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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 / homologyPeptidase M42, domain 2 / M42 glutamyl aminopeptidase / Peptidase M42 / Hydrolases; Acting on peptide bonds (peptidases); Aminopeptidases / aminopeptidase activity / metallopeptidase activity / proteolysis / metal ion binding / Tetrahedral aminopeptidase
Function and homology information
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: Diego F Gauto / Leandro F Estrozi / Charles D Schwieters / Gregory Effantin / Pavel Macek / Remy Sounier / Astrid C Sivertsen / Elena Schmidt / Rime Kerfah / Guillaume Mas / Jacques-Philippe ...Authors: Diego F Gauto / Leandro F Estrozi / Charles D Schwieters / Gregory Effantin / Pavel Macek / Remy Sounier / Astrid C Sivertsen / Elena Schmidt / Rime Kerfah / Guillaume Mas / Jacques-Philippe Colletier / Peter Güntert / Adrien Favier / Guy Schoehn / Paul Schanda / Jerome Boisbouvier /
Abstract: Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely ...Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enables structure determination of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and ILV methyl groups, and a 4.1 Å resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution cryo-EM data are available.
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
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  • Surface view with fitted model
  • Atomic models: PDB-6f3k
  • Surface level: 0.05
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6f3k
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4179_1.png

emd_4179_2.png

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
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 FIELDBright-field microscopy / Cs: 2.0 mm / Nominal magnification: 20000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
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
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
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
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
FSC plot (resolution estimation)

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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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