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- EMDB-11606: Cryo-EM map of the large glutamate dehydrogenase composed of 180 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-11606
TitleCryo-EM map of the large glutamate dehydrogenase composed of 180 kDa subunits from Mycobacterium smegmatis (open conformation)
Map data
Sample
  • Complex: Cryo-EM map of the large glutamate dehydrogenase composed of 180 kDa subunits from Mycobacterium smegmatis (open conformation)
    • Protein or peptide: NAD-specific glutamate dehydrogenase
Function / homologyNAD-glutamate dehydrogenase, bacteria / NAD-glutamate dehydrogenase / Bacterial NAD-glutamate dehydrogenase, catalytic domain / glutamate dehydrogenase / glutamate catabolic process to 2-oxoglutarate / glutamate dehydrogenase (NAD+) activity / Aminoacid dehydrogenase-like, N-terminal domain superfamily / NAD(P)-binding domain superfamily / NAD-specific glutamate dehydrogenase
Function and homology information
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria) / Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.19 Å
AuthorsLazaro M / Melero R / Huet C / Lopez-Alonso JP / Delgado S / Dodu A / Bruch EM / Abriata LA / Alzari PM / Valle M / Lisa MN
Funding support Spain, 1 items
OrganizationGrant numberCountry
Spanish Ministry of Science, Innovation, and UniversitiesPGC2018-098996-B-100 Spain
CitationJournal: Commun Biol / Year: 2021
Title: 3D architecture and structural flexibility revealed in the subfamily of large glutamate dehydrogenases by a mycobacterial enzyme.
Authors: Melisa Lázaro / Roberto Melero / Charlotte Huet / Jorge P López-Alonso / Sandra Delgado / Alexandra Dodu / Eduardo M Bruch / Luciano A Abriata / Pedro M Alzari / Mikel Valle / María-Natalia Lisa /
Abstract: Glutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180 kDa subunits (L-GDHs) contain long N- ...Glutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180 kDa subunits (L-GDHs) contain long N- and C-terminal segments flanking the catalytic core. Despite the relevance of L-GDHs in bacterial physiology, the lack of structural data for these enzymes has limited the progress of functional studies. Here we show that the mycobacterial L-GDH (mL-GDH) adopts a quaternary structure that is radically different from that of related low molecular weight enzymes. Intersubunit contacts in mL-GDH involve a C-terminal domain that we propose as a new fold and a flexible N-terminal segment comprising ACT-like and PAS-type domains that could act as metabolic sensors for allosteric regulation. These findings uncover unique aspects of the structure-function relationship in the subfamily of L-GDHs.
History
DepositionAug 12, 2020-
Header (metadata) releaseJun 9, 2021-
Map releaseJun 9, 2021-
UpdateOct 6, 2021-
Current statusOct 6, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0104
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0104
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7a1d
  • Surface level: 0.0104
  • Imaged by UCSF Chimera
  • Download
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11606.png

Downloads & links

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Map

FileDownload / File: emd_11606.map.gz / Format: CCP4 / Size: 209.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.0104 / Movie #1: 0.0104
Minimum - Maximum-0.024270207 - 0.049838196
Average (Standard dev.)0.00012095307 (±0.0014329612)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions380380380
Spacing380380380
CellA=B=C: 402.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z380380380
origin x/y/z0.0000.0000.000
length x/y/z402.800402.800402.800
α/β/γ90.00090.00090.000
start NX/NY/NZ727265
NX/NY/NZ157157169
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS380380380
D min/max/mean-0.0240.0500.000

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Supplemental data

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Mask #1

Fileemd_11606_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_11606_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11606_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Cryo-EM map of the large glutamate dehydrogenase composed of 180 ...

EntireName: Cryo-EM map of the large glutamate dehydrogenase composed of 180 kDa subunits from Mycobacterium smegmatis (open conformation)
Components
  • Complex: Cryo-EM map of the large glutamate dehydrogenase composed of 180 kDa subunits from Mycobacterium smegmatis (open conformation)
    • Protein or peptide: NAD-specific glutamate dehydrogenase

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Supramolecule #1: Cryo-EM map of the large glutamate dehydrogenase composed of 180 ...

SupramoleculeName: Cryo-EM map of the large glutamate dehydrogenase composed of 180 kDa subunits from Mycobacterium smegmatis (open conformation)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant strain: BL21(DE3)
Molecular weightTheoretical: 705 KDa

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Macromolecule #1: NAD-specific glutamate dehydrogenase

MacromoleculeName: NAD-specific glutamate dehydrogenase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: glutamate dehydrogenase
Source (natural)Organism: Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155
Molecular weightTheoretical: 176.341859 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MHHHHHHENL YFQGAASMIR RLSVAFLSTY RGPQADAPGV TSTGPLAVAA HDDLVSDDLV AAHYRLASMR APGETKAAVY PGDAGSGAA LQIVTDQAPM LVDSVTVLLH RHGIAYTAIM NPVFRVRRGL DGELLDVRPA AEAAPGDGAD ECWILVPITA A ADGEALTE ...String:
MHHHHHHENL YFQGAASMIR RLSVAFLSTY RGPQADAPGV TSTGPLAVAA HDDLVSDDLV AAHYRLASMR APGETKAAVY PGDAGSGAA LQIVTDQAPM LVDSVTVLLH RHGIAYTAIM NPVFRVRRGL DGELLDVRPA AEAAPGDGAD ECWILVPITA A ADGEALTE ATRLVPGILA EARQIGLDSG AMIAALHGLA NDLATDLEGH FPNAERKEVA ALLRWLADGH FVLLGYQQCV VG DGNAEVD PASRLGVLRL RNDVLPPLTD SDDLLVLAQA TMPSYLRYGA YPYIVVVRES PGASRVIEHR FVGLFTVAAM NAN ALEIPL ISRRVEEALA MAHRDPSHPG QLLRDIIQTI PRPELFALSS KQLLEMALAV VDLGSRRRTL LFLRADHLAH FVSC LVYLP RDRYTTAVRL EMQDILVREL GGAGIDYSAR VSESPWAVVH FTVRLPEGTA ADSVDTSLEN ESRIQDLLTE ATRNW GDRM ISAAAAASIS PAALEHYAHA FPEDYKQAFA PQDAIADISL IEALQDDSVK LVLADTAEDR VWKLTWYLGG HSASLS ELL PMLQSMGVVV LEERPFTLRR TDGLPVWIYQ FKISPHPSIP HAPDAEAQRD TAQRFADAVT AIWHGRVEID RFNELVM RA GLTWQQVVVL RAYAKYLRQA GFPYSQSHIE SVLNENPHTT RSLIDLFEAL FDPSQETDGR RDAQGAAAAV AADIDALV S LDTDRVLRAF ANLIEATLRT NYFVARPDSA RARNVLAFKL NPLVIKELPL PRPKFEIFVY SPRVEGVHLR FGFVARGGL RWSDRREDFR TEILGLVKAQ AVKNAVIVPV GAKGGFVVKR PPTLTGDAAA DREATRAEGV ECYRLFISGL LDVTDNVDKA TGAVVTPPE VVRRDGEDAY LVVAADKGTA TFSDIANEVA KSYGFWLGDA FASGGSIGYD HKAMGITAKG AWESVKRHFR E MGVDTQTQ DFTVVGIGDM SGDVFGNGML LSKHIRLVAA FDHRDIFLDP NPDAGRSWDE RKRLFDLPRS SWADYDKSLI SE GGGVYSR QQKSIPISPQ VRTALGLDAD VEELTPPALI KAILKAPVDL LWNGGIGTYI KAETEADADV GDRANDQIRV CGN QVRAKV IGEGGNLGVT ALGRIEFDLA GGRINTDALD NSAGVDCSDH EVNIKILIDS AVTAGKVTPE ERTELLLSMT DEVG ELVLA DNRDQNDLMG TSRANAASLL SVHARMIKDL VDNRGLNREL EALPSEKEIR RRADAGIGLT SPELATLMAH VKLAL KDDV LASDLPDQEV FASRLPYYFP TRLREELHGE IRSHQLRREI ITTMLVNDLV DTAGISYAYR ITEDVGVGPV DAVRSY VAI NAIFGIGDVW RRIRAAGDAG VPTSVTDRMT LDLRRLVDRA GRWLLNYRPQ PLAVGAEINR FGAKVAALTP RMSEWLR GD DKAIVSKEAG DFASHGVPED LAYHIATGLY QYSLLDVIDI ADIVDREPDE VADTYFALMD HLGADALLTA VSRLSRDD R WHSLARLAIR DDIYGSLRAL CFDVLAVGEP DENGEEKIAE WETTNSSRVT RARRTLTEIY KDGEQDLATL SVAARQIRS MTRTSGTGTT G

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 6
GridModel: Quantifoil / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.2600000000000002 µm / Calibrated defocus min: 0.67 µm / Calibrated magnification: 47170 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-20 / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: MotionCorr2 (ver. 2_1.4.0)
Initial angle assignmentType: COMMON LINE / Software - Name: EMAN
Final 3D classificationNumber classes: 2 / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.19 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 63715
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsModel fitting into the cryo-EM map was performed using the programs UCSF Chimera (Pettersen et al., 2004), Namdinator (Kidmose et al., 2019), phenix.real_space_refine (Afonine et al., 2018) and Coot (Emsley, Lohkamp, Scott, & Cowtan, 2010). Residues 500-1588 from the crystal structure of Se-Met mL-GDH180 (PDB code 7JSR) were fitted into the cryo-EM map. Se-methionine residues were replaced by methionine residues using Coot (Emsley et al., 2010) and the model was finally refined employing phenix.real_space_refine (Afonine et al., 2018) with NCS and secondary structure restraints.
RefinementProtocol: OTHER
Output model

PDB-7a1d:
Cryo-EM map of the large glutamate dehydrogenase composed of 180 kDa subunits from Mycobacterium smegmatis (open conformation)

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