localises L18 in the central protuberance. The basic scattering function U(q)2 is a representative of the scattering of the ribosomal matrix, and thus it should be almost the same for every sample. We note that the shape of U(q)2 from this particle is a bit di¡erent from the other measurements (Fig. 3A). The basic scattering curve V(q)2 shows that the shape of the labelled protein is not su¤ciently described by a sphere. The minimum map shows only one deep minimum (Rvalue 3.310) up to 5% higher R-values (Figs. 3B and 5C). For higher R-values multiple positions show up. 3.6. In situ structure determination of L24 in the 50S ribosome The determination of the position of L24, which was so far not localised by other methods in E. coli ribosomes, turned out to give a markedly stable result even though the protein belongs to the smaller proteins investigated (molecular mass 13.541 kDa). The radius of gyration î which agrees well with the small was found to be 12 þ 3 A mass of L24. From crystallographic data [12] the RG (L24) î. was calculated to be approx. 15 A The protein is localised in a central position, the distance to the centre of mass of the 50S subunit is only î . The determination of the angles gave 33 þ 6 A a = 1.40 þ 0.34 and P = 5.87 þ 0.17 [rad]. As can be seen in Fig. 3C, the basic scattering curves are very well modelled. The minimum map indicates an unequivocal localisation of L24 (Figs. 3D and 5D; minimum R-value 1.379). 3.7. In situ structure determination of L25 in the 50S ribosome Protein L25 is so far the smallest protein whose in situ structure is determined by spin-contrast variation. Its molecular mass of 10.693 kDa corresponds to a theoretical î . From NMR and crysradius of gyration of approx. 11 A tal structures of L25 [37,38] it can be seen that L25 is slightly elongated while it interacts with the 5S rRNA. The dimensions of the protein were estimated from the î U25 A î U45 A î . With the procrystal structure to be 25 A î was calgramme CRYSOL a radius of gyration of 13 A culated. Our spin-contrast measurements of the in situ î. radius of gyration of L25 gave the value RG = 13 þ 1 A Model calculation with an elongated shape of L25 led to î U(20 þ 5) slightly smaller dimensions of (20 þ 5) A î î AU(35 þ 6) A. The protein was localised at a distance of
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î in the central protuberance of the 50S subunit. 93 þ 9 A The angles a and P were determined to be a = 1.55 þ 0.09 and P = 1.41 þ 0.07 [rad]. Fig. 4A shows that for almost the whole q-range of the basic scattering functions a good agreement between measured and modelled data was found. More surprising was the ¢nding that a second equally possible position of L25 shows up as displayed in the minimum map (Figs. 4B and 5E). This position is below the L7/l12 stalk and hardly touches the ribosome. Furthermore the minimum of the R-value is not very pronounced (smallest R-value 3.465). Other possible positions, for example near L2, were detected. A possible reason for this ambiguity is that with this small protein the limits of the technique are reached because even with the increase of the contrast by polarisation dependent scattering the scattering contribution of labels smaller than 0.5% of the total mass is almost not distinguishable from the matrix and solvent scattering. 4. Discussion Relative positions of proteins can be determined by cross-linking and IEM methods. These methods resulted in the ¢rst map of the 50S proteins [39]. An alternative to the structure determination by biochemical means is neutron scattering which was used to develop a protein map of the 30S subunit [11]. Both techniques together with high resolution X-ray crystallography of the protein and 30S subunit crystals produced a well resolved view of the rRNA and protein distribution in the 30S subunit [13]. The recent crystallographic map of the large subunit of î localised 27 proteins H. marismortui ribosomes at 2.4 A
17
and revealed their in situ structure [12]. Here we present a 50S map with the positions of 14 out of the 33 proteins of the E. coli large subunit of ribosomes (Fig. 4C, Table 5). The map was determined independently by spin-contrast variation and contained seven more proteins than our previous map [16]. A striking feature is that most of the proteins investigated by spin-contrast variation are elongated, only L1 and L24 are largely spherical. In the following paragraphs we will discuss our ¢ndings in comparison with the results of IEM and crystallography. 4.1. Resolution of the methods The IEM positions are determined with a precision of î , in some cases even better. It is obvious that approx. 40 A in the crystallographic map we see the position with a 2.4 î resolution, the best precision reached at the moment. A However, protein L1 and the L7/L12 stalk were not seen in the crystal map but could be localised in the electron microscope. For the neutron scattering analysis we used a î resolution. cryoelectron microscopy structure with 40 A The standard deviations were calculated taking into account systematic and statistical errors. 4.2. Species di¡erences of the ribosomes For IEM and spin-contrast variation ribosomes from E. coli were investigated, and the crystal structure was obtained from the archea H. marismortui ribosomes. These ribosomes di¡er in their composition: the archeal ribosome does not contain proteins L9, L17, L20 and L25. Furthermore the sequence identities between the ortho-
Table 4 Comparison of the protein positions and radii of gyration determined by spin-contrast variation and crystallography [12] î ) SANS î ) cryst. [12] î ) towards IEM î ) towards cryst. Protein RG (A RG (A Distances (A Distances (A L1 L2 L3 L4 L5 L13 L14 L17 L18 L20 L22 L23 L24 L25
24 þ 1 22 þ 1 23 þ 2b 17 þ 2b 17 þ 1 not measured 15 þ 3 24 þ 2 15 þ 1 18 þ 2 21 þ 2 23 þ 2 12 þ 3 13 þ 1
18.8a 22.2 23.6 26.4d 17.0 14.9 13.8c no data available 18.4 no data available 17.1 11.5 15.5 12.8a
15 45 30 15 53 53 94 38 59 60 51 10 not detected 38
not 51 75 28 36 38 56 not 10 not 53 49 93 not
detected
detected detected
detected
The distance between the positions was determined as follows : the neutron scattering coordinate system was rotated by 45³ around the z-axis, followed by a 90³ rotation around the y-axis. Then the coordinates of the centre of mass of the proteins were translated into the coordinate system of the crystallographic structure. The distance was then calculated from the centre of mass of the crystallographic protein positions with respect to the translated neutron scattering positions. The coordinate systems of immunoelectron microscopy and neutron scattering were identical. a Structure of L1: PDB entry 1AD2, structure of L25 : PDB entry 1DFU. b î and RG (L4) = 19 þ 2 A î (private communications by R.P. May). RG (L3) = 35 þ 2 A c î using PDB entry 1WHI. Corresponding to RG = 13.9 A d î. Structure of isolated L4: PDB entry 1DMG gives RG (L4) = 17 A
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Table 5 Comparison of the coordinates of protein positions determined by crystallography (H. marismortui) [12] and spin-contrast variation (E. coli) Protein
H. marismortui X
L1 L2 L3 L4 L5 L13 L14 L17 L18 L20 L22 L23 L24 L25
E. coli Y
Z
120.4 43.8 22.9 91.6 22.8 86.6
134.6 158.4 111.9 24.6 123.0 149.4
61.4 139.9 37.6 108.1 134.2 143.3
63.4
17.4
83.0
29.4 68.4 20.1
161.5 181.7 152.9
87.1 38.7 29.8
X
Y
Z
109.4 110.4 67.4 42.4 60.4 50.4 82.4 45.4 62.4 0.4 59.4 29.4 67.4 79.4
61.5 109.5 166.5 101.5 30.5 99.5 103.5 171.5 19.5 101.5 121.5 156.5 140.5 42.5
37.1 17.9 68.9 19.9 90.9 123.9 174.9 87.9 72.9 71.9 70.9 22.9 108.9 95.9
The coordinates refer to the center of mass of the proteins with respect to the center of mass and the coordinate system of the PDB structure 1FFK [12].
logue proteins of E. coli and H. marismortui are in the range of 24^37% (L1 (29%), L2 (36%), L3 (25%), L4 (24%), L5 (34%), L13 (26%), L14 (37%), L18 (25%), L23 (35%) (BLAST search)). Other proteins correspond to eucaryotic ribosomal proteins but have no counterpart in procaryotic ribosomes. All these di¡erences could shift the relative positions of those proteins of the H. marismortui ribosome that could be compared with E. coli proteins, partially explaining the di¡erences of the corresponding positions described below. Proteins L5, L18 and L25 are the so-called 5S rRNA binding proteins that are located in the central protuberance of the 50S subunit together with the 5S rRNA. IEM investigations had positioned these proteins near to the intersubunit interface, whereas our analysis places these proteins more towards the back of the central protuber-
ance (CP). This coincides well with the distribution of rRNA as presented by Mu«ller et al. [40], and is also in agreement with the relative positioning of the CP proteins that were also found mainly in the cytoplasmic region of the central protuberance as proposed by Svergun and Nierhaus [6]. A comparison of the positions of the three proteins proposed by IEM with those determined by neutron scattering shows that the deviation between the corî for responding positions of each method is approx. 53 A î protein L5, approx. 59 A for protein L18 and approx. 38 î in the case of L25 (Table 4). The corresponding values A for the distances between the neutron scattering and the corresponding crystal positions [12] agree better : L5 was î away from the crystallographic posifound approx. 36 A î (see also Tables 4 and 5 and Fig. 6). tion, L18 about 10 A All three CP proteins are next to each other, probably
Fig. 6. Comparison of the protein positions obtained by neutron scattering and crystallography [12]. The spheres symbolise the centre of mass of the protein positions determined by spin-contrast variation. The crystallographic structures of the respective proteins are highlighted in the same colour. Front view (A) is left (interface side towards the 30S subunit), rear view (C) right (cytoplasmic side with the exit of the polypeptide channel). The picture in the middle (B) shows the subunit from the L1 side (90³ rotation).
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close in contact. L25 was found on the `left' side of the strand identi¢ed as part of the 5S rRNA in the central protuberance which is contrary to the results published previously [40,41]. L5 was found on the back side of the CP next to L18. This position agrees well with the correî structure [12]. The quality sponding position in the 2.4 A of the calculated solutions for the positions of the three CP proteins is rather di¡erent. In the case of L5 a deep minimum was obtained (Fig. 2B). For R-values of up to 10% higher than the best ¢t further possible positions show up in a similar distance to the centre of mass of the ribosome, mainly near the L7/L12 stalk or in a symmetric location (Fig. 5A). In the case of proteins L18 and L25 the minimum is not as deep as for protein L5 (Figs. 3B and 4B). The distance to the centre of mass was determined precisely, but the determination of the angles a and P turned out to be more complicated (L18, Fig. 5C). With L25 the limits of the SANS procedure applied here seems to be reached, since a second position on the L7/L12 stalk side of the ribosome was calculated with an equally good R-value. Other positions can be found in symmetric locations (Fig. 5E). Protein L14 was found near the L7/L12 stalk which does not correspond with the position found by IEM [4] î ). In the 2.4 A î structure L14 is 56 A î (distance approx. 94 A apart from our position [12] (Fig. 6). Again the determination of the distance to the centre of mass was unique. The angles a and P gave several other positions in the CP, near L1 and on the back side of the ribosome (Fig. 5B). Since all these positions have R-values about 10% higher than the minimal value, they are less likely. L24 is located near the intersubunit interface, but is not exposed at the interface. The representation of additional possible positions for higher R-values is shown in Fig. 5D. A symmetric location near the centre of mass of the ribosome was also detected. The distance between L24 from î H. marismortui and our position was calculated to be 93 A (Fig. 6). An overall comparison of the protein positions obtained from neutron scattering with recently published high resolution structures shows that in some respects the similarities are markedly consistent, while for other aspects striking discrepancies are seen. For example, the radii of gyration as determined by neutron scattering are very similar to the corresponding values derived from the crystal structure or from isolated proteins (exception : proteins L1, L4 and L23, see Table 4). The protein positions of L4 and L18 coincide within the limits of the experimental and statistical error with the crystallographic structure, and there is a quite good agreement for the positions of L2, L5, L13, L14, L22 and L23 (Table 4). The relatively large deviations from the crystal positions concerning L2 and L14 might be partially due to the fact that L1, L9 (important for L2) and the L7/L12 stalk (important for L14) are absent in the crystal structure.
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19
For L17 and L20 no comparison with the crystal map can be made since these proteins do not exist in that structure. Our data compare very well with the corresponding data from the older IEM map except for proteins L14, L18 and L20 (Table 4). Proteins L3 and L24 are found at rather di¡erent locations inside the ribosome. Note that with classical contrast variation methods L3 was also found on the right hand side of the subunit (crown view [16]). It has to be seen whether or not the di¡erences are `real', i.e. at least partially due to domain di¡erences of the respective organisms E. coli and H. marismortui, respectively, or, alternatively, are caused by drawbacks of the low resolution method of neutron scattering.
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BBAEXP 93541 10-7-01
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