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- EMDB-9592: Structure of NAD+-bound leucine dehydrogenase from Geobacillus st... -

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Basic information

Entry
Database: EMDB / ID: 9592
TitleStructure of NAD+-bound leucine dehydrogenase from Geobacillus stearothermophilus by cryo-EM
Map dataThe holo form of GstLDH
SampleBinary complex of leucine dehydrogenase with NAD+:
Leucine dehydrogenase / ligand
Function / homologyGlutamate/phenylalanine/leucine/valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal / Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain / Glutamate/phenylalanine/leucine/valine dehydrogenase, bacterial/archaeal / Leu/Phe/Val dehydrogenases active site / NAD(P)-binding domain superfamily / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Glu/Leu/Phe/Val dehydrogenase, dimerisation domain / Glu / Leu / Phe / Val dehydrogenases active site. / oxidoreductase activity, acting on the CH-NH2 group of donors, NAD or NADP as acceptor ...Glutamate/phenylalanine/leucine/valine dehydrogenase / Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal / Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain / Glutamate/phenylalanine/leucine/valine dehydrogenase, bacterial/archaeal / Leu/Phe/Val dehydrogenases active site / NAD(P)-binding domain superfamily / Glutamate/Leucine/Phenylalanine/Valine dehydrogenase / Glu/Leu/Phe/Val dehydrogenase, dimerisation domain / Glu / Leu / Phe / Val dehydrogenases active site. / oxidoreductase activity, acting on the CH-NH2 group of donors, NAD or NADP as acceptor / cellular amino acid metabolic process / nucleotide binding / Leucine dehydrogenase
Function and homology information
SourceGeobacillus stearothermophilus 10 (bacteria)
Methodsingle particle reconstruction / cryo EM / 3.2 Å resolution
AuthorsYamaguchi H / Kamegawa A / Nakata K / Kashiwagi T / Mizukoshi T / Fujiyoshi Y / Tani K
CitationJournal: J. Struct. Biol. / Year: 2018
Title: Structural insights into thermostabilization of leucine dehydrogenase from its atomic structure by cryo-electron microscopy.
Authors: Hiroki Yamaguchi / Akiko Kamegawa / Kunio Nakata / Tatsuki Kashiwagi / Toshimi Mizukoshi / Yoshinori Fujiyoshi / Kazutoshi Tani
Abstract: Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD-dependent oxidoreductase that catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH of Geobacillus stearothermophilus (GstLDH) is a ...Leucine dehydrogenase (LDH, EC 1.4.1.9) is a NAD-dependent oxidoreductase that catalyzes the deamination of branched-chain l-amino acids (BCAAs). LDH of Geobacillus stearothermophilus (GstLDH) is a highly thermostable enzyme that has been applied for the quantification or production of BCAAs. Here the cryo-electron microscopy (cryo-EM) structures of apo and NAD-bound LDH are reported at 3.0 and 3.2 Å resolution, respectively. On comparing the structures, the two overall structures are almost identical, but it was observed that the partial conformational change was triggered by the interaction between Ser147 and the nicotinamide moiety of NAD. NAD binding also enhanced the strength of oligomerization interfaces formed by the core domains. Such additional interdomain interaction is in good agreement with our experimental results showing that the residual activity of NAD-bound form was approximately three times higher than that of the apo form after incubation at 80 °C. In addition, sequence comparison of three structurally known LDHs indicated a set of candidates for site-directed mutagenesis to improve thermostability. Subsequent mutation analysis actually revealed that non-conserved residues, including Ala94, Tyr127, and the C-terminal region, are crucial for oligomeric thermostability.
Validation ReportPDB-ID: 6ach

SummaryFull reportAbout validation report
DateDeposition: Jul 26, 2018 / Header (metadata) release: Dec 26, 2018 / Map release: Dec 26, 2018 / Last update: Dec 26, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
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  • Surface view colored by radius
  • Surface level: 0.05
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  • Surface view with fitted model
  • Atomic models: : PDB-6ach
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

Fileemd_9592.map.gz (map file in CCP4 format, 28312 KB)
Projections & slices

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Others
AxesZ (Sec.)Y (Row.)X (Col.)
192 pix
1.23 Å/pix.
= 236.544 Å
192 pix
1.23 Å/pix.
= 236.544 Å
192 pix
1.23 Å/pix.
= 236.544 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.232 Å
Density
Contour Level:0.05 (by author), 0.05 (movie #1):
Minimum - Maximum-0.22664541 - 0.4768642
Average (Standard dev.)0.000025061747 (0.016380014)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions192192192
Origin0.00.00.0
Limit191.0191.0191.0
Spacing192192192
CellA=B=C: 236.544 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2321.2321.232
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z236.544236.544236.544
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.2270.4770.000

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Supplemental data

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Sample components

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Entire Binary complex of leucine dehydrogenase with NAD+

EntireName: Binary complex of leucine dehydrogenase with NAD+ / Number of components: 3

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Component #1: protein, Binary complex of leucine dehydrogenase with NAD+

ProteinName: Binary complex of leucine dehydrogenase with NAD+ / Recombinant expression: No
MassExperimental: 305 kDa
SourceSpecies: Geobacillus stearothermophilus 10 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, Leucine dehydrogenase

ProteinName: Leucine dehydrogenase / Number of Copies: 8 / Recombinant expression: No
MassTheoretical: 40.58402 kDa
SourceSpecies: Geobacillus stearothermophilus 10 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: ligand, NICOTINAMIDE-ADENINE-DINUCLEOTIDE

LigandName: NICOTINAMIDE-ADENINE-DINUCLEOTIDE / Number of Copies: 8 / Recombinant expression: No
MassTheoretical: 0.663425 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.5 mg/ml / pH: 10.5
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

ImagingMicroscope: JEOL KYOTO-3000SFF
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 2.7 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: D4 (2*4 fold dihedral) / Number of projections: 92034
3D reconstructionSoftware: RELION / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Output model

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