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X-ray crystallography is the most powerful tool for obtaining structural information about protein molecules, affording accurate and precise positions for all of the atoms in the protein except for hydrogen. However, hydrogen species play crucial roles in the physiological functions of enzymes, including molecular recognition through hydrogen bonding and catalytic reactions involving proton transfer. Neutron crystallography enables direct identification of the positions of hydrogen species. [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F is an enzyme that catalyzes the reversible oxidation of molecular hydrogen. It contains a bimetallic Ni–Fe active site for the catalytic reaction and three Fe–S clusters for electron transfer. Previous X-ray structure analyses of the enzyme under various oxidation conditions have revealed that the active site changes its coordination structure depending on the redox state. In the inactive air-oxidized form, an oxygen species was identified between the Ni and Fe atoms, whereas in the active H2-reduced form, subatomic-resolution X-ray structure analysis and single-crystal EPR analyses indicated a hydride ligand between the two metal atoms. However, the assignment of the hydride moiety by X-ray crystallography remains controversial, and the proton transfer pathways in the molecule are still ambiguous. To allow neutron diffraction experiments, large crystals of [NiFe]-hydrogenase were prepared by the vapor diffusion method with the macroseeding technique according to the two-dimensional phase diagram (protein concentration vs. precipitant concentration). Neutron diffraction data were collected at approximately 2.0 A resolution at cryogenic temperature using a gas-stream cooling system to trap short-lived intermediates in the catalytic reaction.

X-ray crystallography is the most powerful tool for obtaining structural information about protein molecules, affording accurate and precise positions for all of the atoms in the protein except for hydrogen. However, hydrogen species play crucial roles in the physiological functions of enzymes, including molecular recognition through hydrogen bonding and catalytic reactions involving proton transfer. Neutron crystallography enables direct identification of the positions of hydrogen spe-cies. [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F is an enzyme that catalyzes the reversible oxidation of molecular hydrogen. It contains a bimetallic Ni–Fe active site for the catalytic reaction and three Fe–S clusters for electron transfer. Previous X-ray structure analyses of the enzyme under various oxidation conditions have revealed that the active site changes its coordination structure depending on the redox state. In the inactive air-oxidized form, an oxygen species was identified between the Ni and Fe atoms, whereas in the active H2-reduced form, subatomic-resolution X-ray structure analysis and single-crystal EPR analyses indicated a hydride ligand between the two metal atoms. However, the assignment of the hydride moiety by X-ray crystallography remains controversial, and the proton transfer pathways in the molecule are still ambiguous. To allow neutron diffraction experiments, large crystals of [NiFe]-hydrogenase were prepared by the vapor diffusion method with the macroseeding technique according to the two-dimensional phase diagram (protein concentration vs. precipitant concentration). Neutron diffraction data were collected at approximately 2.0 Å resolution at cryogenic tem-perature using a gas-stream cooling system to trap short-lived intermediates in the catalytic reaction.

[NiFe]-ヒドロゲナーゼはNi-Fe活性部位、Mg中心、および鉄-硫黄クラスターを有し、水素の合成・分解を両方向に触媒できる。Desulfovibrio vulgaris Miyazaki F株由来[NiFe]-ヒドロゲナーゼのNi-Fe活性部位は４つのシステイン残基由来の硫黄原子により配位された2つの八面体型配位錯体構造を形成しており、酸化型（不活性状態：Ni-AおよびNi-B）ではNi-Fe間には酸素種（OH-イオン）が架橋していると考えられている。また、酸化型結晶中の一部の酵素分子において，Niイオンが本来の位置から欠落していると考えられることを、最近実施したX線結晶構造解析から確認している。酸化型の活性中心の詳細を明らかにするために、本研究では中性子およびX線結晶構造解析を実施した。 NiおよびFe原子の吸収端近傍で収集したX線回折データから、結晶中の約30 %の酵素分子においてNiイオンがNi-Fe活性部位の本来の位置から移動し、新たに平面四角型錯体構造を形成していることを明らかにした。一方で、2 Å分解能で実施した中性子結晶構造解析からは、架橋したOH-／OD-イオンを明瞭に確認できなかった。この結果は、重水環境で結晶化したにもかかわらず大多数がOH-イオンのままで架橋しており、さらにある程度自由に回転できるコンフォメーションを持つことにより、水素原子が持つ負の散乱長密度分布により酸素原子の散乱長密度分布が打ち消されていることを示唆していた。, 第22回日本蛋白質科学会年会

A membrane-bound hydrogenase from Desulfovibrio vulgaris Miyazaki F is a metalloenzyme that contains a binuclear Ni–Fe complex in its active site and mainly catalyzes the oxidation of molecular hydrogen to generate a proton gradient in the bacterium. The active-site Ni–Fe complex of the aerobically purified enzyme shows its inactive oxidized form, which can be reactivated through reduction by hydrogen. Here, in order to understand how the oxidized form is reactivated by hydrogen and further to directly evaluate the bridging of a hydride ligand in the reduced form of the Ni–Fe complex, a neutron structure determination was undertaken on single crystals grown in a hydrogen atmosphere. Cryogenic crystallography is being introduced into the neutron diffraction research field as it enables the trapping of short-lived intermediates and the collection of diffraction data to higher resolution. To optimize the cooling of large crystals under anaerobic conditions, the effects on crystal quality were evaluated by X-rays using two typical methods, the use of a cold nitrogen gas stream and plunge-cooling into liquid nitrogen, and the former was found to be more effective in cooling the crystals uniformly than the latter. Neutron diffraction data for the reactivated enzyme were collected at the Japan Photon Accelerator Research Complex under cryogenic conditions, where the crystal diffracted to a resolution of 2.0 angstroms. A neutron diffraction experiment on the reduced form was carried out at Oak Ridge National Laboratory under cryogenic conditions and showed diffraction peaks to a resolution of 2.4 angstroms.

Cytochrome c oxidase (CcO) reduces O2 to water, coupled with a proton-pumping process. The structure of the O2-reduction site of CcO contains two reducing equivalents, Fea32+ and CuB1+, and suggests that a peroxide-bound state (Fea33+–O−–O−–CuB2+) rather than an O2-bound state (Fea32+–O2) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fea32+–O2, whereas Fea33+–O−–O−–CuB2+ is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Å resolution. A 1.70 Å Fe–O distance of the ferryl center could best be described as Fea34+ = O2−, not as Fea34+–OH−. The distance suggests an ∼800-cm−1 Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fea33+–O−–O−–CuB2+ state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.

Significance DNA damage causes loss of or alterations in genetic information, resulting in cell death or mutations. Ionizing radiations produce local, multiple DNA damage sites called clustered DNA damage. In this study, a complete protocol was established to analyze the damage complexity of clustered DNA damage, wherein damage-containing genomic DNA fragments were selectively concentrated via pulldown, and clustered DNA damage was visualized by atomic force microscopy. It was found that X-rays and Fe ion beams caused clustered DNA damage. Fe ion beams also produced clustered DNA damage with high complexity. Fe ion beam–induced complex DNA double-strand breaks (DSBs) containing one or more base lesion(s) near the DSB end were refractory to repair, implying their lethal effects.

研究用原子炉JRR-3は約10年の運転停止期間を終え、2021年2月に再稼働を果たし、7月から供用運転を再開した。量研はJRR-3炉室に2台の生体高分子用中性子単結晶回折装置（BIX-3,4）を保有している。BIX-3,4は単色中性子を用いた高精度な回折データ収集が可能という特徴を有しており、これまでに計21個の中性子構造がPDBに登録されている（JRR-3運転停止前は全世界の約1/3に相当）。 運転停止期間中に、Si(111)とSi(311)結晶を遠隔で切り替えることができるモノクロメータシステムを導入した。これにより、従来の測定限界であった1.4Å分解能を超えてサブオングストロームの回折データ収集が可能となった。将来的に放射光X線を相補的に用いた超高精度構造解析による量子レベルでの機能解明に供することを目指している。 BIX-3,4は測定可能な格子定数が100Å（3軸）である。この限界を突破することを目指して、BIX-3,4のうち1台を炉室からビームホールに移設し、冷中性子（長波長）を用いることで従来よりも大型の格子結晶（165Å：3軸）からのデータ収集を可能とする計画を進行中である。現在、予算化と並行して計算機シミュレーション等からの性能評価を実施している。 今年度は11月でJRR-3は運転停止となるが、次年度からは4月から12月までの連続運転（1サイクル25日 x 7サイクル）が計画されている。BIX-3,4については、産官学いずれのユーザーにとっても利用しやすい運用を検討中で、本発表においても種々の要望をお聞きし、それを反映したいと考えている。 今後は大強度陽子加速器施設J-PARCに設置された茨城県生命物質構造解析装置（iBIX）と両輪で構造生物学分野における中性子利用の拡大を目指していきたい。, 日本結晶学会令和3年度年会

Radiation-induced local multiple damage to DNA (cluster DNA damage) is thought to be closely related to the biological effects of radiation. We developed a new protocol to visualize DNA damage in genomic DNA extracted from TK6 cells irradiated with a low-dose ionizing radiation by atomic force microscopy (AFM). This was realized by isolating and concentrating the DNA molecules with lesions from the whole irradiated DNA molecules also containing intact ones. We firstly applied the method to Fe ion- and X-irradiated cells. As a result, we observed a clustered DNA damage in X- and Fe irradiated genomic DNA, whereas did not found it in Fenton’s regent-treated DNA. In addition, Fe-ion beams, a high-LET radiation, produced various types of clustered DNA damage with higher complexity than those of X-rays. They also produced multiple clustered DNA damage sites in DNA. Moreover, we found that base damage or cluster damage were repaired by base excision repair (BER) mechanisms, but a flanking DSB (DSB + base damage) was repaired by homologous recombination (HR) or non homologous end joining(NHEJ)., 日本放射線影響学会第64回大会

[NiFe]-ヒドロゲナーゼはNi-Fe活性部位、Mg中心、および鉄-硫黄クラスターを有し、水素の合成・分解を両方向に触媒できる。これまでに行われたDesulfovibrio vulgaris Miyazaki F株由来[NiFe]-ヒドロゲナーゼのX線結晶構造解析から、Ni-Fe活性部位は４つのシステイン残基由来の硫黄原子により配位された2つの八面体型配位錯体構造を形成していることが明らかになった。さらに、酸化型（不活性状態）ではNi-Fe間には酸素種が、還元型（活性状態）ではヒドリドが架橋されていることが、それぞれX線結晶構造解析と単結晶EPR解析から示された。ヒドリドについては、0.89Å分解能のX線結晶構造解析によってもその存在が示唆されている。しかし、X線散乱能が弱い水素についての回折実験結果に基づいていることから信憑性に懸念がある。さらに、酵素触媒反応に関与するプロトンの輸送経路は不明なままである。 現在、酸化型・還元型の双方でNi-Fe間に架橋されている原子種およびその状態、およびプロトン輸送経路を水素原子の直接観察に長けた中性子回折により明らかにすることに取り組んでいる。すでに、酸化型および還元型（複数条件で調製）の大型結晶を作製し、2つの中性子施設（J-PARC/MLFおよび米国SNS）において100K下で回折実験を実施し、いずれも2 Å分解能前後の良質な回折データを収集済みである。現在、同一結晶から取得したＸ線回折データを相補的に用いた構造精密化に取り組んでいる。, 第21回日本蛋白質科学会年会

タンパク質などの生体高分子を測定対象とするBIX-3, BIX-4（原子力機構・JRR-3炉室設置。JRR-3は2021年6月供用再開予定）は、直接観察された水素原子や水和水の構造情報から、タンパク質の機能発現に必須なプロトン化状態の決定や低障壁水素結合の生体高分子における初めての観察などの成果を上げてきた。その有用性にも関わらず、中性子を用いた構造機能研究は構造生物学分野においてまだ限定的であるが、回折装置を高性能化することで試料対象や回折データ測定可能領域を広げることができれば、より普遍的な手法としての今後の発展が大きく期待できる。 そこで、＜1＞BIX-3,4ではモノクロメータ（遮蔽体内に格納）としてSi(111)結晶を用いている（BIX-3,4: 分解能d-min=1.5, 1.4Å）。これをSi(311)結晶に変更することにより短波長中性子を得て、装置仕様分解能としてサブÅレベルまでの高分解能化を実現した（遠隔操作で随時切り替え可能）。＜2＞近年、JRR-3ビームホールが有する冷中性子（長波長）ビームラインが高強度化されている。長波長ビームは大型の単位格子を持つ結晶からのブラッグ反射の分離に有利に働く。そこで、BIX-3,4の一方のビームホール移設を検討しており、得られる中性子強度利得および反射分離能について計算機シミュレーション等から性能評価を行っている。本発表では、これまでの高性能化の実施・評価結果を報告する。, 第21回日本蛋白質科学会年会

The elucidation of the protein-protein interaction, especially among membrane proteins and protein complexes, is one of the most important research fields in life science. Such proteins have large molecular weights, and the lattice lengths of their crystals have large values. Cold neutrons contribute to improve the difficulty in separating Bragg peaks from those crystals. JRR-3 has three cold neutron beamlines in the beam hall facility, in which neutron guides have been recently upgraded to supermirror guides (m=3). The neutron intensity gain by the upgrading has also been estimated. A diffractometer, to be installed at the cold neutron beamline, will be equipped a feature to choose a wavelength appropriate for data collection from crystals with a large unit cell. While BIX-3 and BIX-4 diffractometers with the Neutron Imaging Plate detector are now located in the reactor hall of JRR-3, this detector is more suitable to be used in the beam hall with a lower gamma-ray background. As the effectively usable angular divergence is limited for the single crystal diffraction method, neutron beam within the divergence of 1.0 degree at the cold beamline was simulated by the McStas program. From the calculation the peak wavelength of the spectrum at C1-3 beam port (a candidate port for the installation) was shifted from 0.4 nm to 0.29 nm, and the gain was one order of magnitude at the wavelength of 0.29 nm by the upgrading. The performance of the diffractometer will be discussed., MLZ Conference 2021: Neutrons for Life Sciences

タンパク質などの生体高分子を測定対象とするBIX-3, BIX-4（原子力機構・JRR-3炉室設置。JRR-3は2020年度末運転再開予定）は、これまでにDNAオリゴマーや創薬標的タンパク質など21構造（JRR-3運転停止前は全世界の約1/3に相当）を中性子結晶回折法により明らかにしている。そこで直接観察された水素原子や水和水の構造情報から、タンパク質の機能発現に必須なプロトン化状態の決定や低障壁水素結合の生体高分子における初めての観察などの成果を上げてきた。その有用性にも関わらず、中性子を用いた構造機能研究は構造生物学分野においてまだ限定的であるが、回折装置を高性能化することで試料対象や回折データ測定可能領域を広げることができれば、量子生命分野への適応も含めた今後の発展が大きく期待できる。そこで、①短波長中性子を用いることによる測定可能な回折データ分解能dminの高分解能化を実施するとともに、②長波長中性子ビームラインを利用した高分子量タンパク質（大型単位格子結晶）を測定可能にする回折装置設置の検討を進めている。 ①：BIX-3,4では試料に照射する単色化中性子を得るために、シリコン完全結晶弾性湾曲モノクロメータとしてSi(111)結晶を用いている（BIX-3,4: 分解能dmin=1.5, 1.4Å）。このモノクロメータをSi(311)結晶モノクロメータに変更することで、より短波長な中性子を得る。これによって、測定可能な分解能をサブオングストロームレベルまでの高分解能化を実現した（BIX-3,4: dmin=0.8, 0.7Å）。また、遮蔽体内に格納されたモノクロメータをJRR-3運転中でも随時変更可能にするため、遠隔操作で切り替え可能とした。②：近年、JRR-3ビームホールが有する冷中性子（長波長）ビームラインが高強度化されている。また、ビームホールはバックグラウンドが低い点でも測定データ精度に対し有利である。よって、BIX-3,4の片方をビームホールに移設することを検討している。単結晶回折ではビームラインにおける全ビーム中での有効利用可能な角度分散成分が限定される。そのため、検討装置が利用する長波長ビーム内の角度分散の利得成分を計算機シミュレーションし、高分子量タンパク質（大型単位格子結晶）に対する検討装置の測定性能を評価している。 本発表では、これまでの高性能化の実施・評価結果を報告する。, 量子生命科学会第2回大会

放射線は飛跡に沿って分子を電離することから，照射された細胞では，飛跡と重なるDNA部位に高密度な損傷（クラスターダメージ）が生じると考えられている。しかし，実験的に高密度損傷の存在を実証した研究はなかった。そこで申請者はこれまでに、原子間力顕微鏡（AFM）を用いクラスター損傷を個別に可視化・検出方法を確立し、放射線を照射したプラスミドに生じた損傷の解析を行い、LETの増加によりクラスターダメージを伴う損傷が増加している事を明らかにし、これについて前年度の学会で報告した。その後さらに系を発展させ、AFMによるDNA損傷の可視化分析技術を発展的に展開し、放射線を照射した細胞や腫瘍中のDNAでも検討する方法を確立し、細胞や腫瘍中に生じた塩基損傷、クラスターダメージ、DSB末端に塩基損傷を含む新たなDNA損傷の観察を可能にした。さらに、本発表では、この系を軸に損傷毎の修復速度を解析した。, 量子生命科学会第二回大会

A membrane-bound hydrogenase fromDesulfovibrio vulgarisMiyazaki F is a metalloenzyme that contains a binuclear Ni–Fe complex in its active site and mainly catalyzes the oxidation of molecular hydrogen to generate a proton gradient in the bacterium. The active-site Ni–Fe complex of the aerobically purified enzyme shows its inactive oxidized form, which can be reactivated through reduction by hydrogen. Here, in order to understand how the oxidized form is reactivated by hydrogen and further to directly evaluate the bridging of a hydride ligand in the reduced form of the Ni–Fe complex, a neutron structure determination was undertaken on single crystals grown in a hydrogen atmosphere. Cryogenic crystallography is being introduced into the neutron diffraction research field as it enables the trapping of short-lived intermediates and the collection of diffraction data to higher resolution. To optimize the cooling of large crystals under anaerobic conditions, the effects on crystal quality were evaluated by X-rays using two typical methods, the use of a cold nitrogen-gas stream and plunge-cooling into liquid nitrogen, and the former was found to be more effective in cooling the crystals uniformly than the latter. Neutron diffraction data for the reactivated enzyme were collected at the Japan Photon Accelerator Research Complex under cryogenic conditions, where the crystal diffracted to a resolution of 2.0 Å. A neutron diffraction experiment on the reduced form was carried out at Oak Ridge National Laboratory under cryogenic conditions and showed diffraction peaks to a resolution of 2.4 Å.

T4 phage lysozyme is an inverting glycoside hydrolase that degrades the murein of bacterial cell walls by cleaving the β-1,4-glycosidic bond. The substitution of the catalytic Thr26 residue to a histidine converts the wild type from an inverting to a retaining enzyme, which implies that the original general acid Glu11 can also act as an acid/base catalyst in the hydrolysis. Here, we have determined the neutron structure of the perdeuterated T26H mutant to clarify the protonation states of Glu11 and the substituted His26, which are key in the retaining reaction. The 2.09-A resolution structure shows that the imidazole group of His26 is in its singly protonated form in the active site, suggesting that the deprotonated Nɛ2 atom of His26 can attack the anomeric carbon of bound substrate as a nucleophile. The carboxyl group of Glu11 is partially protonated and interacts with the unusual neutral state of the guanidine moiety of Arg145, as well as two heavy water molecules. Considering that one of the water-binding sites has the potential to be occupied by a hydronium ion, the bulk solvent could be the source for the protonation of Glu11. The respective protonation states of Glu11 and His26 are consistent with the bond lengths determined by an unrestrained refinement of the high-resolution X-ray structure of T26H at 1.04-A resolution. The detail structural information, including the coordinates of the deuterium atoms in the active site, provides insight into the distinctively different catalytic activities of the mutant and wild type enzymes.

Cytochrome c oxidase (CcO) reduces O(2) to water, coupled with a proton-pumping process. The structure of the O(2)-reduction site of CcO contains two reducing equivalents, Fe(a)(3)(2+) and Cu(B)(1+), and suggests that a peroxide-bound state (Fe(a)(3)(3+)–O(−)–O(−)–Cu(B)(2+)) rather than an O(2)-bound state (Fe(a)(3)(2+)–O(2)) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fe(a)(3)(2+)–O(2), whereas Fe(a)(3)(3+)–O(−)–O(−)–Cu(B)(2+) is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Å resolution. A 1.70 Å Fe–O distance of the ferryl center could best be described as Fe(a)(3)(4+) = O(2−), not as Fe(a)(3)(4+)–OH(−). The distance suggests an ∼800-cm(−1) Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fe(a)(3)(3+)–O(−)–O(−)–Cu(B)(2+) state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.

UDP-glucose: anthocyanidin 3-O-glucosyltransferase (UGT78K6) from Clitoria ternatea catalyzes the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin. After the acylation of the 3-O-glucosyl residue, the 3′- and 5′-hydroxyl groups of the product are further glucosylated by a glucosyltransferase in the biosynthesis of ternatins, which are anthocyanin pigments. To understand the acceptor-recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 A, 2.55 A, 2.70 A, and 1.75 A, respectively. The enzyme recognition of unstable anthocyanidin aglycones was initially observed in this structural determination. The anthocyanidin- and flavonol-acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The 3-hydroxyl groups of the acceptor substrates were located at hydrogen-bonding distances to the Ne2 atom of the His17 catalytic residue, supporting a role for glucosyl transfer to the 3-hydroxyl groups of anthocyanidins and flavonols. However, the molecular orientations of these three acceptors are different from those of the known flavonoid glycosyltransferases, VvGT1 and UGT78G1. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180° rotation about the O1–O3 axis of the flavonoid backbones observed in VvGT1 and UGT78G1; consequently, the 5- and 7-hydroxyl groups are protected from glucosylation. These substrate recognition schemes are useful to understand the unique reaction mechanism of UGT78K6 for the ternatin biosynthesis, and suggest the potential for controlled synthesis of natural pigments.

T4 phage lysozyme (T4L) is an endoacetylmuramidase that degrades the murein of the bacterial cell wall by cleaving the β-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine. We previously reported that the substitution of the catalytic Thr26 with the nucleophilic His converts the wild-type (WT) T4L from an inverting to a retaining glycosidase, in which the β-configuration of the substrate is retained in the product. We also found that the Thr26His (T26H) mutant can catalyze a transglycosylation reaction more effectively than hydrolysis, although the WT-T4L has no transglycosidase activity. To clarify the role of the substituted His26 in transglycosylation and to investigate its relationship to the neighboring acidic residue Asp20 using neutron crystallography, a perdeuterated recombinant protein of the T26H mutant (d-T26H) was prepared for crystallization. The perdeuteration would reduce the crystal-size requirement by one order of magnitude and enable better visualization of the protonation and hydration states of the catalytic residues. The perdeuterated form was produced in Escherichia coli cells cultured in deuterated-rich media. After purification, the d-T26H mutant was crystallized under deuterated conditions and grown to a volume of 0.12 mm using a macroseeding technique. A preliminary neutron-diffraction experiment at 100 K at the FRM II research reactor (Munich, Germany) gave diffraction spots of up to 2.8 A resolution after a 1.5 h exposure.