+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-40970 | |||||||||
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タイトル | DSBU crosslinked nNOS-CaM oxygenase homodimer | |||||||||
マップデータ | nNOS-CaM-DSBU map | |||||||||
試料 |
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キーワード | Complex / CYTOSOLIC PROTEIN | |||||||||
機能・相同性 | 機能・相同性情報 Nitric oxide stimulates guanylate cyclase / negative regulation of hepatic stellate cell contraction / positive regulation of adenylate cyclase-activating adrenergic receptor signaling pathway / retrograde trans-synaptic signaling by nitric oxide / synaptic signaling by nitric oxide / negative regulation of iron ion transmembrane transport / ROS and RNS production in phagocytes / azurophil granule / negative regulation of vasoconstriction / positive regulation of sodium ion transmembrane transport ...Nitric oxide stimulates guanylate cyclase / negative regulation of hepatic stellate cell contraction / positive regulation of adenylate cyclase-activating adrenergic receptor signaling pathway / retrograde trans-synaptic signaling by nitric oxide / synaptic signaling by nitric oxide / negative regulation of iron ion transmembrane transport / ROS and RNS production in phagocytes / azurophil granule / negative regulation of vasoconstriction / positive regulation of sodium ion transmembrane transport / response to nitric oxide / nitric oxide metabolic process / postsynaptic specialization, intracellular component / positive regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / Ion homeostasis / negative regulation of cytosolic calcium ion concentration / peptidyl-cysteine S-nitrosylation / cadmium ion binding / positive regulation of the force of heart contraction / negative regulation of potassium ion transport / negative regulation of calcium ion transport / calyx of Held / behavioral response to cocaine / negative regulation of serotonin uptake / regulation of neurogenesis / nitric-oxide synthase (NADPH) / sodium channel regulator activity / response to vitamin E / regulation of postsynaptic membrane potential / postsynaptic density, intracellular component / negative regulation of insulin secretion / nitric oxide mediated signal transduction / nitric-oxide synthase activity / multicellular organismal response to stress / xenobiotic catabolic process / arginine catabolic process / NADPH binding / striated muscle contraction / regulation of sodium ion transport / nitric oxide-cGMP-mediated signaling / sarcoplasmic reticulum membrane / T-tubule / cellular response to epinephrine stimulus / : / nitric oxide biosynthetic process / negative regulation of blood pressure / photoreceptor inner segment / response to nutrient levels / response to hormone / sarcoplasmic reticulum / secretory granule / response to activity / positive regulation of long-term synaptic potentiation / establishment of localization in cell / cell periphery / female pregnancy / phosphoprotein binding / response to lead ion / response to nicotine / potassium ion transport / establishment of protein localization / response to organic cyclic compound / sarcolemma / cellular response to growth factor stimulus / response to peptide hormone / Z disc / cellular response to mechanical stimulus / response to estrogen / vasodilation / calcium-dependent protein binding / calcium ion transport / FMN binding / positive regulation of peptidyl-serine phosphorylation / NADP binding / flavin adenine dinucleotide binding / ATPase binding / response to heat / scaffold protein binding / nuclear membrane / response to ethanol / negative regulation of neuron apoptotic process / transmembrane transporter binding / mitochondrial outer membrane / response to lipopolysaccharide / dendritic spine / postsynaptic density / cytoskeleton / response to hypoxia / calmodulin binding / membrane raft / negative regulation of cell population proliferation / glutamatergic synapse / synapse / dendrite / heme binding / negative regulation of apoptotic process / perinuclear region of cytoplasm / positive regulation of DNA-templated transcription / enzyme binding / positive regulation of transcription by RNA polymerase II 類似検索 - 分子機能 | |||||||||
生物種 | Rattus norvegicus (ドブネズミ) | |||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.14 Å | |||||||||
データ登録者 | Lee K / Pospiech TH / Southworth D | |||||||||
資金援助 | 米国, 1件
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引用 | ジャーナル: J Biol Chem / 年: 2024 タイトル: Mapping interactions of calmodulin and neuronal NO synthase by crosslinking and mass spectrometry. 著者: Dana Felker / Kanghyun Lee / Thomas H Pospiech / Yoshihiro Morishima / Haoming Zhang / Miranda Lau / Daniel R Southworth / Yoichi Osawa / 要旨: Neuronal nitric oxide synthase (nNOS) is a homodimeric cytochrome P450-like enzyme that catalyzes the conversion of L-arginine to nitric oxide in the presence of NADPH and molecular oxygen. The ...Neuronal nitric oxide synthase (nNOS) is a homodimeric cytochrome P450-like enzyme that catalyzes the conversion of L-arginine to nitric oxide in the presence of NADPH and molecular oxygen. The binding of calmodulin (CaM) to a linker region between the FAD/FMN-containing reductase domain, and the heme-containing oxygenase domain is needed for electron transfer reactions, reduction of the heme, and NO synthesis. Due to the dynamic nature of the reductase domain and low resolution of available full-length structures, the exact conformation of the CaM-bound active complex during heme reduction is still unresolved. Interestingly, hydrogen-deuterium exchange and mass spectrometry studies revealed interactions of the FMN domain and CaM with the oxygenase domain for iNOS, but not nNOS. This finding prompted us to utilize covalent crosslinking and mass spectrometry to clarify interactions of CaM with nNOS. Specifically, MS-cleavable bifunctional crosslinker disuccinimidyl dibutyric urea was used to identify thirteen unique crosslinks between CaM and nNOS as well as 61 crosslinks within the nNOS. The crosslinks provided evidence for CaM interaction with the oxygenase and reductase domain residues as well as interactions of the FMN domain with the oxygenase dimer. Cryo-EM studies, which gave a high-resolution model of the oxygenase domain, along with crosslink-guided docking provided a model of nNOS that brings the FMN within 15 Å of the heme in support for a more compact conformation than previously observed. These studies also point to the utility of covalent crosslinking and mass spectrometry in capturing transient dynamic conformations that may not be captured by hydrogen-deuterium exchange and mass spectrometry experiments. | |||||||||
履歴 |
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-構造の表示
添付画像 |
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-ダウンロードとリンク
-EMDBアーカイブ
マップデータ | emd_40970.map.gz | 141.4 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-40970-v30.xml emd-40970.xml | 16.1 KB 16.1 KB | 表示 表示 | EMDBヘッダ |
画像 | emd_40970.png | 92.4 KB | ||
Filedesc metadata | emd-40970.cif.gz | 6.3 KB | ||
その他 | emd_40970_half_map_1.map.gz emd_40970_half_map_2.map.gz | 139.2 MB 139.2 MB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-40970 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-40970 | HTTPS FTP |
-関連構造データ
関連構造データ | 8t1kMC 8t1jC M: このマップから作成された原子モデル C: 同じ文献を引用 (文献) |
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類似構造データ | 類似検索 - 機能・相同性F&H 検索 |
-リンク
EMDBのページ | EMDB (EBI/PDBe) / EMDataResource |
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「今月の分子」の関連する項目 |
-マップ
ファイル | ダウンロード / ファイル: emd_40970.map.gz / 形式: CCP4 / 大きさ: 149.9 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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注釈 | nNOS-CaM-DSBU map | ||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 0.912 Å | ||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||
詳細 | EMDB XML:
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-添付データ
-ハーフマップ: nNOS-CaM-DSBU half map A
ファイル | emd_40970_half_map_1.map | ||||||||||||
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注釈 | nNOS-CaM-DSBU half map A | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: nNOS-CaM-DSBU half map B
ファイル | emd_40970_half_map_2.map | ||||||||||||
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注釈 | nNOS-CaM-DSBU half map B | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-試料の構成要素
-全体 : DSBU crosslinked CaM-bound nNOS homodimer
全体 | 名称: DSBU crosslinked CaM-bound nNOS homodimer |
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要素 |
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-超分子 #1: DSBU crosslinked CaM-bound nNOS homodimer
超分子 | 名称: DSBU crosslinked CaM-bound nNOS homodimer / タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1 |
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由来(天然) | 生物種: Rattus norvegicus (ドブネズミ) |
-分子 #1: Nitric oxide synthase 1
分子 | 名称: Nitric oxide synthase 1 / タイプ: protein_or_peptide / ID: 1 / コピー数: 2 / 光学異性体: LEVO / EC番号: nitric-oxide synthase (NADPH) |
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由来(天然) | 生物種: Rattus norvegicus (ドブネズミ) |
分子量 | 理論値: 160.769562 KDa |
組換発現 | 生物種: Escherichia coli (大腸菌) |
配列 | 文字列: MEENTFGVQQ IQPNVISVRL FKRKVGGLGF LVKERVSKPP VIISDLIRGG AAEQSGLIQA GDIILAVNDR PLVDLSYDSA LEVLRGIAS ETHVVLILRG PEGFTTHLET TFTGDGTPKT IRVTQPLGPP TKAVDLSHQP SASKDQSLAV DRVTGLGNGP Q HAQGHGQG ...文字列: MEENTFGVQQ IQPNVISVRL FKRKVGGLGF LVKERVSKPP VIISDLIRGG AAEQSGLIQA GDIILAVNDR PLVDLSYDSA LEVLRGIAS ETHVVLILRG PEGFTTHLET TFTGDGTPKT IRVTQPLGPP TKAVDLSHQP SASKDQSLAV DRVTGLGNGP Q HAQGHGQG AGSVSQANGV AIDPTMKSTK ANLQDIGEHD ELLKEIEPVL SILNSGSKAT NRGGPAKAEM KDTGIQVDRD LD GKSHKAP PLGGDNDRVF NDLWGKDNVP VILNNPYSEK EQSPTSGKQS PTKNGSPSRC PRFLKVKNWE TDVVLTDTLH LKS TLETGC TEHICMGSIM LPSQHTRKPE DVRTKDQLFP LAKEFLDQYY SSIKRFGSKA HMDRLEEVNK EIESTSTYQL KDTE LIYGA KHAWRNASRC VGRIQWSKLQ VFDARDCTTA HGMFNYICNH VKYATNKGNL RSAITIFPQR TDGKHDFRVW NSQLI RYAG YKQPDGSTLG DPANVQFTEI CIQQGWKAPR GRFDVLPLLL QANGNDPELF QIPPELVLEV PIRHPKFDWF KDLGLK WYG LPAVSNMLLE IGGLEFSACP FSGWYMGTEI GVRDYCDNSR YNILEEVAKK MDLDMRKTSS LWKDQALVEI NIAVLYS FQ SDKVTIVDHH SATESFIKHM ENEYRCRGGC PADWVWIVPP MSGSITPVFH QEMLNYRLTP SFEYQPDPWN THVWKGTN G TPTKRRAIGF KKLAEAVKFS AKLMGQAMAK RVKATILYAT ETGKSQAYAK TLCEIFKHAF DAKAMSMEEY DIVHLEHEA LVLVVTSTFG NGDPPENGEK FGCALMEMRH PNSVQEERKS YKVRFNSVSS YSDSRKSSGD GPDLRDNFES TGPLANVRFS VFGLGSRAY PHFCAFGHAV DTLLEELGGE RILKMREGDE LCGQEEAFRT WAKKVFKAAC DVFCVGDDVN IEKPNNSLIS N DRSWKRNK FRLTYVAEAP DLTQGLSNVH KKRVSAARLL SRQNLQSPKF SRSTIFVRLH TNGNQELQYQ PGDHLGVFPG NH EDLVNAL IERLEDAPPA NHVVKVEMLE ERNTALGVIS NWKDESRLPP CTIFQAFKYY LDITTPPTPL QLQQFASLAT NEK EKQRLL VLSKGLQEYE EWKWGKNPTM VEVLEEFPSI QMPATLLLTQ LSLLQPRYYS ISSSPDMYPD EVHLTVAIVS YHTR DGEGP VHHGVCSSWL NRIQADDVVP CFVRGAPSFH LPRNPQVPCI LVGPGTGIAP FRSFWQQRQF DIQHKGMNPC PMVLV FGCR QSKIDHIYRE ETLQAKNKGV FRELYTAYSR EPDRPKKYVQ DVLQEQLAES VYRALKEQGG HIYVCGDVTM AADVLK AIQ RIMTQQGKLS EEDAGVFISR LRDDNRYHED IFGVTLRTYE VTNRLRSESI AFIEESKKDA DEVFSS UniProtKB: Nitric oxide synthase 1 |
-分子 #2: ZINC ION
分子 | 名称: ZINC ION / タイプ: ligand / ID: 2 / コピー数: 1 / 式: ZN |
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分子量 | 理論値: 65.409 Da |
-分子 #3: ARGININE
分子 | 名称: ARGININE / タイプ: ligand / ID: 3 / コピー数: 2 / 式: ARG |
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分子量 | 理論値: 175.209 Da |
Chemical component information | ChemComp-ARG: |
-分子 #4: 5,6,7,8-TETRAHYDROBIOPTERIN
分子 | 名称: 5,6,7,8-TETRAHYDROBIOPTERIN / タイプ: ligand / ID: 4 / コピー数: 2 / 式: H4B |
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分子量 | 理論値: 241.247 Da |
Chemical component information | ChemComp-H4B: |
-分子 #5: PROTOPORPHYRIN IX CONTAINING FE
分子 | 名称: PROTOPORPHYRIN IX CONTAINING FE / タイプ: ligand / ID: 5 / コピー数: 2 / 式: HEM |
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分子量 | 理論値: 616.487 Da |
Chemical component information | ChemComp-HEM: |
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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解析 | 単粒子再構成法 |
試料の集合状態 | particle |
-試料調製
緩衝液 | pH: 7.5 |
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凍結 | 凍結剤: ETHANE |
-電子顕微鏡法
顕微鏡 | TFS GLACIOS |
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撮影 | フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 平均電子線量: 62.0 e/Å2 |
電子線 | 加速電圧: 200 kV / 電子線源: FIELD EMISSION GUN |
電子光学系 | 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / 最大 デフォーカス(公称値): 2.0 µm / 最小 デフォーカス(公称値): 1.0 µm |
-画像解析
初期モデル | モデルのタイプ: OTHER |
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最終 再構成 | 解像度のタイプ: BY AUTHOR / 解像度: 3.14 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 使用した粒子像数: 294924 |
初期 角度割当 | タイプ: MAXIMUM LIKELIHOOD |
最終 角度割当 | タイプ: MAXIMUM LIKELIHOOD |