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- PDB-8k3h: Cryo-EM structure of PseP with NAD at 2.86 angstrom resolution -

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Basic information

Entry
Database: PDB / ID: 8k3h
TitleCryo-EM structure of PseP with NAD at 2.86 angstrom resolution
ComponentsPseP
KeywordsLYASE / altemicidin / pyridoxal 5'-phosphate / PLP-dependent enzyme
Function / homologyNICOTINAMIDE-ADENINE-DINUCLEOTIDE
Function and homology information
Biological speciesPseudomonas fluorescens (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsMori, T. / Awakawa, T. / Adachi, N. / Abe, I.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP21am0101071 Japan
Japan Agency for Medical Research and Development (AMED)JP22ama121001 Japan
CitationJournal: To Be Published
Title: Cryo-EM structure of PseP with NAD at 2.86 angstrom resolution
Authors: Mori, T. / Awakawa, T. / Adachi, N. / Abe, I.
History
DepositionJul 15, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 17, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: PseP
B: PseP
hetero molecules


Theoretical massNumber of molelcules
Total (without water)183,2304
Polymers181,9032
Non-polymers1,3272
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein PseP


Mass: 90951.523 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas fluorescens (bacteria) / Production host: Streptomyces lividans TK24 (bacteria)
#2: Chemical ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE


Mass: 663.425 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: NAD*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: PseP / Type: COMPLEX
Details: PLP-dependent enzyme in the biosynthesis of altemicidin
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.09 MDa / Experimental value: YES
Source (natural)Organism: Pseudomonas fluorescens (bacteria)
Source (recombinant)Organism: Streptomyces lividans TK24 (bacteria)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono-disperse.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 105000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 49 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3006

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Processing

EM software
IDNameVersionCategory
1RELIONparticle selection
2SerialEMimage acquisition
4CTFFIND4CTF correction
10RELION3.1.3initial Euler assignment
11RELION3.1.3final Euler assignment
12RELION3.1classification
13RELION3.1.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2478450
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 462772 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027638
ELECTRON MICROSCOPYf_angle_d0.45610424
ELECTRON MICROSCOPYf_dihedral_angle_d5.8561178
ELECTRON MICROSCOPYf_chiral_restr0.0391184
ELECTRON MICROSCOPYf_plane_restr0.0041332

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