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- PDB-6r8n: STRUCTURE DETERMINATION OF THE TETRAHEDRAL AMINOPEPTIDASE TET2 FR... -

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Basic information

Entry
Database: PDB / ID: 6r8n
TitleSTRUCTURE 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
ComponentsTetrahedral aminopeptidase
KeywordsPEPTIDE BINDING PROTEIN / PEPTIDASE / PROTEIN QUALITY CONTROL / OLIGOMER / AMINOPEPTIDASE
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 OT3 (archaea)
MethodELECTRON MICROSCOPY / SOLUTION NMR / single particle reconstruction / simulated annealing / cryo EM / Resolution: 4.1 Å
AuthorsColletier, J.-P. / Gauto, D. / Estrozi, L. / Favier, A. / Effantin, G. / Schoehn, G. / Boisbouvier, J. / Schanda, P.
Funding support France, 6items
OrganizationGrant numberCountry
French National Research AgencyANR-17-CE11-0018-01 France
French National Research AgencyANR-10-INSB-05-02 France
French National Research AgencyANR-10-LABX-49-01 France
European Research CouncilStG-2012-311318-ProtDyn2Function France
European Research CouncilCoG-2010-260887 France
European Research CouncilStG-311318 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
DepositionApr 2, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 14, 2019Provider: repository / Type: Initial release
Revision 1.1Aug 21, 2019Group: Data collection / Category: pdbx_nmr_software / Item: _pdbx_nmr_software.name
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.3Sep 13, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Tetrahedral aminopeptidase
B: Tetrahedral aminopeptidase
C: Tetrahedral aminopeptidase
D: Tetrahedral aminopeptidase
E: Tetrahedral aminopeptidase
F: Tetrahedral aminopeptidase
G: Tetrahedral aminopeptidase
H: Tetrahedral aminopeptidase
I: Tetrahedral aminopeptidase
J: Tetrahedral aminopeptidase
K: Tetrahedral aminopeptidase
L: Tetrahedral aminopeptidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)470,42236
Polymers468,85212
Non-polymers1,57024
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, microscopy, CryoEM, negative staining EM
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)1 / 100structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein
Tetrahedral aminopeptidase / TET aminopeptidase / Leucyl aminopeptidase / PhTET2


Mass: 39071.027 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus horikoshii OT3 (archaea) / Gene: frvX, PH1527
Variant: ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3
Production host: Escherichia coli (E. coli)
References: UniProt: O59196, Hydrolases; Acting on peptide bonds (peptidases); Aminopeptidases
#2: Chemical...
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: Zn

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Experimental details

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Experiment

Experiment
Method
ELECTRON MICROSCOPY
SOLUTION NMR
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic2RFDR 3D
323isotropic2DARR
232isotropic33D 1H-13C NOESY aliphatic
344isotropic3CCdream (CC)
354isotropic3NCO
364isotropic3NCOCX
374isotropic3NCACX
384isotropic3CONCACB
394isotropic3CANCOCX
1105isotropic2hCANH
1115isotropic4hCONH
1125isotropic2hcoCAcoNH

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Sample preparation

ComponentName: TETRAHEDRAL AMINOPEPTIDASE TET2 / Type: COMPLEX
Details: The structure was built using combined SOLID-STATE NMR, SOLUTION-STATE NMR AND EM DATA, and then refined using phenix.real_space_refine
Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Pyrococcus horikoshii OT3 (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: Blotting time 2s, force 1, drain time 0.
Details
TypeSolution-IDContentsDetailsLabelSolvent system
solid120 mM TRIS, 50 mM non sodium chloride, 100 % H2Oprotein solution at 10 mg/ml was supplemented by methyl-pentanediol, resulting in microcrystalline precipitate. Labeling 2H15N and 13C at methyls of ILV2H15N_ILVCHD2100 % H2O
solution250 mM Tris, 50 mM NaCl, 100% D2O2H15N_ILV-CH3100% D2O
solid320 mM Tris, 50 mM NaCl, 100% H2Oprotein solution at 10 mg/ml was supplemented by methyl-pentanediol, resulting in microcrystalline precipitate. Labeling 13C at all carbons of LKP residuesLKP-labeled100% H2O
solid420 mM Tris, 50 mM NaCl, 100% H2OH2O13C15N TET2100% H2O
solid520 mM TRIS, 50 mM NaCl, 100% H2O2H13C15N100% H2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
20 mMTRISnone1
50 mMsodium chloridenon1
50 mMTrisnone2
50 mMNaClnone2
20 mMTrisnone3
50 mMNaClnone3
20 mMTrisnone4
50 mMNaClnone4
20 mMTRISnone5
50 mMNaClnone5
Sample conditions
Conditions-IDIonic strengthLabelpHPressure (kPa)Temperature (K)Temperature err
150 mM50 kHz MAS7.5 1 atm300 K2
250 mMsolution8 1 atm328 K
350 mM15kHz MAS7.5 1 atm300 K

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Data collection

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 20000 X / Calibrated magnification: 27773 X / Calibrated defocus min: 8000 nm / Calibrated defocus max: 30000 nm / Cs: 2 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 90
Image scansMovie frames/image: 40
NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCE IIBrukerAVANCE II6002
Varian INOVAVarianINOVA8003
Varian INOVAVarianINOVA6004

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
1RELION1.4particle selection
2Latitude3image acquisition
4CTFFIND3CTF correction
7iMODFIT1.44model fitting
9RELION1.4initial Euler assignment
11RELION1.4classification
12RELION1.43D reconstruction
13X-PLOR4.39model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 30407
SymmetryPoint symmetry: T (tetrahedral)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27130 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
NMR software
NameVersionDeveloperClassification
TopSpin3.2Bruker Biospincollection
VnmrJAgilentcollection
CYANA3.97Guntert, Mumenthaler and Wuthrichstructure calculation
CcpNmr AnalysisCCPNchemical shift assignment
CcpNmr AnalysisCCPNpeak picking
Xplor-NIH2.44.8G. Marius Clore , Guillermo Bermejo, , John Kuszewski, Charles D. Schwieters, and Nico Tjandrastructure calculation
RefinementMethod: simulated annealing / Software ordinal: 7
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 1

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