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基本情報
登録情報 | データベース: PDB / ID: 2vdc | ||||||
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タイトル | THE 9.5 A RESOLUTION STRUCTURE OF GLUTAMATE SYNTHASE FROM CRYO-ELECTRON MICROSCOPY AND ITS OLIGOMERIZATION BEHAVIOR IN SOLUTION: FUNCTIONAL IMPLICATIONS. | ||||||
![]() | (GLUTAMATE SYNTHASE [NADPH] ...) x 2 | ||||||
![]() | OXIDOREDUCTASE / AMIDOTRANSFERASE / AMMONIA ASSIMILATION / NADP / IRON / ZYMOGEN / NADPH-DEPENDENT GLUTAMATE SYNTHASE / IRON SULPHUR FLAVOPROTEIN / GLUTAMINE AMIDOTRANSFERASE / GLUTAMATE BIOSYNTHESIS / AMINO-ACID BIOSYNTHESIS | ||||||
機能・相同性 | ![]() glutamate synthase (NADPH) / glutamate synthase (NADPH) activity / glutamate synthase (NADH) activity / L-glutamate biosynthetic process / ammonia assimilation cycle / 3 iron, 4 sulfur cluster binding / 4 iron, 4 sulfur cluster binding / metal ion binding 類似検索 - 分子機能 | ||||||
生物種 | ![]() | ||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 9.5 Å | ||||||
![]() | Cottevieille, M. / Larquet, E. / Jonic, S. / Petoukhov, M.V. / Caprini, G. / Paravisi, S. / Svergun, D.I. / Vanoni, M.A. / Boisset, N. | ||||||
![]() | ![]() タイトル: The subnanometer resolution structure of the glutamate synthase 1.2-MDa hexamer by cryoelectron microscopy and its oligomerization behavior in solution: functional implications. 著者: Magali Cottevieille / Eric Larquet / Slavica Jonic / Maxim V Petoukhov / Gianluca Caprini / Stefano Paravisi / Dmitri I Svergun / Maria A Vanoni / Nicolas Boisset / ![]() 要旨: The three-dimensional structure of the hexameric (alphabeta)(6) 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, ...The three-dimensional structure of the hexameric (alphabeta)(6) 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, small angle x-ray scattering, and molecular modeling, providing for the first time a molecular model of this complex iron-sulfur flavoprotein. In the hexameric species, interprotomeric alpha-alpha and alpha-beta contacts are mediated by the C-terminal domain of the alpha subunit, which is based on a beta helical fold so far unique to glutamate synthases. The alphabeta protomer extracted from the hexameric model is fully consistent with it being the minimal catalytically active form of the enzyme. The structure clarifies the electron transfer pathway from the FAD cofactor on the beta subunit, to the FMN on the alpha subunit, through the low potential [4Fe-4S](1+/2+) centers on the beta subunit and the [3Fe-4S](0/1+) cluster on the alpha subunit. The (alphabeta)(6) hexamer exhibits a concentration-dependent equilibrium with alphabeta monomers and (alphabeta)(2) dimers, in solution, the hexamer being destabilized by high ionic strength and, to a lower extent, by the reaction product NADP(+). Hexamerization seems to decrease the catalytic efficiency of the alphabeta protomer only 3-fold by increasing the K(m) values measured for l-Gln and 2-OG. However, it cannot be ruled out that the (alphabeta)(6) hexamer acts as a scaffold for the assembly of multienzymatic complexes of nitrogen metabolism or that it provides a means to regulate the activity of the enzyme through an as yet unknown ligand. | ||||||
履歴 |
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Remark 700 | SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AJ" IN EACH CHAIN ON SHEET RECORDS BELOW ... SHEET DETERMINATION METHOD: DSSP THE SHEETS PRESENTED AS "AJ" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 8-STRANDED BARREL THIS IS REPRESENTED BY A 9-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "BH" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 8-STRANDED BARREL THIS IS REPRESENTED BY A 9-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "CJ" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 7-STRANDED BARREL THIS IS REPRESENTED BY A 8-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "DH" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 7-STRANDED BARREL THIS IS REPRESENTED BY A 8-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "EJ" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 7-STRANDED BARREL THIS IS REPRESENTED BY A 8-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEETS PRESENTED AS "FH" IN EACH CHAIN ON SHEET RECORDS BELOW IS ACTUALLY AN 7-STRANDED BARREL THIS IS REPRESENTED BY A 8-STRANDED SHEET IN WHICH THE FIRST AND LAST STRANDS ARE IDENTICAL. THE SHEET STRUCTURE OF THIS MOLECULE IS BIFURCATED. IN ORDER TO REPRESENT THIS FEATURE IN THE SHEET RECORDS BELOW, TWO SHEETS ARE DEFINED. |
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構造の表示
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構造ビューア | 分子: ![]() ![]() |
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PDBx/mmCIF形式 | ![]() | 2 MB | 表示 | ![]() |
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PDB形式 | ![]() | 1.6 MB | 表示 | ![]() |
PDBx/mmJSON形式 | ![]() | ツリー表示 | ![]() | |
その他 | ![]() |
-検証レポート
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
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-関連構造データ
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リンク
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集合体
登録構造単位 | ![]()
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要素
-GLUTAMATE SYNTHASE [NADPH] ... , 2種, 12分子 ABCDEFGHIJKL
#1: タンパク質 | 分子量: 161615.875 Da / 分子数: 6 / 断片: RESIDUES 37-1508, ALPHA SUBUNIT / 由来タイプ: 組換発現 由来: (組換発現) ![]() 株: SP7 / プラスミド: PET11A / 発現宿主: ![]() ![]() #2: タンパク質 | 分子量: 49367.531 Da / 分子数: 6 / 断片: RESIDUES 27-482, BETA SUBUNIT / 由来タイプ: 組換発現 由来: (組換発現) ![]() 株: SP7 / プラスミド: PET11A / 発現宿主: ![]() ![]() |
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-非ポリマー , 6種, 42分子 ![](data/chem/img/OMT.gif)
![](data/chem/img/FMN.gif)
![](data/chem/img/AKG.gif)
![](data/chem/img/F3S.gif)
![](data/chem/img/SF4.gif)
![](data/chem/img/FAD.gif)
![](data/chem/img/FMN.gif)
![](data/chem/img/AKG.gif)
![](data/chem/img/F3S.gif)
![](data/chem/img/SF4.gif)
![](data/chem/img/FAD.gif)
#3: 化合物 | ChemComp-OMT / #4: 化合物 | ChemComp-FMN / #5: 化合物 | ChemComp-AKG / #6: 化合物 | ChemComp-F3S / #7: 化合物 | ChemComp-SF4 / #8: 化合物 | ChemComp-FAD / |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
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試料調製
構成要素 | 名称: AZOSPIRILLUM BRASILENSE GLUTAMATE SYNTHASE / タイプ: COMPLEX |
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緩衝液 | 名称: 25 MM HEPES/KOH, 1 MM EDTA, 1 MM DTT / pH: 7.5 / 詳細: 25 MM HEPES/KOH, 1 MM EDTA, 1 MM DTT |
試料 | 濃度: 9.25 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES |
試料支持 | 詳細: HOLEY CARBON |
急速凍結 | 装置: HOMEMADE PLUNGER / 凍結剤: ETHANE / 詳細: NLIQUID ETHANE |
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電子顕微鏡撮影
顕微鏡 | モデル: JEOL 2010UHR / 日付: 2005年5月10日 詳細: CC =1.1 MM, FOCAL -LEN-1.9 M. MICROGRAPH WITH ANISOTROPIC OR NO DIFFRACTION RINGS WERE REMOVED USING THE ENHANCED POWER SPECTRA SORTING METHOD (JONIC S,SORZANO CO,COTTEVIEILLE M, LARQUET E, ...詳細: CC =1.1 MM, FOCAL -LEN-1.9 M. MICROGRAPH WITH ANISOTROPIC OR NO DIFFRACTION RINGS WERE REMOVED USING THE ENHANCED POWER SPECTRA SORTING METHOD (JONIC S,SORZANO CO,COTTEVIEILLE M, LARQUET E, BOISSET N.,A NOVEL METHOD FOR IMPROVEMENT OF VISUALIZATION OF POWER SPECTRA FOR SORTING CRYO-ELECTRON MICROGRAPHS AND THEIR LOCAL AREAS. J STRUCT BIOL. 2007, 157(1),156-67.) |
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電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 50000 X / 最大 デフォーカス(公称値): 3200 nm / 最小 デフォーカス(公称値): 1700 nm / Cs: 0.5 mm |
試料ホルダ | 温度: 93.15 K / 傾斜角・最大: 0 ° / 傾斜角・最小: 0 ° |
撮影 | 電子線照射量: 10 e/Å2 / フィルム・検出器のモデル: KODAK SO-163 FILM |
画像スキャン | デジタル画像の数: 151 |
放射波長 | 相対比: 1 |
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解析
EMソフトウェア |
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CTF補正 | 詳細: WIENER FILTERING OF VOLUMES FROM FOCAL SERIES | ||||||||||||
対称性 | 点対称性: I (正20面体型対称) | ||||||||||||
3次元再構成 | 手法: RANDOM CONICAL TILT SERIES (CRYOEM) AND REFINEMENT BY PROJECTION MATCHING AND CTF CORRECTION USING WIENER FILTERING OF VOLUMES FROM FOCAL SERIES 解像度: 9.5 Å / 粒子像の数: 12800 / ピクセルサイズ(公称値): 1.59 Å 詳細: THE CHAINS A,B,C,D,E,F OF THE COMPLEX CORRESPOND TO THE ALPHA SUBUNITS, SOLVED BY X-RAY (PDB CODE 1EA0). THREE DISORDERED REGIONS OF THIS X-RAY STRUCTURE (CHAINS A,B,C,D,E, F), IE RESIDUES ...詳細: THE CHAINS A,B,C,D,E,F OF THE COMPLEX CORRESPOND TO THE ALPHA SUBUNITS, SOLVED BY X-RAY (PDB CODE 1EA0). THREE DISORDERED REGIONS OF THIS X-RAY STRUCTURE (CHAINS A,B,C,D,E, F), IE RESIDUES 305-307, 1172-1179 AND 1194-1202 WERE MODELLED WITH MODELLER. THE CHAINS G,H,I,J,K,L CORRESPOND TO THE HOMOLOGY MODEL OF THE BETA SUBUNIT (GENERATED BY MODELLER). 対称性のタイプ: POINT | ||||||||||||
原子モデル構築 | プロトコル: FLEXIBLE FIT / 空間: REAL 詳細: METHOD--REAL-SPACE LOCAL OPTIMIZATION REFINEMENT PROTOCOL--X-RAY AND HOMOLOGY MODELLING | ||||||||||||
原子モデル構築 | PDB-ID: 2VDC Accession code: 2VDC / Source name: PDB / タイプ: experimental model | ||||||||||||
精密化 | 最高解像度: 9.5 Å | ||||||||||||
精密化ステップ | サイクル: LAST / 最高解像度: 9.5 Å
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