+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-40969 | |||||||||
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Title | Uncrosslinked nNOS-CaM oxygenase homodimer | |||||||||
Map data | nNOS-CaM map | |||||||||
Sample |
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Keywords | Calmodulin / Complex / CYTOSOLIC PROTEIN | |||||||||
Function / homology | Function and homology information Nitric oxide stimulates guanylate cyclase / negative regulation of hepatic stellate cell contraction / synaptic signaling by nitric oxide / negative regulation of iron ion transmembrane transport / ROS and RNS production in phagocytes / azurophil granule / negative regulation of vasoconstriction / retrograde trans-synaptic signaling by nitric oxide / Ion homeostasis / positive regulation of sodium ion transmembrane transport ...Nitric oxide stimulates guanylate cyclase / negative regulation of hepatic stellate cell contraction / synaptic signaling by nitric oxide / negative regulation of iron ion transmembrane transport / ROS and RNS production in phagocytes / azurophil granule / negative regulation of vasoconstriction / retrograde trans-synaptic signaling by nitric oxide / Ion homeostasis / positive regulation of sodium ion transmembrane transport / positive regulation of adenylate cyclase-activating G protein-coupled receptor signaling pathway / response to nitric oxide / postsynaptic specialization, intracellular component / nitric oxide metabolic process / negative regulation of cytosolic calcium ion concentration / peptidyl-cysteine S-nitrosylation / behavioral response to cocaine / postsynaptic density, intracellular component / cadmium ion binding / positive regulation of the force of heart contraction / negative regulation of potassium ion transport / calyx of Held / negative regulation of calcium ion transport / negative regulation of serotonin uptake / sodium channel regulator activity / regulation of neurogenesis / striated muscle contraction / negative regulation of insulin secretion / response to vitamin E / nitric-oxide synthase (NADPH) / regulation of postsynaptic membrane potential / multicellular organismal response to stress / xenobiotic catabolic process / nitric-oxide synthase activity / negative regulation of peptidyl-serine phosphorylation / nitric oxide mediated signal transduction / arginine catabolic process / NADPH binding / regulation of sodium ion transport / response to organonitrogen compound / cellular response to epinephrine stimulus / sarcoplasmic reticulum membrane / T-tubule / photoreceptor inner segment / nitric oxide biosynthetic process / negative regulation of blood pressure / response to hormone / response to nutrient levels / response to activity / response to nicotine / sarcoplasmic reticulum / muscle contraction / secretory granule / female pregnancy / positive regulation of long-term synaptic potentiation / cell periphery / establishment of localization in cell / phosphoprotein binding / response to lead ion / sarcolemma / establishment of protein localization / potassium ion transport / response to organic cyclic compound / response to peptide hormone / cellular response to growth factor stimulus / Z disc / vasodilation / cellular response to mechanical stimulus / response to estrogen / calcium-dependent protein binding / calcium ion transport / FMN binding / flavin adenine dinucleotide binding / positive regulation of peptidyl-serine phosphorylation / NADP binding / ATPase binding / response to heat / scaffold protein binding / response to ethanol / nuclear membrane / negative regulation of neuron apoptotic process / mitochondrial outer membrane / transmembrane transporter binding / response to lipopolysaccharide / dendritic spine / postsynaptic density / response to hypoxia / cytoskeleton / calmodulin binding / membrane raft / negative regulation of cell population proliferation / dendrite / glutamatergic synapse / synapse / 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 Similarity search - Function | |||||||||
Biological species | Rattus norvegicus (Norway rat) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.7 Å | |||||||||
Authors | Lee K / Pospiech TH / Southworth D | |||||||||
Funding support | United States, 1 items
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Citation | Journal: J Biol Chem / Year: 2024 Title: Mapping interactions of calmodulin and neuronal NO synthase by crosslinking and mass spectrometry. Authors: Dana Felker / Kanghyun Lee / Thomas H Pospiech / Yoshihiro Morishima / Haoming Zhang / Miranda Lau / Daniel R Southworth / Yoichi Osawa / Abstract: 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. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_40969.map.gz | 118 MB | EMDB map data format | |
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Header (meta data) | emd-40969-v30.xml emd-40969.xml | 16 KB 16 KB | Display Display | EMDB header |
Images | emd_40969.png | 86.5 KB | ||
Filedesc metadata | emd-40969.cif.gz | 6.3 KB | ||
Others | emd_40969_half_map_1.map.gz emd_40969_half_map_2.map.gz | 116.2 MB 116.2 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-40969 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-40969 | HTTPS FTP |
-Related structure data
Related structure data | 8t1jMC 8t1kC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_40969.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | nNOS-CaM map | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.059 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: nNOS-CaM half map A
File | emd_40969_half_map_1.map | ||||||||||||
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Annotation | nNOS-CaM half map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: nNOS-CaM half map B
File | emd_40969_half_map_2.map | ||||||||||||
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Annotation | nNOS-CaM half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Native CaM-bound nNOS homodimer
Entire | Name: Native CaM-bound nNOS homodimer |
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Components |
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-Supramolecule #1: Native CaM-bound nNOS homodimer
Supramolecule | Name: Native CaM-bound nNOS homodimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) |
-Macromolecule #1: Nitric oxide synthase 1
Macromolecule | Name: Nitric oxide synthase 1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: nitric-oxide synthase (NADPH) |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) |
Molecular weight | Theoretical: 160.769562 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MEENTFGVQQ IQPNVISVRL FKRKVGGLGF LVKERVSKPP VIISDLIRGG AAEQSGLIQA GDIILAVNDR PLVDLSYDSA LEVLRGIAS ETHVVLILRG PEGFTTHLET TFTGDGTPKT IRVTQPLGPP TKAVDLSHQP SASKDQSLAV DRVTGLGNGP Q HAQGHGQG ...String: 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 |
-Macromolecule #2: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Macromolecule #3: ARGININE
Macromolecule | Name: ARGININE / type: ligand / ID: 3 / Number of copies: 2 / Formula: ARG |
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Molecular weight | Theoretical: 175.209 Da |
Chemical component information | ChemComp-ARG: |
-Macromolecule #4: 5,6,7,8-TETRAHYDROBIOPTERIN
Macromolecule | Name: 5,6,7,8-TETRAHYDROBIOPTERIN / type: ligand / ID: 4 / Number of copies: 2 / Formula: H4B |
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Molecular weight | Theoretical: 241.247 Da |
Chemical component information | ChemComp-H4B: |
-Macromolecule #5: PROTOPORPHYRIN IX CONTAINING FE
Macromolecule | Name: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 5 / Number of copies: 2 / Formula: HEM |
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Molecular weight | Theoretical: 616.487 Da |
Chemical component information | ChemComp-HEM: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 |
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Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 56.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: OTHER |
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Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 138304 |