PROTEINS: Structure, Function, and Genetics 24:269-271 (1996) Crystallization and Preliminary X-Ray Characterization of the Methanothermus fervidus Histones HMfA and HMfB Klaas Decanniere,' Kathleen Sandman: John N. Reeve: and Udo Heinemann' 'Forschungsgruppe Kristallographie, Max-Delbruck-Centrumfur Molekulare Medizin, 0-13122 Berlin, Germany; and 2Department of Microbiology, The Ohio State University, Columbus, Ohio 43210 HMfA and HMfB are histone ABSTRACT proteins from the thermophilic archaeon Methanothermus fervidus. They wrap DNA into nucleosome-like structures and appear to represent the basic core histone fold. HMfA was crystallized in space groups P4,2,2 and P2,2,2,. HMfB crystallized in space group P2,2,2, while a selenomethionine-substituted variant, SeMet-HMfB,yielded crystals in C222,. In all crystal forms HMfA, HMfB, or SeMetHMfB may be present as homodimers. 0 1996 Wiley-Liss, Inc. Key words: Archaea, hyperthermophiles, histone fold, X-ray diffraction, anomalous dispersion INTRODUCTION Methanothermus fervidus is a methanogenic archaeon that grows optimally a t 83°C. It contains two small proteins, HMfA and HMfB, which bind double-stranded DNA in ~ i t r o , ' - ~forming structures that resemble eukaryal nucleosomes. They bind to DNA as dimers or tetramers, and both homodimers and HMfA-HMfB heterodimers have been observed to exist in vivo5 The amino acid sequences of HMfA and HMfB differ at only 12 of 68 or 69 positions, respectively. Sequence alignments reveal homologies between HMfMHMfB and the eukaryal core histones HZA, H2B, H3, and H4.l The closest match is observed between HMfB and the consensus sequence for the C-terminal part of H4,, which share 29% of identical and 55% of conserved residues. The similarity to histone H4 is particularly high in three regions that adopt a n a-helical conformation and form the histone-fold motif within the structured core of the histone ~ c t a m e r .HMfA ~ and HMfB therefore appear to represent the basic core histone motif.' Here we report the crystallization and preliminary crystal data for HMfA and HMfB. A variant of HMfB in which residues Met-1 and Met-35 have been replaced by selenomethionine residues (SeMet-HMfB) has also been produced and crystallized. 0 1996 WILEY-LISS, INC. MATERIALS AND METHODS Generation of Selenomethionine Variant, SeMet-HMfB E . coli JM105 was made awotrophic for methionine by P1 transduction of metC162::TnlO (tet') from E . coli NK6027 (obtained from the E . coli Genetic Stock Center), and the resulting strain was transformed with the hmfB expression vector pKS323 All cultures were grown with 20 pg/ml ampicillin and 10 pg/ml tetracycline. An overnight culture of E . coli JM105-metC162::TnlO (pKS323) was grown in M9 medium" supplemented with 5 pg methionine/ml, and a 100 ml starter culture was grown in M9 containing 5% LB and 50 pg D,L-selenomethionine/ml (Sigma, St. Louis, MO) by inoculation with a 100-fold dilution of the overnight culture. After 5 h growth at 37°C the starter culture (OD,,, of 0.3) was diluted into a 10 L fermentor containing M9 and 50 pg selenomethionine/ml. When the culture reached a n OD,,, of 0.2 (-17 h later), isoptopyl P-D-thiogalacto pyranoside (IPTG) was added to 0.4 mM to induce the synthesis of SeMet-HMfB. The culture was harvested 6 h later. The total yields from two 20 L cultures were 11.5 g of E. coli cells (wet weight) and 5 mg of purified SeMet-HMfB. Protein Purification and Characterization Cell growth and purification of HMfA was as described," except that the growth medium was Superbroth." Recombinant HMfB (rHMfB) was purified as describedg except that the heparin sepharose column was equilibrated with 30 mM potassium citrate, 50 mM Tris, pH 8.0, and the rHMfB was eluted with a 30-200 mM gradient of potassium citrate in 50 mM Tris, pH 8.0. Amino acid compositions were determined directly for rHMfB and SeMet-HMfB. In SeMet-HMfB > 99% of the methionyl residues in rHMfB was replaced by selenomethionyl residues. Received June 22, 1995; revision accepted August 28, 1995. Address reprint requests to Udo Heinernann, Forschungsgruppe Kristallographie, Max-Delbruck-Centrum fur Molekulare Medizin, Robert-RGssle-Str. 10, D-13122 Berlin, Germany. 270 K. DECANNIERE ET AL. TABLE I. Crystallization Conditions Reservoir Crystallization time (days) Max. crystal size (mm) HMfA tetragonal 0.1 M Na-acetate, pH 4.6, 2.2 M NaCl HMfA orthorhombic 2.6 M (NH,)2S04 HMfB 0.15 M cacodylate, pH 6.5, 0.3 M Zn-acetate, 17% (w/v) PEG 8000 SeMet-HMfB 0.1 M MES pH 6.5, 0.2 M (NH4),S0,, 30% (wiv) PEG monomethyl ether 5000 2-3 1-2 1-2 7 0.4 X 0.4 0.6 X 0.2 x 0.2 x 0.4 0.3 X 0.5 X 0.7 0.15 x 0.3 x 0.5 TABLE 11. Crystallographic Characterization Space group a (A) b (A) c (A) VM (A3/Da) Protein monomers per asymmetric unit* HMfA tetragonal P4,2,2 41.24 41.24 81.19 2.26 1 HMfA orthorhombic p212121 33.68 52.65 67.22 1.95 2 HMfB P2,2,2 31.24 39.41 61.47 2.47 1 SeMet-HMfB (2222, 29.88 59.94 75.50 2.21 1 *Chosen such as to yield reasonable V, values. Growth and Preliminary Characterization of Crystals All crystallization experiments used the hanging drop vapor diffusion method at room temperature. HMfA and HMfB were concentrated to 10-20 mglml in their buffer solutions (approximately 100 mM potassium citrate, 50 mM Tris-HC1, pH 8.0). Initial crystallization conditions were established by mixing 1 pl of protein solution with 1 pl of precipitant solution and equilibration with the precipitant solution in an extended sparse matrix screen.l3,l4 For X-ray diffraction experiments, crystals were mounted in sealed glass capillaries. In most cases the crystals proved sensitive to changes of their mother liquor and were thus mounted straight from the drop. Diffraction data sets were collected on 180 mm or 300 mm MarResearch (Hamburg, Germany) imaging plate systems. For in-house experiments, CuK, radiation was produced by Rigaku-Denki (Tokyo, Japan) RU H2R direct-drive rotating-anode generators. For use with the small imaging plate, the generator was operated at 44 kV, 110 mA with 0.3 mm focal spot size and graphite monochromator. For use with the large plate, operation conditions were 44 kV, 68 mA with 0.2 mm focus, monochromatization by Ni-coated double-focusing mirrors (Charles Supper, Natick, MA) and filtering through a 17.5 pm Ni foil. Some data sets were collected at beamlines X31 or BW6 of the EMBL outstation a t the Deutsche Elektronensynchrotron (DESY, Hamburg). Primary data were processed with DENZ015 and scaled with the programs SCALA and AGROVATA of the CCP4 program package.16 The Laue symmetry and screw axes were verified by precession photography. RESULTS AND DISCUSSION After refinement of the crystallization conditions found by the initial screen, proteins HMfA, HMfB, and SeMet-HMfB formed single crystals suitable for high-resolution structure analysis (Table I). Crystallographic parameters for the M. fervidus histones are summarized in Table 11. The two different crystallization conditions for HMfA give rise to tetragonal and orthorhombic crystal forms. Unfortunately, SeMet-HMfB does not crystallize isomorphously with HMfB. However, the crystal packing may be similar, since space group P2,2,2 of HMf33 is a subgroup of C222, found for SeMet-HMfB, and lattice parameters a, b, c of the C222, cell are close to a', c', 2b' of the P2,2,2 cell, respectively. Alternatively, c may correspond to the b'ic' diagonal. The packing parameters V , are in the expected range,17 between 1.95and 2.47 A31Da, if a protein dimer is assumed to occupy the asymmetric unit in orthorhombic HMfA and a monomer i n all other crystals. We note that symmetric dimers with crystallographic dyad axes may be present in all crystal forms in which the asymmetric unit contains a protein monomer. X-Ray diffraction data sets were collected either using in-house facilities or at DESY, Hamburg (Table 111).Completeness and Rmergevalues (Rmerge= ZlZ,, 12&, where Zi,jare the measurements contributing to the mean reflection intensity, <Ii>>, for the entire data sets and for the highest shells, indicate reasonable data up to the diffraction limits given. It is hoped that anomalous dispersion measurements using the selenium atoms of SeMet-HMfB will help solve the phase problem for the structure analysis by established procedures.18,19 271 CRYSTALLIZATION OF ARCHAEAL HISTONES TABLE 111. X-Ray Diffraction Data X-ray source Wavelength (A) Detector Whole data set Resolution limit (A) Completeness (%) HMfA tetragonal RU H2B HMfA orthorhombic DESY, BW6 HMfB RU H2B SeMet-HMfB DESY, X31 1.5418 IP 180 mm 1.030 IP 180 mm 1.5418 IP 300 mm 0.877 IP 180 mm 2.1 84.6 7.4 1.9 90.4 6.0 1.9 96.5 5.3 2.1 97.2 8.6 2.15-2.10 59.4 23.7 1.96-1.90 85.4 19.3 1.97-1.90 97.2 19.6 2.17-2.10 90.2 15.8 Rrnerge Highest shell Resolution limits (A) Completeness (%) Rmerze (%) ACKNOWLEDGMENTS X-Ray diffraction experiments were in part performed a t the EMBL Outstation at DESY, Hamburg, with kind help from G. Evans. The help of J.J. Muller and A. Knespel in setting up the X-ray laboratory at the MDC is gratefully acknowledged. We thank Don Ordaz, OSU Fermentation Facility, John Lowbridge, OSU Biochemical Instrumentation Center, and A. Rampersaud for gift of P1 lysates and protocols. 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