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- EMDB-29657: Semi-synthetic CoA-alpha-Synuclein Constructs Trap N-terminal Ace... -
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Basic information
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Title | Semi-synthetic CoA-alpha-Synuclein Constructs Trap N-terminal Acetyltransferase NatB for Binding Mechanism Studies | |||||||||
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Function / homology | ![]() N-terminal peptidyl-glutamine acetylation / N-terminal methionine Nalpha-acetyltransferase NatB / N-terminal peptidyl-aspartic acid acetylation / N-terminal peptidyl-glutamic acid acetylation / NatB complex / N-terminal protein amino acid acetylation / peptide alpha-N-acetyltransferase activity / negative regulation of mitochondrial electron transport, NADH to ubiquinone / neutral lipid metabolic process / regulation of phospholipase activity ...N-terminal peptidyl-glutamine acetylation / N-terminal methionine Nalpha-acetyltransferase NatB / N-terminal peptidyl-aspartic acid acetylation / N-terminal peptidyl-glutamic acid acetylation / NatB complex / N-terminal protein amino acid acetylation / peptide alpha-N-acetyltransferase activity / negative regulation of mitochondrial electron transport, NADH to ubiquinone / neutral lipid metabolic process / regulation of phospholipase activity / negative regulation of monooxygenase activity / regulation of acyl-CoA biosynthetic process / negative regulation of dopamine uptake involved in synaptic transmission / negative regulation of norepinephrine uptake / positive regulation of glutathione peroxidase activity / positive regulation of SNARE complex assembly / positive regulation of hydrogen peroxide catabolic process / supramolecular fiber / negative regulation of transporter activity / mitochondrial membrane organization / negative regulation of chaperone-mediated autophagy / regulation of reactive oxygen species biosynthetic process / positive regulation of protein localization to cell periphery / regulation of synaptic vesicle recycling / negative regulation of platelet-derived growth factor receptor signaling pathway / negative regulation of exocytosis / regulation of glutamate secretion / response to iron(II) ion / regulation of norepinephrine uptake / SNARE complex assembly / positive regulation of neurotransmitter secretion / dopamine biosynthetic process / regulation of locomotion / positive regulation of inositol phosphate biosynthetic process / synaptic vesicle priming / regulation of macrophage activation / negative regulation of microtubule polymerization / synaptic vesicle transport / dopamine uptake involved in synaptic transmission / dynein complex binding / regulation of dopamine secretion / positive regulation of receptor recycling / protein kinase inhibitor activity / negative regulation of thrombin-activated receptor signaling pathway / response to type II interferon / cuprous ion binding / positive regulation of exocytosis / synaptic vesicle exocytosis / positive regulation of endocytosis / kinesin binding / cysteine-type endopeptidase inhibitor activity involved in apoptotic process / response to magnesium ion / synaptic vesicle endocytosis / regulation of presynapse assembly / negative regulation of serotonin uptake / alpha-tubulin binding / phospholipid metabolic process / supramolecular fiber organization / axon terminus / mitochondrial ATP synthesis coupled electron transport / inclusion body / fatty acid metabolic process / cellular response to copper ion / cellular response to epinephrine stimulus / Hsp70 protein binding / response to interleukin-1 / adult locomotory behavior / negative regulation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of release of sequestered calcium ion into cytosol / SNARE binding / excitatory postsynaptic potential / long-term synaptic potentiation / phosphoprotein binding / protein tetramerization / regulation of transmembrane transporter activity / synapse organization / microglial cell activation / negative regulation of protein kinase activity / regulation of long-term neuronal synaptic plasticity / protein destabilization / ferrous iron binding / tau protein binding / positive regulation of protein serine/threonine kinase activity / PKR-mediated signaling / receptor internalization / phospholipid binding / activation of cysteine-type endopeptidase activity involved in apoptotic process / synaptic vesicle membrane / positive regulation of inflammatory response / positive regulation of peptidyl-serine phosphorylation / actin cytoskeleton / actin binding / cellular response to oxidative stress / histone binding / cell cortex / growth cone / chemical synaptic transmission / neuron apoptotic process / negative regulation of neuron apoptotic process / transcription cis-regulatory region binding Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.39 Å | |||||||||
![]() | Gardner SM / Marmorstein R | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Semi-synthetic CoA-α-Synuclein Constructs Trap N-terminal Acetyltransferase NatB for Binding Mechanism Studies. Authors: Buyan Pan / Sarah Gardner / Kollin Schultz / Ryann M Perez / Sunbin Deng / Marie Shimogawa / Kohei Sato / Elizabeth Rhoades / Ronen Marmorstein / E James Petersson Abstract: N-terminal acetylation is a chemical modification carried out by N-terminal acetyltransferases (NATs). A major member of this enzyme family, NatB, acts on much of the human proteome, including α- ...N-terminal acetylation is a chemical modification carried out by N-terminal acetyltransferases (NATs). A major member of this enzyme family, NatB, acts on much of the human proteome, including α-synuclein (αS), a synaptic protein that mediates vesicle trafficking. NatB acetylation of αS modulates its lipid vesicle binding properties and amyloid fibril formation, which underlies its role in the pathogenesis of Parkinson's disease. Although the molecular details of the interaction between human NatB (hNatB) and the N-terminus of αS have been resolved, whether the remainder of the protein plays a role in interacting with the enzyme is unknown. Here we execute the first synthesis, by native chemical ligation, of a bisubstrate inhibitor of NatB consisting of coenzyme A and full-length human αS, additionally incorporating two fluorescent probes for studies of conformational dynamics. We use cryo-electron microscopy (cryo-EM) to characterize the structural features of the hNatB/inhibitor complex and show that, beyond the first few residues, αS remains disordered when in complex with hNatB. We further probe changes in the αS conformation by single molecule Förster resonance energy transfer (smFRET) to reveal that the C-terminus expands when bound to hNatB. Computational models based on the cryo-EM and smFRET data help to explain the conformational changes and their implications for hNatB substrate recognition and specific inhibition of the interaction with αS. Beyond the study of αS and NatB, these experiments illustrate valuable strategies for the study of challenging structural biology targets through a combination of protein semi-synthesis, cryo-EM, smFRET, and computational modeling. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 51.6 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 16.9 KB 16.9 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 9.9 KB | Display | ![]() |
Images | ![]() | 48.5 KB | ||
Others | ![]() ![]() | 95.5 MB 95.5 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 901 KB | Display | ![]() |
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Full document | ![]() | 900.5 KB | Display | |
Data in XML | ![]() | 18.3 KB | Display | |
Data in CIF | ![]() | 23.5 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8g0lMC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 0.86 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #1
File | emd_29657_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_29657_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Ternary complex of NAA20, NAA25 and CoA-alpha-Synuclein
Entire | Name: Ternary complex of NAA20, NAA25 and CoA-alpha-Synuclein |
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Components |
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-Supramolecule #1: Ternary complex of NAA20, NAA25 and CoA-alpha-Synuclein
Supramolecule | Name: Ternary complex of NAA20, NAA25 and CoA-alpha-Synuclein type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3 |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: N-alpha-acetyltransferase 20
Macromolecule | Name: N-alpha-acetyltransferase 20 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO EC number: N-terminal methionine Nalpha-acetyltransferase NatB |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 20.390133 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MTTLRAFTCD DLFRFNNINL DPLTETYGIP FYLQYLAHWP EYFIVAEAPG GELMGYIMGK AEGSVAREEW HGHVTALSVA PEFRRLGLA AKLMELLEEI SERKGGFFVD LFVRVSNQVA VNMYKQLGYS VYRTVIEYYS ASNGEPDEDA YDMRKALSRD T EKKSIIPL PHPVRPEDIE |
-Macromolecule #2: N-alpha-acetyltransferase 25, NatB auxiliary subunit
Macromolecule | Name: N-alpha-acetyltransferase 25, NatB auxiliary subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 112.444258 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MATRGHVQDP NDRRLRPIYD YLDNGNNKMA IQQADKLLKK HKDLHCAKVL KAIGLQRTGK QEEAFTLAQE VAALEPTDDN SLQALTILY REMHRPELVT KLYEAAVKKV PNSEEYHSHL FMAYARVGEY KKMQQAGMAL YKIVPKNPYY FWSVMSLIMQ S ISAQDENL ...String: MATRGHVQDP NDRRLRPIYD YLDNGNNKMA IQQADKLLKK HKDLHCAKVL KAIGLQRTGK QEEAFTLAQE VAALEPTDDN SLQALTILY REMHRPELVT KLYEAAVKKV PNSEEYHSHL FMAYARVGEY KKMQQAGMAL YKIVPKNPYY FWSVMSLIMQ S ISAQDENL SKTMFLPLAE RMVEKMVKED KIEAEAEVEL YYMILERLGK YQEALDVIRG KLGEKLTSEI QSRENKCMAM YK KLSRWPE CNALSRRLLL KNSDDWQFYL TYFDSVFRLI EEAWSPPAEG EHSLEGEVHY SAEKAVKFIE DRITEESKSS RHL RGPHLA KLELIRRLRS QGCNDEYKLG DPEELMFQYF KKFGDKPCCF TDLKVFVDLL PATQCTKFIN QLLGVVPLST PTED KLALP ADIRALQQHL CVVQLTRLLG LYHTMDKNQK LSVVRELMLR YQHGLEFGKT CLKTELQFSD YYCLLAVHAL IDVWR ETGD ETTVWQALTL LEEGLTHSPS NAQFKLLLVR IYCMLGAFEP VVDLYSSLDA KHIQHDTIGY LLTRYAESLG QYAAAS QSC NFALRFFHSN QKDTSEYIIQ AYKYGAFEKI PEFIAFRNRL NNSLHFAQVR TERMLLDLLL EANISTSLAE SIKSMNL RP EEDDIPWEDL RDNRDLNVFF SWDPKDRDVS EEHKKLSLEE ETLWLRIRSL TLRLISGLPS LNHPVEPKNS EKTAENGV S SRIDILRLLL QQLEATLETG KRFIEKDIQY PFLGPVPTRM GGFFNSGCSQ CQISSFYLVN DIYELDTSGL EDTMEIQER IENSFKSLLD QLKDVFSKCK GDLLEVKDGN LKTHPTLLEN LVFFVETISV ILWVSSYCES VLRPYKLNLQ KKKKKKKETS IIMPPVFTS FQDYVTGLQT LISNVVDHIK GLETHLIALK LEELILEDTS LSPEERKFSK TVQGKVQSSY LHSLLEMGEL L KKRLETTK KLKI |
-Macromolecule #3: Alpha-synuclein
Macromolecule | Name: Alpha-synuclein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 641.799 Da |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MDVFM |
-Macromolecule #4: CARBOXYMETHYL COENZYME *A
Macromolecule | Name: CARBOXYMETHYL COENZYME *A / type: ligand / ID: 4 / Number of copies: 1 / Formula: CMC |
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Molecular weight | Theoretical: 825.57 Da |
Chemical component information | ![]() ChemComp-CMC: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 4 mg/mL |
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Buffer | pH: 7.5 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 5470 / Average electron dose: 43.9 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |