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データを開く
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基本情報
登録情報 | データベース: PDB / ID: 6hbc | ||||||
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タイトル | Structure of the repeat unit in the network formed by CcmM and Rubisco from Synechococcus elongatus | ||||||
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![]() | PROTEIN BINDING / CcmM / M58 / M35 / SSUL domain / Rubisco / Carboxysome | ||||||
機能・相同性 | ![]() structural constituent of carboxysome shell / carboxysome / photorespiration / ribulose-bisphosphate carboxylase / carbon fixation / ribulose-bisphosphate carboxylase activity / reductive pentose-phosphate cycle / photosynthesis / monooxygenase activity / magnesium 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: 2.78 Å | ||||||
![]() | Wang, H. / Yan, X. / Aigner, H. / Bracher, A. / Nguyen, N.D. / Hee, W.Y. / Long, B.M. / Price, G.D. / Hartl, F.U. / Hayer-Hartl, M. | ||||||
![]() | ![]() タイトル: Rubisco condensate formation by CcmM in β-carboxysome biogenesis. 著者: H Wang / X Yan / H Aigner / A Bracher / N D Nguyen / W Y Hee / B M Long / G D Price / F U Hartl / M Hayer-Hartl / ![]() ![]() 要旨: Cells use compartmentalization of enzymes as a strategy to regulate metabolic pathways and increase their efficiency. The α- and β-carboxysomes of cyanobacteria contain ribulose-1,5-bisphosphate ...Cells use compartmentalization of enzymes as a strategy to regulate metabolic pathways and increase their efficiency. The α- and β-carboxysomes of cyanobacteria contain ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-a complex of eight large (RbcL) and eight small (RbcS) subunits-and carbonic anhydrase. As HCO can diffuse through the proteinaceous carboxysome shell but CO cannot, carbonic anhydrase generates high concentrations of CO for carbon fixation by Rubisco. The shell also prevents access to reducing agents, generating an oxidizing environment. The formation of β-carboxysomes involves the aggregation of Rubisco by the protein CcmM, which exists in two forms: full-length CcmM (M58 in Synechococcus elongatus PCC7942), which contains a carbonic anhydrase-like domain followed by three Rubisco small subunit-like (SSUL) modules connected by flexible linkers; and M35, which lacks the carbonic anhydrase-like domain. It has long been speculated that the SSUL modules interact with Rubisco by replacing RbcS. Here we have reconstituted the Rubisco-CcmM complex and solved its structure. Contrary to expectation, the SSUL modules do not replace RbcS, but bind close to the equatorial region of Rubisco between RbcL dimers, linking Rubisco molecules and inducing phase separation into a liquid-like matrix. Disulfide bond formation in SSUL increases the network flexibility and is required for carboxysome function in vivo. Notably, the formation of the liquid-like condensate of Rubisco is mediated by dynamic interactions with the SSUL domains, rather than by low-complexity sequences, which typically mediate liquid-liquid phase separation in eukaryotes. Indeed, within the pyrenoids of eukaryotic algae, the functional homologues of carboxysomes, Rubisco adopts a liquid-like state by interacting with the intrinsically disordered protein EPYC1. Understanding carboxysome biogenesis will be important for efforts to engineer CO-concentrating mechanisms in plants. | ||||||
履歴 |
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構造の表示
ムービー |
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構造ビューア | 分子: ![]() ![]() |
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PDBx/mmCIF形式 | ![]() | 237.3 KB | 表示 | ![]() |
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PDB形式 | ![]() | 189.8 KB | 表示 | ![]() |
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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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1 | ![]()
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要素
#1: タンパク質 | 分子量: 10550.686 Da / 分子数: 1 / 断片: SSUL domain 1 / 由来タイプ: 組換発現 由来: (組換発現) ![]() 遺伝子: ccmM, Synpcc7942_1423 / プラスミド: pHue / 発現宿主: ![]() ![]() | ||
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#2: タンパク質 | 分子量: 52516.605 Da / 分子数: 2 / 断片: Rubisco large subunit / 由来タイプ: 組換発現 由来: (組換発現) ![]() 遺伝子: cbbL, rbcL, Synpcc7942_1426 / プラスミド: pET11a / 発現宿主: ![]() ![]() #3: タンパク質 | 分子量: 13349.196 Da / 分子数: 2 / 断片: Rubisco small subunit / 由来タイプ: 組換発現 由来: (組換発現) ![]() 遺伝子: Synpcc7942_1427 / 発現宿主: ![]() ![]() |
-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: Repeat unit in the CcmM-Rubisco network consisting of a SSUL domain from CcmM and each two RbcL and two RbcS chains from Rubisco タイプ: COMPLEX 詳細: CcmM contains a succession of three highly similar SSUL domains (residues 225-313, 340-428 and 455-539, respectively), which bind to cleft on the surface of Rubisco. Rubisco is a hexadecamer ...詳細: CcmM contains a succession of three highly similar SSUL domains (residues 225-313, 340-428 and 455-539, respectively), which bind to cleft on the surface of Rubisco. Rubisco is a hexadecamer of eight RbcL and eight RbcS subunits. The complex has D4 symmetry. The SSUL-RbcL2-RbcS2 repeat units can have one of two orientations (up or down). Thus Rubisco complexes saturated with SSUL domains can have four different configurations (uuuu, uuud, uudd, udud). In reality, some SSUL binding sites are probably left unoccupied. The network is formed by flexible linkers connecting the SSUL domains in CcmM, which then interlink Rubisco hexadecamers. Entity ID: all / 由来: RECOMBINANT | |||||||||||||||||||||||||
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分子量 | 実験値: NO | |||||||||||||||||||||||||
由来(天然) | 生物種: ![]() Organelle: Carboxysome | |||||||||||||||||||||||||
由来(組換発現) | 生物種: ![]() ![]() | |||||||||||||||||||||||||
緩衝液 | pH: 8 / 詳細: Solutions were made fresh | |||||||||||||||||||||||||
緩衝液成分 |
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試料 | 濃度: 5 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: This sample contained 10 nm gold beads and was not monodisperse | |||||||||||||||||||||||||
試料支持 | グリッドの材料: COPPER / グリッドのサイズ: 300 divisions/in. / グリッドのタイプ: Quantifoil R2/1 | |||||||||||||||||||||||||
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 90 % / 凍結前の試料温度: 298 K / 詳細: blot for 3 seconds before plunging |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD |
撮影 | 平均露光時間: 0.15 sec. / 電子線照射量: 1.05 e/Å2 フィルム・検出器のモデル: GATAN K2 QUANTUM (4k x 4k) |
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解析
EMソフトウェア |
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CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||
対称性 | 点対称性: C1 (非対称) | ||||||||||||
3次元再構成 | 解像度: 2.78 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 78916 / 対称性のタイプ: POINT | ||||||||||||
原子モデル構築 | プロトコル: AB INITIO MODEL / 空間: RECIPROCAL / Target criteria: Average Fourier shell correlation 詳細: The cryoEM density for the repeat unit was masked by Refmac to the coordinates and converted into structure factors by Refmac. The model was adjusted with coot. This model was submitted to ...詳細: The cryoEM density for the repeat unit was masked by Refmac to the coordinates and converted into structure factors by Refmac. The model was adjusted with coot. This model was submitted to restrained refinement with Refmac against the structure factors. |