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- EMDB-4179: Combined solid-state NMR, solution-state NMR and EM data for stru... -

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Basic information

Entry
Database: EMDB / ID: EMD-4179
TitleCombined solid-state NMR, solution-state NMR and EM data for structure determination of the tetrahedral aminopeptidase TET2 from P. horikoshii
Map dataCryoEM map of the H78C/H248C mutant tetrahedral aminopeptidase from P. horikoshii
Sample
  • Complex: tetrahedral amino-peptidase from P. horikoshii
    • Protein or peptide: Tetrahedral aminopeptidase
  • Ligand: ZINC ION
Keywordspeptidase / protein quality control / oligomer / aminopeptidase / PEPTIDE BINDING PROTEIN
Function / homology
Function and homology information


Hydrolases; Acting on peptide bonds (peptidases); Aminopeptidases / aminopeptidase activity / metallopeptidase activity / proteolysis / metal ion binding
Similarity search - Function
Peptidase M42, domain 2 / : / M42 glutamyl aminopeptidase / Peptidase M42
Similarity search - Domain/homology
Tetrahedral aminopeptidase
Similarity search - Component
Biological speciesPyrococcus horikoshii (archaea) / Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsGauto DF / Estrozi LF / Schwieters CD
Funding support France, 3 items
OrganizationGrant numberCountry
European Research Council311318
French National Research AgencyANR-10-INSB-05-02 France
French National Research AgencyANR-10-LABX-49-01 France
CitationJournal: Nat Commun / Year: 2019
Title: Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex.
Authors: Gauto DF / Estrozi LF / Schwieters CD / Effantin G / Macek P / Sounier R / Sivertsen AC / Schmidt E / Kerfah R / Mas G / Colletier JP / Guntert P / Favier A / Schoehn G / Schanda P / Boisbouvier J
History
DepositionNov 28, 2017-
Header (metadata) releaseMar 14, 2018-
Map releaseMar 14, 2018-
UpdateSep 13, 2023-
Current statusSep 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6f3k
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6f3k
  • Imaged by Jmol
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol

Downloads & links

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Map

FileDownload / File: emd_4179.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM map of the H78C/H248C mutant tetrahedral aminopeptidase from P. horikoshii
Voxel sizeX=Y=Z: 0.97 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.17549673 - 0.21628784
Average (Standard dev.)0.0005608964 (±0.0140974475)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-99
Dimensions200200200
Spacing200200200
CellA=B=C: 194.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.970.970.97
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z194.000194.000194.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-13-52-30
NX/NY/NZ576659
MAP C/R/S123
start NC/NR/NS-100-100-99
NC/NR/NS200200200
D min/max/mean-0.1750.2160.001

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Supplemental data

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Sample components

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Entire : tetrahedral amino-peptidase from P. horikoshii

EntireName: tetrahedral amino-peptidase from P. horikoshii
Components
  • Complex: tetrahedral amino-peptidase from P. horikoshii
    • Protein or peptide: Tetrahedral aminopeptidase
  • Ligand: ZINC ION

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Supramolecule #1: tetrahedral amino-peptidase from P. horikoshii

SupramoleculeName: tetrahedral amino-peptidase from P. horikoshii / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: H78C/H248C mutant
Source (natural)Organism: Pyrococcus horikoshii (archaea)
Molecular weightTheoretical: 490 KDa

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Macromolecule #1: Tetrahedral aminopeptidase

MacromoleculeName: Tetrahedral aminopeptidase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Aminopeptidases
Source (natural)Organism: Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) (archaea)
Strain: ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3
Molecular weightTheoretical: 39.071027 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEVRNMVDYE LLKKVVEAPG VSGYEFLGIR DVVIEEIKDY VDEVKVDKLG NVIAHKKGEG PKVMIAAHMD QIGLMVTHIE KNGFLRVAP IGGVDPKTLI AQRFKVWIDK GKFIYGVGAS VPPHIQKPED RKKAPDWDQI FIDIGAESKE EAEDMGVKIG T VITWDGRL ...String:
MEVRNMVDYE LLKKVVEAPG VSGYEFLGIR DVVIEEIKDY VDEVKVDKLG NVIAHKKGEG PKVMIAAHMD QIGLMVTHIE KNGFLRVAP IGGVDPKTLI AQRFKVWIDK GKFIYGVGAS VPPHIQKPED RKKAPDWDQI FIDIGAESKE EAEDMGVKIG T VITWDGRL ERLGKHRFVS IAFDDRIAVY TILEVAKQLK DAKADVYFVA TVQEEVGLRG ARTSAFGIEP DYGFAIDVTI AA DIPGTPE HKQVTHLGKG TAIKIMDRSV ICHPTIVRWL EELAKKHEIP YQLEILLGGG TDAGAIHLTK AGVPTGALSV PAR YIHSNT EVVDERDVDA TVELMTKALE NIHELKI

UniProtKB: Tetrahedral aminopeptidase

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration1 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time 2s, force 1, drain time 0..
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 90 / Average exposure time: 4.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.5 µm / Calibrated defocus min: 0.8 µm / Calibrated magnification: 25773 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal magnification: 20000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 30407
Startup modelType of model: OTHER / Details: RIco webserver rico.ibs.fr
Final reconstructionApplied symmetry - Point group: T (tetrahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 27130
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)

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Atomic model buiding 1

RefinementProtocol: OTHER
Output model

PDB-6f3k:
Combined solid-state NMR, solution-state NMR and EM data for structure determination of the tetrahedral aminopeptidase TET2 from P. horikoshii

PDB-6r8n:
STRUCTURE DETERMINATION OF THE TETRAHEDRAL AMINOPEPTIDASE TET2 FROM P. HORIKOSHII BY USE OF COMBINED SOLID-STATE NMR, SOLUTION-STATE NMR AND EM DATA 4.1 A, FOLLOWED BY REAL_SPACE_REFINEMENT AT 4.1 A

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