J. Mol. Biol. (1992) 224, 253-264

Comparison of X-ray Structures of the Nucleosome Core Particle in Two Different Hydration States Mark-M.

Struck, Aaron Klug

MRC Laboratory of Molecular Biology Hills Road, Cambridge CB2 2&H, U.K.

and Timothy J. Richmond lnstitut fiir Molekularbiologie und Biophysik Eidgen&Gwhe Technische Hochschule Hiinggerberg CH-8093 Ziirich, Switzerland (Received 24 May

1991; accepted 14 November 1991)

The X-ray structure of the nucleosome core particle was determined at 7 b resolution using crystals containing mixed-sequence DNA and 21 “/b to 27% of 1,6-hexanediol (partially dehydrated crystals). The alcohol was added to the crystals after growth to overcome the non-isomorphism of the crystals and improve the quality of their X-ray diffraction. Here, we report the structure of the nucleosome core particle from these crystals in the absence of the alcohol 1,6-hexanediol at 9 a resolution. The structure, under conditions of nearly full hydration, has been solved by multiple isomorphous replacement methods employing multiple heavy-atom compounds identical to those used for the partially dehydrat’ed structure. The electron density of particles in the two crystal structures is well-correlated throughout the maps and structural elements of the DNA superhelix and histone proteins are generally similar, e.g. the DNA bends sharply at positions + 1 and +4 double-helical turns from the DNA center. These results rule out the occurrence of gross structural changes in the 7 Lh structure due to addition of alcohol. The parts of the nucleosome core particle structure, which are dissimilar between the two forms, can be attributed to differences in molecular packing induced by the addition of 1,6-hexanediol. In contrast to the structure seen in the partially dehydrated crystals, the fully hydrated crystals show a particle in which the H2A-H2B dimers are symmetrically related by the dyad axis found in the H3-H4 tetramer region. However, in the fully hydrated crystals, t’he first and last double-helical turns of DNA superhelix are not related by dyad symmetry, and one of these segments has reduced contact with the adjacent H2A-H2B dimer. Kp;ywords:

chromatin;

histones: hydration;

1. Introduction The nucleosome is a histone protein-DNA complex that plays a fundamental role in the organization of chromatin (Kornberg, 1977; McGhee & Felsenfeld, 1980; Klug & Butler, 1983; Pederson et al.. 1986; Wu et a,l., 1986; van Holde, 1989; Widom, 1989). Upon micrococcal nuclease treatment of chromatin, a relatively stable nucleosome core particle (206 kDa), containing 146( +2) base-pairs of DNA and one histone octamer, is released (No11 & Kornberg, 1977; Lutter, 1978; Cockell et al., 1983). The histone octamer is made of pairs of the histones H2A, H2B, H3 and H4 (Thomas & Kornberg, 0022-2838/92/050253-

12 $03.00/0

nucleosome; X-ray crystallography

1975a,b; Albright et al., 1979) that exist in solution at physiological ionic strength as H2A-H2B dimers and H3-H4 tetramers (Kornberg & Thomas, 1974). The linker DNA, which connects to adjacent nucleosomes (Kornberg, 1977), and histone Hl are removed during nuclease digestion. The nucleosome core particle, which lacks linker DNA and histone Hl, is a universal part of eukaryotic chromatin (Klug & Butler, 1983), exhibiting similar unit cell dimensions when isolated and crystallized from various organisms (Finch et aE., 1981). Furthermore, the primary arrangement of the histones along the DNA in the nucleosome core particle is highly similar whether isolated from transcriptionally i%: 1992 Academlr

Prexs Limited

active or inactive chromatin prepared from animal, plant or yeast cells. as shown by histone--D?u’A crosslinking experiments (Bavykin et al.. 1985). Structural studies on nucleosome core particle crystals by X-rag diffraction, electron microscopy and neutron diffraction have resulted in a wealth of structural information (e.g. see Finch et al.; 1977, 1980, 1981; Rent’lep et wl., 1981, 1984). The most detailed three-dimensional electron density was obtained by X-ra)- diffraction of nucleosome core particle crystals at 7 A resolution (1 A = 0.1 nm) fin the multiple isomorphous replacement method (Richmond et al.. 1984). The use of mult,iple hearyatom compounds for the phase determination process was an important step in the structure solution avoiding the use of models in the phasing of the X-ray dat’a (Richmond et aE., 1984: O’Halloran ef nl., 1987). Crystals were grown from nucleosome core particles derived from beef kidney according to Finch et nl. (1981) and were treated with 21 to 27’& 1,Ghexanediol to partially dehydrate the crystals. The addition of the alcohol was used to adjust the O-axis length of crystals grown from different preparations to a unique value by exploiting the dependence of the b-axis length on the concentration of alcohol (Richmond et al., 1984). However, studies on the unwinding of naked DNA by dehydration (Lee et al.. 1981) indicate that the screw angle may be increased by up t’o 3 o/;, under the conditions used by the h-axis adjustment procedure. In this regard, the results from cryo-enzymological studies on va.rious proteins using organic cryoprotectives suggest that only minor changes would occur in protein structure on the addition of alcohol (Douzou et al.. 1974: Fink & Cartwright, 1981; Fink & Petsko. 1981). Alt’hough several studies of the effects of partial dehydration on histone and nucleosome core particle structure have been made using salt as the dehydration agent (e.g. see Tager et nl.. 1985), the use of alcohol has not been examined in detail previously. The electron density map of the nucleosome core particle at 7 A determined from partially dehydrated crystals shows that approximately 1.8 turns of a left-handed DNA superhelix are wound around the histone octamer. Some of the secondary structural elements of the hist’one proteins could be discerned, and boundaries for the different protein domains were inferred using the available histoneDNA crosslinking data (Richmond et al., 1984; Kelyavsky et al., 1980). The overall dimensions of the part’icle are 110 A x 110 L%x 57 A, and the pitch of the DSA superhelix varies along its path from 25 to 30 8. An overall molecular dyad symmetry is absent in the crystals, most likely due to a distortion arising from” interparticle associations mediated by histone H2A and DNA. However, the halves of the central histone H3-H4 tetramer are related by a molecular dyad axis. Importantly, the DNA in the crystal was found to be most tightly bent at positions + 1 and + 4, symmetrically disposed about t,he H3-DNA binding site at the center of the nucleosomal DNA. The distortions of the DNA from a smooth path in the region approximately one

double-helical t,urn away from the I)SA (,Pntt’r ww also detected I,)- the react,ivit.y (11’ thch I)NA Ici singlet oxygen (Hogan et d., 1987), and are possibl! explained in part by contacts with a cluster of basic* amino acids from histone H4 (Ebralidse of nl.. 198X). Two models of the nucleosomc> core partic.le havta been proposed since the determination of t,hrb nucleosome core particle structure by Ric*hmond cjf al. (1984). On the basis of their histone octamer structure determination. Kurlingamr it al. (1985) have proposed a structure of the histone octamer in the absence of DNA remarkably different in size and shape from the structure of the histone octarner determined by Klug et nl. (1980) and observed in the nucleosome core particle crystal structure t1> Richmond rt al. (1984). Suggest’ions on how these differences might be explained have been put forward (Klug et al.. 1985; Moudrianakis vt ~6.. 1985a,b; Uberbacher di Hunick. 19856). Another model of the nucleosome (aore particle has been presented by Uberbachrr & Kunick (1985a) on the basis of X-ray intensity measurements from native crystals alone and model building. These aut,hors propose that the nuclrosome core part~icle has an overall dyad symmetry and find no evidence for asymmet,rit positions of the H2A hist)ones in the particle (TTberbacher & Bunick, 1985a. 1989). The aim of the present study using untreated crystals was t’o determine the extent to which the presence of alcohol in the crystals has disturbed the nucleosome

Although addition

core

particle

Wucture

seen at

the space group 1’2,2,2, is maintained of alcohol, the (aeli dimensions

7 X.

on

of n = 111 A, h = 198 a. c = 110.5 A of thcl fully hydrated crystals shrink to n = 107.5 ,r\, 6 = 184 A.

c = 1105 A when soaked in 247; 1.Chrxanediol, 7 ‘30 to 89~ reduction

of t.he b-axis

dimension.

a \I:(

have used cryst,als prepared identically with t.hose used for the earlier X-ray study (Richmond V/ r1,1., 1984), but have not made t,he post-growt,h addition of alcohol. The structnre determinatjion from these “fully-hydrated” crystals was possible to !f A resolution. A comparison of the t,wo carystal forms reveals the differences in interactions that (*an occur in the presence and absence of the al(~ohol 1.(i-hexanediol.

2. Materials and Methods (a) Preparation

of n2.dx~sorn~ core purticks

Nucleosome core particles were prepared as described by Lutter (1978) and Finch et al. (1981). Briefly, long chromatin was prepared from chicken erythrocyte nuclei by light digestion with micrococcal nuclease. The linker histones H5 and Hl were removed from chromatin by column chromatography on Sepharose 48 in 065 M-NaCI. Nucleosome core particles were obtained by digestion of this stripped chromatin with micrococcal nuclease. Subsequent column chromatography on Biogel ASM separated nucleosome core particle monomers from other particles. Chicken erythrocyte chromatin was chosen as the source of nucleosome core particles rather than beef kidney chromatin as used in the earlier study (Richmond

X-ray Structures

qf the Nucleosome

et al., 1984) because larger yields per preparation are possible. The X-ray intensity patterns and cell dimensions from the principle projections of crystals from both sources under the same hydration state show that the structures are very similar (Finch et al., 1981; T. J. Richmond & J. T. Finch, unpublished results).

(b) (‘rystallization

of nucleosome core particles

Nucleosome core particles were crystallized by the batch method (Blundell & Johnson, 1976) in siliconized glass tubes at a concentration of 4 mg/ml over a period of 2 to 4 weeks at 27°C. according to Finch et al. (1981) and Rhodes et al. (1989). The concentrations of salts were 20 to 60 mM-&Cl,, 40 to 60 mM-KCl, llW3d 10 mM-potassium cacodylate (pH 6.0).

Richmond, T. Bailey, A. R. Faruqi & F. Mallet at the MRC (unpublished results), was used to collect the X-ray intensities from 50 A to 9 A. The detector was operat,ed at 2.2 kV with maximal counting rates of 103 &s/mm per s and an effective time resolution of 5 ps. The spatial resolution for the full width of the peaks was 250 pm (A. R. Faruqi, personal communication). The data were collected using the w-scan mode with 0.02” steps. with a maximum of 7 reflections collected at a time. The allowed X-ray-induced crystal degradation of 200/Owas calculated on the basis of a set of 5 reference reflections that were regularly measured. The fluctuation of the primary X-ray beam intensity was monitored with an ionization chamber. and the recorded X-ray int’ensities were normalized for fluctuations and filament decay. The j-circle diffractometer was also used in single counter mode to accurately collect t,he lowest-order reflections.

(c) Ikrivntization of nucleosome cow particle crystals Preparation of the multiple heavy-atom compounds was as described by O’Halloran et al. (1987). Heavy-atom cryst,als were prepared for TAMMf derivative (tetrakis(acetoxymercuri)methane) and PIP (di-p-iodobis (ethylenediamine)~di-platinum (II) nitrate) in the absence of 1,6-hexanediol in the following way. For the TAMM derivative, crystals were harvested into a solution containing 2 mM-TAMM. 20 mu-glycylglycine. 30 mM30 mm-KCl. 10 mM-potassium cacodylate MnCl,. (pH 60). The soaking time was between 36 to 72 h at 20°C. For bhe PIP derivative crystals, crystals were harvested into a solution containing @l mM-PIP, 30 mM-MnCl,, 30 mM-KCl, 10 mw-potassium cacodylate (pH 60). The soaking time was between 2 to 7 days at

20°C. (d) Mounting

of nucleosome core particle crystals

The procedure for mounting nucleosome core particle crystals was as described by Richmond et al. (1984). except that crystals were not treated with 1,6-hexanediol. Crystals were immersed in a solution containing @34/, (w/v) low-gelling temperature agarose, 30 mm-MnCl,, 30 mM-KCl, 10 mw-potassium cacodylate (pH 60) in a glass X-ray capillary. Heavy atom compounds were not included for the derivatized crystals. The X-ray capillaries were tapered at one end to provide a good fit to the rod-shaped rrystals and thereby reduced X-ray absorption. This method of mounting keeps crystals under conditions of constant hydration. The crystal is gelled in place in the capillary at 4”C, the temperature used for data collection. (e) X-ray data collection Data were collected on an Elliott GX13 rotating anode generator run at 40 kV, 80 mA and 2Oy; bias setting. Pin-hole collimation in conjunction with a spot focus size of 200 pm was sufficient to resolve the spacing of the b*-axis. The X-ray radiation was filtered by a 0015 mm thick nickel filter to obtain characteristic CuKa X-ray radiation. The crystal-to-detector distance was 410 mm and the source-to-crystal distance was 279 mm. A linear. position-sensitive detector (Faruqi, 1988) mounted on a 5-circle diffractometer (Banner et al., 1977), build by T. J. t Abbreviations used: TAMM, tetrakis(acetoxymercuri)methane; PIP, di-p-iodobis (ethylenediamine)-di-platinum (II) nitrate.

255

Core Particle

(f) X-ray data prowssing The measured X-ray intensities were corrected with X-ray absorption, induced crystal degradation and the Lorentz-polarization factors using programs included in the CCP4 suite. Profile fitting of reflections was carried out with a program written by T. J. Richmond (unpublished results) using Bayesian statistics as suggested by S. French (1978; Oatley & French, 1982). This profile fitting procedure incorporates information from several sources otherwise neglected. e.g. expected and observed properties of peak shape, local behavior of the background and instrument stability. The profile fitting program was well suited for fitting both strong and weak reflections and was capable of fitting peaks that were only partially recorded due to offsets in the data collection scan. ;211 other programs used for data processing and calculation of Patterson and Fourier maps are part of the CCP4 program package, except for a local scaling program (T. J. Richmond, unpublished results).

3. Results (a) Structure determination of the fully

hydrated

nucleosome core particlp The space group of the crystals is P2,212,, with one nucleosome core particle per asymmetric unit. The unit cell axes are given in Table 1. The a and caxes length are similar for all crystal types. The b-axis length agrees well between the TAMM derivative and the native crystals, but the PIP derivative has a somewhat’ shorter b-axis. When compared with the b-axis dimension from native crystals, the non-isomorphism in the b-axis is 150/,. For the 7 ,& structure from the partially dehydrated crystals, the non-isomorphism was est,imated t.o be I(& following treatment with 1,6-hexanediol to shrink and adjust the b-axis of the crystals. The data for

Table 1 Cnit cell dimensions (Lg)

a Native

TAMM derivative PIP derivative

111.41+@24 111.20+@24 111.39*0.42

h 198.76+(>35 198.55f083 19593&1.35

c 110~70+031 110.48+0.15 I 10.50 + 0.29

Table 2 Data

collection

Table 4

statistic8 TAMM derivative

Sative Total number of measurements No. of independent reflections No. of centric reflections Average I Sigmat Relative sigma1 Lr,,§

Phasing

10,158 2649 588 729 115.2 0.158 6067

PIP derivative

7559 2632 579

12,030 2646 584 408 81.9 OC‘?Ol 0.093

566

156.2 0.276 9064

Resolution range: 50 A to 9 A. t Root-mean-square (I - 1,“s) for each data set. $ Sigma/Z,,,. for each data set. ii Rne,, = Zll- I,JU

Crystal form Resolution range Derivative Phasing power: J%,iӤ

Fulls hvdrated s5-i A TAMM PIP I ,09 I.27 0.1 x 023

Table 3 Heavy-atom

parameters

Site no.

Relative occupancy

5

!I

z

R

1 2 1 2 3 4

0.69 0235 1.0 058% 0392 0.287

-9072 0454 - 0.065 - 0.226 -0.092 0441

-0.101 -0.124 -0.156 -0135 -0.145 - 0.327

9021 0009 0.470 6328 0.331 0.186

30 30 80 60 50 50

Partially

dehydratrtit 35-7 -\ TAMM 1’11’ I ,55 I.72 WI-1 (P20

t Richmond et al. (1984). 1 Phasing power is Af/E, where Af is the root-mean-square (r.m.s.) calculated heavy-atom contribution to the structure factor and E is the multiple isomorphous replacement roottmeansquare lack of closure error.

derivative

and native

2594 independent reflections was 6416 and 0415 for the two enantiomorphs. The difference between these values is too small to be taken as a reliable indicator of the correct handedness of the heavyatom co-ordinates. However, the native Fourier map with the higher figure of merit contained a right-handed DNA double helix and a left-handed DNA superhelix, as was observed from the partially dehydrated crystals. The phasing power and the average change in intensity due to the addition of the multiple heavy-atom compounds for both derivatives of the fully hydrated crystal data (9 A) is compared to the partially dehydrated crystal data in Table 4. The average change in intensity is slightly higher for the data extending to 9 A as compared to that to 7 A, and reflects the difference in scattering power in these different resolution shells. The phasing power for the TAMM and PTP derivative is lower for the structure at 9 A than for the structure at 7 A (O’Halloran et al., 1987) and is probably due to the greater non-isomorphism in the b-axis dimension in the 9 A structure. For the final calculat’ion of the native Fourier map we used the X-ray diffraction data from 30 A to 9 a and centroid phases (Blow & Crick, 1959). The first contour in the electron density maps is at 55”jo of the difference between the maximum and minimum density values. (b) Uructure fully

TAMM TAMM PIP PIP PIP PIP

stat8tic.s

where I, and I, are the heavyatom intensity, respectively.

the fully hydrated crystals were measured from crystals using predominantly one large batch of core particles to avoid adjustment of the cell dimensions with addition of alcohol. A summary of the data collection statistics is given in Table 2. The merging R-factor for the (R,,,ers) indicated that the corrected intensities X-ray measurements were generally good, with the PIP derivative data being the poorest. These data were used to calculate Patterson maps both in projection and in three dimensions that yielded the co-ordinates of the main heavv-atom sites in both derivatives. Minor substitution sites were found in difference Patterson and difference Fourier maps and collectively refined with the main sites. The heavy-atom data set scale factors, and the three coordinates and occupancy of each heavy-atom site, were refined for each derivative data set. The heavy-atom temperature factors were adjusted to values between 20 and 100 to give the highest. correlation coefficients of observed and calculated heavy-atom contribution. The refined heavy-atom parameters used in the phase calculation are given in Table 3. The phases for calculating a native Fourier map were determined on the basis of the measured isomorphous and anomalous X-ray diffraction. The scattering factors for the multiple heavy-atom groups were calculated using the formula given by O’Halloran et al. (1987). The mean figure of merit. calculated according to Blow & Crick (1959), for the

Derivative

and wding

interpretation

hydrated

from

thr

crystals

The sections of the electron densit,y map displayed in Figure 1 demonstrate that’ the DNA superhelix is visible, and that the two chains of the double helix could be traced. As in the earlier study, the locations where the minor groove face outward are labeled; for example, the center of the nucleosomal DNA is labeled position 0, the end of the superhelix with the lowest value of the z co-ordinate in the map is labeled -7, and the end with the highest value is labeled + 7 (Richmond et al., 1984). The interpretation of DNA in the electron density map led t,o a complete outline of the DNA super-

X-ray Structures of the Nudeosome Core Particle

257

Figure 1. H2A’-H2RZ dimer region of the fully hydrated nucleosome core particle structure. The electron density sections correspond to a thickness of 11.6 A, or somewhat less than 025 of the thickness of the particle, and are marked in divisions of 91 of the unit cell dimensions along the a (vertical) and b (horizontal) axes. The colored density corresponds to parts of the DNA superhelix (brown with black outline) and histone proteins H2A (yellow with white outline), H2R (red), H3 (blue), and H4 (green) of one particle. The particle is centered in the Figure and the view is nearly parallel to the axis of the DNA superhelix. The DNA is labeled wherever the minor groove faces away from particle. The DNA duplex proceeds from one terminus at the lower left (labeled 7) downward in the map to form a lefthanded superhelix that leaves these sections at the top of the Figure after approximately 45 double helical turns. Only a small part of the upper face of the DNA at position 0 can be seen in these sections.

helix (Fig. 2) and delineation of the protein interior of the molecule. On average, the DNA is approxiand exhibits an average mately 20 A in diameter repeat of near 35 8. The pitch of the approximately

Figure 2. Outline of the DNA double helix of the fully hydrated nucleosome core particle. The numbers indicate positions of the DNA where the minor groove is facing toward the outside of the particle in accord with Richmond eb &E. (1984). The dyad axis of symmetry (not shown) passes through the double helix at position 0 and between turns of the superhelix near position + 3.8 for the upper turn (a) and -3.8 for the lower turn (b). The outline of the double-helical DNA was obtained by marking the center of electron density corresponding to the positions where the phosphodiester chains pass through each section of the map. These ordered series of points were fit with the curves shown. The crossover of strancls at. positions -4 is ambiguous.

1.8 turns of superhelix is variable between 25 to 30 A, although the limit of the measurement is about & 1 A. These observations are in agreement with the structure of the nucleosome core particle from partially dehydrated crystals (Richmond et at., 1984). The largest deviations from a smooth path occur at positions + 1 and +4, which may be identified with positions of DNA bending or kinking. In both the 7 A and the 9 A structures the bend in the double helix at position - 1 appears, in the views down the superhelix axis, distributed towards position -2, although a significant component of the bend at sites + 1 is not observed in this projection because the axis of bending is not parallel to the superhelix axis. In both cases the electron density is weak in the - 1 region, but this appearance is more pronounced in the 9 A fully hydrated structure. The overall shape of the superhelix is consistent with molecular 2-fold symmetry with the notable exception that the dyad symmetry does not hold for the +7

DNA

ends (Figs 1 and 2). The double

helix

(a)

(b) Figure 3. Positions of histones H2A’, H2AZ and DNA termini in the 2 crystal forms. The molecular components for 2 adjacent particles are indicated in the electron density by the same color scheme as used for Fig. 1. The region that most) differs between the 2 crystal forms is enclosed in the white rectangles. The same region is indicated in the particle packing diagrams (Fig. 4(a) and (c)), and the orientation of the reference particle (R) in (a) is the same as for the particle in Fig. 1. (a) Fully hydrated crystals. The + 7 end of the DNA superhelix (labeled 7) bends away from the histone H2A2 domain of its own particle and appears to contact an extension (arrow) from the H2A’ domain of another particle. (b) Partially dehydrated crystals. In contrast to the structure seen in (a), the + 7 end of the DNA follows the surface of the H2A2 domain of the same particle and appears to cont,act the -7 DNA terminus of an adjacent particle.

X-ray Structures

of the Nucleosome

turn between positions + 6 and + 7 is in-phase with the minor-major groove alterations of the superhelix beginning at site 0, but in contrast to the segment at the -7 DNA end and at the sites +7 in the partially dehydrated crystals, it bends gently away from the center of the particle. Furthermore, the DNA at + 7 is better defined in the 9 A map as compared to the 7 A map. This may be a consequence of crystal preparations containing a more unique length of DNA in the former case, or the variable locations of the DNA (and H2A-H2B dimer) in the presence of alcohol for the latter case. The DNA double helix is generally better defined in the partially dehydrated crystals studied at higher resolution, as expected. Smaller deviations from the 2-fold symmetry, other than those noted, are not reliably interpreted due to the lack of high resolution data and the occurrence of non-isomorphism. The boundary of the histone proteins with DNA is well defined; the interface between histone the interface between the molecules, including H2A-H2B dimer and the H3-H4 tetramer, however, is less clear but can be inferred by comparison with the 7 A structure. Equivalent histone proteins in the two electron density maps have been compared and show similarity in their gross size, shape and relative locations within the core particle structure (Struck, 1989). The ultimate designation requires a higher resolution structure of the nucleosome core particle (Richmond et al., 1988). Hist,one H3 is uniquely labeled by the binding of t’he TAMM compound to cysteine 110 of the two histone H3 molecules (site 1 in Table 3), suggesting that the cysteine residues are located on the molecular dyad axis and bridged by the TAMM, as determined for the 7 A structure (Richmond et al., 1984). The assignment of histone proteins in the electron densit’y map is in agreement with the crosslinking experiments of Bavykin et al. (1985) and is consistent with an overall 2-fold symmetry. In particular, we observe contacts of the two histone H2A molecules with DNA at sites &@5 that possibly explain the sites of chemical crosslinking found in nucleosome core particles isolated from chromatin (Bavykin et al., 1985) and with intact chromatin in the absence of histone Hl (Belyavsky et al., 1980). However, as for the nucleosome core particle in the partially dehydrated crystals, contacts are not observed between histone H2A and the DNA at sites +4 to f5. It is possible that this contact is made between the N-terminal or C-terminal extensions of histone H2A that are not observed in the electron density map (Cary et al., 1978; Bijhm & Crane-Robinson, 1984). Examination of the two nucleosome core particle structures reveals that of all the histone proteins H2A engages in the most extensive interparticle interactions in the crystals. The greatest difference between the structures from the two crystal forms is in the interparticle interaction of histone H2A2 with the adjacent DNA terminus at site + 7. In the fully hydrated crystals, approximately one turn of the double helix at the end of the superhelix has been

Core Particle

259

displaced from H2A2 in each particle, and the superhelix terminus now abuts H2A’ of an adjacent core particle (Figs 3(a) and 4(a)). Although it appears that little or no contact is made between H2A2 and this region of the double helix, it is possible that one DNA strand is disordered and variably bound to H2A2. Some of the interactions that appear to be lost as compared to the other copy of this histone, H2A’, are made with the DNA of the adjacent particle. In contrast, this terminal turn of the DNA is bent around and makes substantial interactions with H2A2 in the partially dehydrated crystal structure (Figs 3(b) and 4(c)). The H2A’ molecule binds to the central turn of DNA superhelix of an adjacent particle, not to a DNA terminus. In the 9 A structure, H2A’, and in the 7 A structure both copies of H2A, contact the DNA of an adjacent core particle with their surface which is at the largest radius from the superhelix axis. These regions would most likely be in eontact with linker DNA in intact chromatin, as they would be covered by an extension from the termini of the superhelix. Because the DNA at the + 7 terminus in the fully hydrated state has separated from H2A2, the deviations of the two H2A-H2B dimers from the molecular dyad symmetry determined from the H3-H4 tetramer region appear substantially smaller than found in the partially dehydrated crystals. From the outline of the superhelix in the nucleosome core particle seen in the two electron density maps, we have constructed molecule packing models as schematically depicted in Figure 4. The packing of nucleosome core particles in the plane defined by the crystallographic a and b-axes is pseudohexagonal for both crystal forms. The packing of particles depicted in Figure 4(a) and (c) shows that the dyad axis of the nucleosome core particles in the absence of 1,6-hexanediol is inclined by approximately 10” more with respect to the crystallographic b-axis than in the presence of 1,6-hexanediol (9”). In the plane defined by crystallographic b and c-axes, we observe that the particles are staggered relative to each other in the presence of 1,6-hexanediol due to a translation of approximately 7 A, permitting histone H2A’ to contact DNA at position -3.5 of the neighboring particle in the layer (Fig. 4(d)). The contact at - 3.5 occurs in the major groove of the DNA. In contrast in the absence of 1,6-hexanediol, the particles are not staggered relative to each other, but arranged in flat, regular layers, and the predominate contact for H2A’ is with the DNA at position +7 of the particle in the same layer (Fig. 4(b)).

4. Discussion (a) Effects of I$-hexanediol on the structure of the nucleosome core particle

The structure of the nucleosome core particle has been determined from fully hydrated crystals at 9 A by X-ray crystallographic methods and compared to the structure determined from partially dehy-

M.-M.

&Yruck et al.

Figure 4. Molecular packing diagrams of nucleosome core particles in the 2 crystal forms. The molecular packing of particles in the crystallographic &plane and bc-plane are shown for the fully hydrated ((a) and (b)) and partially dehydrated ((c) and (d)) crystals, respectively. Only the DNA component is shown. In thr views down the crystallographic c-axis, nearly parallel to the superhelix axis ((a) and (c)), the disk-like particles are seen t,o br arranged in pseudo-hexagonal layers. The molecular dyad axes are indicated for 2 of the particles (broken red line), and differ in their rotation around the superhelix axis by 10” in the 2 crystal forms. The views down the crystallographic: n-axis show that the particles are arranged in nearly Aat planes in the full-hydrated crystals (b), but are displaced alternatively along the c-axis direction in the partially dehydrated crystals (d). The orientation of the reference particle (R) indicbat,ed in (a) is the same as for the particle in Fig. 1. The region enclosed in the white rectangles in (a) and ((3) is identical t,o that indicated in Fig. 3.

drated crystals at 7 A resolution (Richmond et al., 1984). Overall, the two structures are very similar: the addition of 1,6-hexanediol to the crystals did not have a dramatic effect on the structure of the particle as a whole. The finding may be of some consequence for other structures determined from co-crystals of protein and DNA, since the related alcohol, 2-methyl-2,4-pentanediol, is used routinely crystallization (e.g. macromolecular gtwinowski et al., 1988). The effect of 2-methyl-2:: pentanediol on nucleosome core particle crystals as judged by unit cell parameters is nearly identical to 1,6-hexanediol (T. J. Richmond, unpublished results). A comparison of the fully dehydrated and partially dehydrated crystal forms at higher resolution (35 to 40 8) using crystals containing defined sequence DNA will not be immediately possible, since the addition of alcohol is required to obtain diffraction data beyond 6 A along the b-axis direction (Richmond et al., 1988). Nevertheless, it appears unlikely that substantial conformational changes in the histone proteins will occur, given that the high resolution structures of several proteins, e.g. chymotrypsin, trypsin and papain determined in the presence of similar cryosolvents, are

the same as in aqueous salt solutions (Fink & Cartwright, 1981; Fink & Petsko, 1981). The structural changes seen in these crystallographic studies are only minor and consist primarily of a reduction in the mobility of both backbone and side-chain atoms due to the low temperatures employed rather than an explicit effect of cryosolvents (Frauenfelder et aE., 1979; Walter et aZ.. 1982; Parak et al., 1987). Studies on the unwinding of naked DNA in the presence of various organic solvents have show11 that at the alcohol concentration in the partially dehydrated crystals, the DNA could be unwound by up to 3% in the screw angle (Lee et al, 1981). Tn the nucleosome core particle, the DNA is constrained by histone interactions, and the similarity hetween the two crystal forms confirms that a major change in the DNA structure has not occurred. Generally, the superhelix is better defined in the 7 A map, except at its termini which appear to be partly disordered, probably due to the effect of 1,6-hexanediol. Digestion of nucleosome core particles with DNase 1 in the presence and absence of 1,6-hexanediol revealed that the last turns of the DNA are not identical in their cutting patterns in the two conditions, whereas the cutting pattern from the internal cleavage sites is identical (Richmond et al., 1984).

X-ray Structures of the Nucleosome Core Particle Despite the overall similarity of the structures of the core particle, some structural features differ between the two crystal forms. In the fully hydrated crystals, the molecular dyad axis holds for the entire particle, including the H2A-H2B dimers, with the exception that approximately ten base-pairs from position + 6 to + 7 of the DNA superhelix leave the surface of the histone H2A2. A solvent cavity is apparent between the histone and DNA (Fig. 3(a)). The end of the DNA contacts an apparent extension of H2A’ from the neighboring particle. This region of electron density may be the C terminus of histone H2A’ because when this part of the core particle is specifically removed by proteolysis with plasmin, the unit cell dimensions of the crystals under conditions of full hydration correspond to those for the partially dehydrated crystals (H. C. Thijrgersen, T. J. Richmond & A. Klug, unpublished results). In the presence of alcohol, the overall molecular dyad axis does not extend to the H2A-H2B dimers because of differences in the extensive interparticle association mediated by the copies of histone H2A on opposite faces of each core particle with the DNA of adjacent particles. The +6 to + 7 end of the DNA superhelix is smoothly bent towards the particle and makes contacts with histone H2A2 over its entire last turn. The region of H2AZ immediately adjacent to the end of the DNA binds to the DNA of a neighboring particle. Nevertheless, the halves of the centrally located histone H3-H4 tetramer are still related by molecular dyad symmetry. The tetramer is almost completely buried inside the particle and is therefore shielded from interparticle contacts. A departure of the histone H2A-H2B dimers from molecular dyad symmetry was suggested from the neutron diffraction study at 16 A resolution using crystals of nucleosome core particles grown in an identical manner to those used in this study, and then subsequently soaked in a solution containing 5% 1,6-hexanediol (Finch et al., 1981; Bentley et al., 1984). However, the density in the neutron map assigned to the H2A proteins is closest to the position assigned to H2B in the electron density map. This interpretation may stem from the low resolution of the structure determination combined with the effect of the alcohol at low concentration. The b-axis dimension is changing rapidly at 5% alcohol, which suggests that one copy of the H2A molecules may be difficult to see as a consequence of low occupancy due to multiple positions. (b) The effect of DNA length on the crystal b-a&s lerz.gth We have demonstrated that the b-axis of the nucleosome core particle crystals is correlated with the mean length of the DNA contained in the particles; there is an approximately 4 A increase in the b-axis dimension per additional base-pair (Richmond et al., 1983). The observation was explained for the fully hydrated crystals by a packing model that had opposite ends of the DNA

261

of adjacent core particles in contact. Thus, a variable number of base-pairs at the DNA termini would cause the b-axis to shrink or expand, and the dislocations would be restricted to the crystallographic ab-plane. The stacking of layers along the c-axis would be virtually unaffected. The packing model based on the 9 A map is in qualitative agreement with this explanation (Fig. 4). The revised packing model shows that the ends of the DNA are contacting an adjacent particle, but the exact description of the dislocation is not possible at low resolution. Assuming a separation of 3.4 A between adjacent base-pairs in B-form DNA and using the observed inclination angle of 19” lying in the crystallographic ab-plane for the direction of the double helix axis with the crystallographic b-axis, one calculates that the b-axis length increases by either 6.2 A or 3.1 A for each additional base-pair, depending on whether the termini are rigidly in contact with each other or with another part of the neighboring molecule. The experimental value is approximately 43 A per base-pair (Richmond et al., 1983). (c) The effect of alcohol concentration on the crystal b-axis length The difference between the b-axis dimensions of the fully hydrated and partially dehydrated crystals is approximately 15 A. The decrease of 7 to 8% in length is continuous with increasing alcohol concentration (J. T. Finch & T. J. Richmond, unpublished results). This can be accounted for by the differences in the interaction between histone H2A and the DNA at position -3.5 and + 7 of adjacent particles giving rise to differences in the molecular packing between the two crystal forms. In the partially dehydrated crystal forms, the extensive contact made by H2A’ with the major groove at site +3.5 of the adjacent particle along a 2, screw axis parallel to the b-axis direction requires an approximately 7 A shift in the columns of particles along the c-axis as compared to the fully hydrated form. In addition, a rotation of IO” of the particle around its superhelix axis occurs, aligning the groove and the binding surface of H2A’. The DNA at position +7 conforms with the surface of histone H2A2 of the same nucleosome as well as binding near its end to H2A’ of the adjacent particle (Fig. 3(b)). We estimate that these changes would result in a decrease in the b-axis by approximately 0.5 of one grid spacing (10 A) along the b-axis direction as seen in Figure 3(a). The shift of the particles from Aat to uneven layers perpendicular to the c-axis allows them to pack more closely together in the ab-plane. (d) Relationship to other structural studies The structures of the nucleosome core particle in the two different hydration states discussed here have been determined independently from each other, and have been found to be highly similar

overall. The structures explain the relatively strong protection of the DNA against DNase I digest,ion at positions + 1 and f4 for the nucleosome core particle in solution since these are the sites where the DNA is seen to be most severely bent (Lutter, 1978; Richmond et aZ., 1984). The two crystal structures are also consistent with the contact of histone H2A to the DNA from position - 1 t.o + 1, which is specific to nucleosome core particles, as well as other contacts observed by protein-DNA crosslinking in et al.: 1980). Furthermore. .the solution (Belyavsky 7 il structure has been used to accurately account for the X-ray scattering pattern obtained from oriented chromatin fibers (Widom $ Klug. 1985). Additional evidence that the nucleosome core particle is representative of nucleosomes in chromatin comes from the analysis of the distribution of pyrimidine dimers induced by ultraviolet light in nucleosome core DNA and chromatin (Gale B Smerdon, 1988). This study demonstrates that the average periodicity of 1@3( f O-1) base-pairs per turn and a reduction in the yield of pyrimidine dimers at specific sites along the DNA is the same in isolated nucleosome core particles and intact chromatin, but differs from t’hat obtained for the DNA free of the histone proteins. The histone-DNA interactions in the core particle that account for the organization of the DNA superhelix are apparent,ly independent of higher-order chromatin structure which is lost when core particles are prepared. We cannot’ make a direct comparison of our solvent composition with those used in the studies on another form of core particle crystals (space group P2,) at I5 a resolution (Uberbacher & Runick, 1985a) and subsequently at 8 A (lrberbather & Bunick, 1989), as these latter conditions have not been reported in their entiret,y. We note. however, t’hat’ a large discrepancy exists between the H2A positions seen in our structure as compared to their model. The outer boundary of both H2A molecules in our P2,2,21 crystals extends to the full radius of the particle, while in the interpretation of the P2, crystals. the H2A molecules end at the radius defining the inner boundary of t,he superhelix. The positions of these histones in the nucleosome core particle emerged clearly in the multiple 7‘4 isomorphous replacement’ structure at (Richmond et al., 1984) and that reported here at 9 A resolution. It is possible that the differences in the two studies arise from the methods employed in each case. We have used multiple isomorphous replacement, with multiple heavy-atom groups (O’Halloran et al., 1989), while the other investigators have used model-building and density modification techniques to obtain an electron density map (Uberbacher & Bunick, 1989). The model of the nucleosome core particle proposed by Burlingame et al. (1985) on the basis of their histone octamer X-ray structure is radically different from that seen in the nucleosome core particle structures, and the model based on the histone octamer structure determined by Klug et al. (1980). The reasons for this discrepancy in the struc-

ture of the histone octamer havr hen disc~ussetl (Klug et al., 1985: Moudrianakis et al.. l!E%%,b). Further proposals to explain the differences rcxly on the observat’ion that the histonr oct,amer undergoes conformational transitions dependent on salt con centration and ion species (Uberbacher et a./., 1986; Park & Fasman, 1987: van Holde. 1989). However, under conditions similar to those used to crystallize, the histone octamer. tubular aggregates were obtained that led t’o a low resolution strutbture determined by image analysis of electron micwgraphs (Klug et al.. 1980) that is in good agreement’ with the structure observed in the nucleosomr core particle (Richmond et al.. 1984). Radius of gyration measurements of the ortatner made in solut’ions containing high concentrations of salt to simulate the crystallization conditions must be interpreted with caution since aggregation of the octamer is likely to occur (Cberbacher et al.. 1986). More recent, low angle neutron and X-ray seatt,cring studies have been invoked to suggest a possible role for the flexible histone-terminal regions to acc~ount for the discrepancy (Wood rt al., 1991). (e) ImpZications

for nucleosomes and chromatir~

The crystal structures of the nucleosome core particle (Richmond et al., 1984; this work) and t,hr protein-DNA crosslinking data of histones in et al., 1985) and HI -containing chromatin (Ravykin nucleosomes (Belyavskp et al., 1980) indicate that histone H2A can occupy somewhat different; positions in the nucleosome core particle. In t,he absence of histone Hl, histone H2A becomes susceptible to crosslinking to the DNA between positions - 1 and + 1 in addition to the positions at + 7. The additional sites of crosslinking that appear when histone Hl is removed may reflect intramolecular rearrangement (Belyavsky et al., 1980), or more likely, a reduction of steric blocking bv Hl of DNA sites to contact with the flexible term”ini of histone H2A. The proposal that the linker I)NA would enter/exit the core histones on a trajectory of roughly 50” from the dyad axis position assumes that. t.he sites on histone H2A, which are hound to DNA of neighboring particles in the crystals, would be bound to the next ten base-pairs extending from the 146 base-pairs length found in the core particle (Richmond et al., 1984). The strength of this interaction may be affected b-y the presence of histone Hl or hist,one modifications with the possibility t.hat, it can be tuned tIo the state of gene act.ivity locally. Clearly, the interactions of histone H2A are affected by rather mild differences in the hydration state. To accommodate short linker lengths of 0 to 20 base-pairs in higher-order structure, for example in Saccharomyces cerevisiae (e.g. see Widom & Lowary, 1989), the contacts with H2A at the entry and exit points into the core structure may be substantially different than for longer linker chromatin. This may imply that in short. linker chromatin the ten base-pair extensions from the 146 base-pair length DNA are not bound by

X-ray Structures of the Nucleosome Core Particle histone H2A. Core particles containing defined sequence DNA of lengths longer than 146 base-pairs are being prepared to examine the interactions in this region (T. Rechsteiner & T. J. Richmond, unpublished results). The lack of interaction between one histone H2A molecule and the terminal segment of DNA seen in the fully hydrated crystal form probably results from crystal packing forces under these conditions. One copy of the H2A molecule in the fully hydrated crystals and both copies of the partially dehydrated crystals are in close contact with the DNA in this region. It is unknown which conditions of hydration best represent the state in viva. The disassembly of the nucleosome core may begin at the interface between histone H2A and DNA and/or between the H2A-H2B dimer and the H3-H4 tetramer. The exact response may depend on the nature of other protein factors present, such as histone Hl, HMG proteins, transcription factors, topoisomerase, polymerases and histone chaperone proteins, that, rompet,e with the core histones for DNA binding.

Blundell,

T.

We are grateful to Dr S. *J. Lippard and Dr T. V.

L.

& Johnson, L. N. (1976). Protein Academic Press, London. Bb;hm, L. & Crane-Robinson, C. (1984). Proteases as structural probes for chromatin: the domain structure of histones. Biosci. Rep. 4, 365-386. Burlingame, R. W., Love, W. E., Wang, B.-C., Hamlin. R.. Xuong, N.-H. 6 Moudrianakis, E. N. (1985). Crystallographic structure of the octameric histone core of the nucleosome at a resolution of 3.3 A. Crystallography,

Science, 228, 546-553.

Gary, P. D., Moss, T. & Bradbury, E. M. (1978). High-resolution proton-magnetic-resonance studies of chromatin core particles. Eur. J. Biochem. 89, 475-482. Cockell, M., Rhodes, D. & Klug, A. (1983). Location of the primary sites of micrococcal nuclease cleavage on the nucleosome core. J. Mol. BioZ. 170, 423-446. Douzou. P., Hoa, B. H. G. & Petsko, G. 4. (1974). Protein crystallography at sub-zero temperatures: lysozylnesubstrate complexes in cooled mixted solvents. J. Mol.

BioZ. 96, 367-380.

Ebralidse. K. K., Grachev, S. A. & Mirzabekov, A. D. (1988). A highly basic histone H4 domain bound t,o the sharply bent region of nucleosomal DNA. Nature (London),

O’Halloran for the gift of the heavy-atom compounds TAMM and PIP, to Mrs B. Rushton for help in the preparation of crystals of nucleosome core particles, and to Dr J. Finch for encouragement, and technical advice. M.-M.S. is pleased to acknowledge support by the Studienstiftung des Deutschen Volkes, by Peterhouse, Cambridge, U.K., by the Eidgeniissische Technische Hochnchule and by the Swiss National Foundation.

263

331, 365-367.

Farqui, A. R. (1988). Development and application of multiwire detectors in biological X-ray studies. XucZ. Instr.

Methods,

273, 754-763.

Finch. J. T.. Lutter, L. C., Rhodes, I>.> Brown, R. S., Rushton, B.. Levitt, M. bz Klug, A. (1977). Structure of nucleosome core particles of chromatin Nature (London),

269, 29-36.

Finch, J. T.. Lewit-Bentley, A., Bentley, G. A., Roth, M. 6 Timmins, P. A. (1980). Neutron diffraction from crystals of nucleosome core particles. Phil. Trans. Roy. Sot. Ser. B, 290, 635-638.

References Albright, S. C., Nelson, P. P. & Garrard, W. T. (1979). Histone molar ratios among different electrophoretic forms of mono- and dinucleosomes. J. Biol. Chem. 254, 106551073. Banner, D. W., Evans, P. R., Marsh, D. J. & Phillips, D. C. (1977). A multiple-counter X-ray diffractometer with equatorial geometry. J. Appl. Crystallogr. 10, 45-51. Bavykin, S. G., Usachenko, S. I., Lishanskaya, A. I.. Shick, V. V.. Belyavsky, V. A., Undritsov, I. M., Strokov, A. A., Zalenskaya, I. A. & Mirzabekov. A. D. (1985). Primary organization of nucleosomal core particles is invariable in repressed and active nuclei from animal, plant and yeast cells. Nucl. Acids Res. 13. 3439-3459. Belyavsky, A. V., Bavykin, S. G., Goguadze, E. G. $ Mirzabekov. A. D. (1980). Primary organization of nucleosomes containing all five histones and DNA 175 and 165 base-pairs long. J. Mol. BioZ. 139, 519536. Bentley, G. A., Finch, J. T. & Lewit-Bentley. A. (1981). Neutron diffraction studies on crystals of nucleosome cores using contrast variation. J. Mol. BioZ. 145, 771 -784. Bent,ley. G. A., Lewit-Bentley, A., Finch, J. T., Podjarny. A. D. & Roth. M. (1984). Crystal structure of the nucleosome core particle at 16 A resolution. J. Mol. RioZ. 176, 55-75. Blow. D. M. & (‘rick, F. H. C. (1959). The treatment of errors in the isomorphous replacement method. Acta Crystallogr. 12. 794-802.

Finch, J. T., Brown, R. S., Rhodes, I).. Richmond, T., Rushton. B., Lutter, L. C. & Klug. A. (1981). X-ray diffraction study of a new crystal form of the nucleosome core showing higher resolution. .J. Mol. RioZ. 145. 757-769. Fink, A. 1,. & Cartwright, S. J. (1981). C:ryoenzymology. CRC Grit. Rev. Biochem. 11, 145-207. Fink, A. I,. & Petsko, G. A. (1981). X-ray cryoenzymology. Advan. Enzymol. 52, 177-246. Frauenfelder, H.. Petsko, G. A. & Tsernoglou, D. (1979). Temperat’ure-dependent X-ray diffraction as a probe of protein structural dynamics. LVVature (London), 280, 558-563. French. S. (1978). A Bayesian three-stage model in crystallography. Acta Crystallogr. sect. A. 34, 76% 738. Gale. ?J. M. & Smerdon, M. ,J. (1988). Photofootprint of nucleosome core DNA in intact chromatin having different structural states. J. Mol. BioZ. 204, 949958.

Hogan, M. E., Rooney, T. F. & Austin, R. H. (1987). Evidence for kinks in DNA folding in the nucleosome. Nature (London), 328, 554-557. Klug, A. & Butler, P. J. G. (1983). The st’ructure of nucleosornes and chromatin. Genes: Structure and Expression, Horizons in Biochemistry and Biophysics Series (Kroon, A. M., ed.), John Wiley & Sons, New York. Klug, A., Rhodes, D., Smith, J., Finch. J. T. & Thomas, J. 0. (1980). A low resolution structure for the histone core of the nucleosome. .Va,tuw (London), 287. 509-516. Klug, A.. Finch, .J. T. & Richmond. 7’. .I. (1985).

264

M-M.

Struck

(‘rystallographic structure of the octamer hist)onr core of the nucleosome. Science, 229. 1109.- 1110. Kornberg, R. I). (1977). Structure of chromatin. Avcnu. Rev. Biochem. 46, 931-954. Kornberg, R. D. &, Thomas, J. 0. (1974). (:hromatin structure: oligomers of the histones. Rciencr, 184. 865-868. Lee, (‘. H., Mizusawa, H. & Kakefuda, T. (1981). Unwinding of double-stranded DNA helix b! dehydration. Proc. Nat. Acad. Rci.. (7.A.A. 78. 283% 2842. Lutter, 1,. C. (1978). Kinetic analysis of deoxyribonuclease I cleavages in the nucleosome core: evidence for a DNA superhelix. J. Mol. BioZ. 124, 391-420. McGhee, J. D. t Felsenfeld, G. (1980). Nucleosome structure. Annu. Rev. Biochem. 49, 1115-l 156. Moudrianikas, E. N., Love, W. E.. Wang, B. c’.. Xuong, N. G. & Burlingame. R. W. (1985a). Science. 229, 1110-1112. Moudrianakis, E. N.. Love, W. E. & Burlingame, R. W. (198%). Science, 229, 1113. Nell, M. & Kornberg, R. D. (1977). Action of micrococcal nuclease on chromatin and the location of histone HI. J. Mol. Biol. 109, 393-404. Oatley. S. & French, S. (1982). A profile-fitting method for the analysis of diffractometer intensity data. Acta Crystallogr.

sect. A, 38, 537-549.

O’Halloran. T. V., Lippard, S. ,J.. Richmond, T. ,J. & Klug, A. (1987). Multiple heavy-atom reagents for macromolecular X-ray structure determination application to the nucleosome core particle. J. Mol. Viol. 194, 705-712. Otwinowski, Z., Schevitz, R. W.. Zhang, R.-C., Lawson, C. L.. ,Joachimiak, A., Marmorstein, B. F.? Luisi, R. F. & Sigler, I’. B. (1988). Crystal structure of TRP repressor/operator complex at atomic resolution. Xature (London), 335. 321-329. Parak, F., Hartmann. H., Aumann. K. I).. Reuscher, H., Rennekamp, G.. Bartunik. H. & Steigemann. W. (1987). Low temperature X-ray investigation of structural distributions in myoglobin. Eur. Biophys. J. 15, 237-249. Park. K. & Fasman. G. D. (1987). This histone octamer. a conformationally flexible structure. Biochemistry, 26, 8042%8045. Pederson, D. S., Thoma, F. & Simpson, R. T. (1986). Core particle, fiber, and transcriptionally active chromatin struct,ure. Annu. Rev. Cell Biol. 2, 117-147. Rhodes, I).. Brown. R. S. & Klug, A. (1989). Crystallization of nucleosome core particles. Methods Enzymol. 170, 42&428. Richmond, T. ,J., Finch, ,J. T. & Klug, A. (1983). Studies of nucleosome structure. Cold Spring Harbor Symp. t&znt. Biol. 47. 493-501. Richmond. T. J.. Finch. J. T.. Rushton, B., Rhodes. D. 8r Edited

et, al.

Klug. A. (1984). Structure of the nucleosomtb (‘or(* particle at 7 A resolution. Xaturr flon~don~ I 311. 532-537. Richmond, T. ,J.. Searles, M. A. & Simpson, K). ‘I’. (I!)#). Crystals of the nucleosomr (‘ore particle caontaining defined sequence DNj,4. J. Mol. Biol. 199. 161 170. Struck, M.-M. (1989). Crystallographic studies of nucleosome-core particles containing mixed- and d&wsequence DNA. Ph.D. t,hesis. (‘ambridge I:niversity. I:.K. Thomas. .J. 0. & Kornberg, K. D. (1975~). An wtarner ot histones in chromatin and free in solution. Proc. .Vat. Acad. Sci., U.S.A. 72, 2626-2630. Thomas, .J. 0. & Kornberg. R. D. (19756). ('lravablr cross-links in the analysis of histone+histone assoc~iations. FEBS Letters, 58, 353-358. Uberbacher, E. (1. & Bunick. G. ,J. (198%). X-ray xtruv ture of t’hr nucleosome core particle. ./. Hiomol. Struct. I~ynam. 2, 1033-1055. Uberbacher. E. C. h Bunick. 0. TJ. (19856). Science. 229, 1112~1113. IJberbacher, E. C. & Bunick. C. .J. (1989). Structure of the nueleosome core particle a.t 8 A resolution. J. Hirumot. A’truct. f)ynam. 7. I-18. IJberbacher. E. (‘.. Harp, J. 11.. Wilkinson-Singlry. E. & Bunick. C. ,J. (1986). Shape analysis of the histonr octamer in solution. #ciunce, 232, 1247-1249. van Holde, K. E. (1989). Phromatin. Springer-Verlag, New York. Walter. J.. Steigemann, W.. Singh, T. P.. Bartunik. H., Bode. W. & Huber. R. (1982). On the disordered activation domain in trypsinogen: chemical labeling and low-temperature crystallography. .4 otn Crystalloyr.

sect. B, 38, 1462~-1472.

Widom, .J. (1989). Toward a unified model of rhromatin folding. Annu. Rev. Niophys. Bioen,g. 18. 365-395. Widom, ,J. & Klug, A. (1985). Structure of the X10 A chromatin filament: X-ray diffraction from oriented samples. Cell, 43, 207-213. Widom. J. & Lowary, P. T. (1989). Higher-order structure of Saccharomyces cerevisiae chromatin. f’roc. .Vat. Acad. Sri..

l’.S’.A.

86, 8266-8270.

Wood, M. ,J.. Yau, P.. Imai. B. S., (Goldberg, M. W.. Lambart. S. ,I., Fowler. A. (i.. Baldwin, .I. I-‘.. Godfrey, .J. E.. Moudrianakis, E. N., Koch, M. H. .I.. Ibel. K.. May, R. I’. &, Bradbury, E. M. (1991). Neutron and X-ray scatter studies of the histone ortamer and amino and carboxyl domain trimmed octamcrs. .J. Biol. Chem. 266, 5696-5702. Wu, R. S., Panusz, H. T.. Hatch, C. L. & Bonner. W. M. (1986). Histones and their modifications (‘K(: (‘rit. Rev.

Biochem.

20,

201.-263.

Yager, T. T).. McMurray, (‘. T. & van Holde, K. E. (1985). Salt)-induced release of DNA from nucleosomr (tore particles. Biochemistry, 28, 2271--2281.

by R. Huber

The X-ray structure of the octameric histone core of the nucleosome (Burlingame al., 1985) has been redetermined by Arents. G., Burlingame, R. W., Wang, B.-C., Love, W. E. & Moudrianakis, E. N. (1991). Proc. Nat. Acad. Sci., lJ.S.A. 88, 10148-10152, and has been found in agreement with the original shape and size reported from structural studies on the nucleosome core particle (Klug et al., 1980; Richmond et al., 1984). Note added in proof

et

Comparison of X-ray structures of the nucleosome core particle in two different hydration states.

The X-ray structure of the nucleosome core particle was determined at 7 A resolution using crystals containing mixed-sequence DNA and 21% to 27% of 1,...
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