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- PDB-6ye4: Structure of ExbB pentamer from Serratia marcescens by single par... -

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Basic information

Entry
Database: PDB / ID: 6ye4
TitleStructure of ExbB pentamer from Serratia marcescens by single particle cryo electron microscopy
ComponentsBiopolymer transport protein ExbB
KeywordsMETAL TRANSPORT / membrane protein / iron uptake / proton transfer / TonB complex
Function / homologyTonB-system energizer ExbB type-1 / MotA/TolQ/ExbB proton channel / MotA/TolQ/ExbB proton channel family / transmembrane transporter activity / protein transport / membrane => GO:0016020 / plasma membrane / Chem-PGT / Biopolymer transport protein ExbB
Function and homology information
Biological speciesSerratia marcescens (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBiou, V. / Delepelaire, P. / Coureux, P.D. / Chami, M.
Funding support France, 1items
OrganizationGrant numberCountry
French National Research AgencyANR-11-LABEX-0011-01 France
Citation
Journal: Commun Biol / Year: 2022
Title: Structural and molecular determinants for the interaction of ExbB from Serratia marcescens and HasB, a TonB paralog.
Authors: Valérie Biou / Ricardo Jorge Diogo Adaixo / Mohamed Chami / Pierre-Damien Coureux / Benoist Laurent / Véronique Yvette Ntsogo Enguéné / Gisele Cardoso de Amorim / Nadia Izadi-Pruneyre / ...Authors: Valérie Biou / Ricardo Jorge Diogo Adaixo / Mohamed Chami / Pierre-Damien Coureux / Benoist Laurent / Véronique Yvette Ntsogo Enguéné / Gisele Cardoso de Amorim / Nadia Izadi-Pruneyre / Christian Malosse / Julia Chamot-Rooke / Henning Stahlberg / Philippe Delepelaire /
Abstract: ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The ...ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens (Sm) possesses both TonB and a heme-specific TonB paralog, HasB. ExbB has a long periplasmic extension absent in other bacteria such as E. coli (Ec). Long ExbB's are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbB and HasB. We determined the cryo-EM structures of ExbB and of the ExbB-ExbD complex from S. marcescens. ExbB alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbB extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbB and ExbB, essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the class of inner membrane energy machinery formed by ExbB, ExbD and HasB.
#1: Journal: Biorxiv / Year: 2021
Title: Functional and structural characterization of Serratia marcescens ExbB: determinants of the interaction with HasB/TonB
Authors: Biou, V. / Chami, M. / Coureux, P.D. / Laurent, B. / Ntsogo, Y. / Izadi-Pruneyre, N. / Malosse, C. / Chamot-Rooke, J. / Stahlberg, H. / Delepelaire, P.
#2: Journal: J Struct Biol / Year: 2012
Title: RELION: implementation of a Bayesian approach to cryo-EM structure determination.
Authors: Sjors H W Scheres /
Abstract: RELION, for REgularized LIkelihood OptimizatioN, is an open-source computer program for the refinement of macromolecular structures by single-particle analysis of electron cryo-microscopy (cryo-EM) ...RELION, for REgularized LIkelihood OptimizatioN, is an open-source computer program for the refinement of macromolecular structures by single-particle analysis of electron cryo-microscopy (cryo-EM) data. Whereas alternative approaches often rely on user expertise for the tuning of parameters, RELION uses a Bayesian approach to infer parameters of a statistical model from the data. This paper describes developments that reduce the computational costs of the underlying maximum a posteriori (MAP) algorithm, as well as statistical considerations that yield new insights into the accuracy with which the relative orientations of individual particles may be determined. A so-called gold-standard Fourier shell correlation (FSC) procedure to prevent overfitting is also described. The resulting implementation yields high-quality reconstructions and reliable resolution estimates with minimal user intervention and at acceptable computational costs.
History
DepositionMar 24, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 31, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_2 / em_admin / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update
Revision 1.2May 4, 2022Group: Database references / Category: citation / citation_author
Revision 1.3Jul 10, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _citation.journal_id_ISSN / _em_3d_fitting_list.accession_code ..._citation.journal_id_ISSN / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-10789
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Biopolymer transport protein ExbB
B: Biopolymer transport protein ExbB
C: Biopolymer transport protein ExbB
D: Biopolymer transport protein ExbB
E: Biopolymer transport protein ExbB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)155,82310
Polymers152,0685
Non-polymers3,7555
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, sample appears as a homogeneous peak
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21040 Å2
ΔGint-185 kcal/mol
Surface area54830 Å2
MethodPISA

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Components

#1: Protein
Biopolymer transport protein ExbB / Tol-pal system-associated acyl-CoA thioesterase


Mass: 30413.621 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Details: a 6-histidine tag is present at the C-terminus / Source: (gene. exp.) Serratia marcescens (bacteria) / Gene: exbB, FG174_21755, PWN146_03792 / Plasmid: pBAD24 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A1C3HJ46
#2: Chemical
ChemComp-PGT / (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE / PHOSPHATIDYLGLYCEROL / 1-PALMITOYL-2-OLEOYL-SN-GLYCERO-3-[PHOSPHO-RAC-(1-GLYCEROL)](SODIUM SALT)


Mass: 751.023 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C40H79O10P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Homopentamer of ExbB / Type: COMPLEX
Details: the expressed sequence corresponds to the mature sequence after signal peptide cleavage.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.174 MDa / Experimental value: NO
Source (natural)Organism: Serratia marcescens (bacteria) / Strain: Db11
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pBAD24
Buffer solutionpH: 8 / Details: 20mM Tris-HCl pH 8,0 100mM NaCl 0,0015% LMNG
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris-HCl1
2100 mMSodium chlorideNaCl1
30.0015 % w/vlauryl maltose neopentyl glycolLMNG1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: the sample was monodisperse as evidenced by gel filtration column
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/4
VitrificationInstrument: LEICA EM CPC / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 93 K / Temperature (min): 83 K
Image recordingAverage exposure time: 7 sec. / Electron dose: 55.95 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3122
Details: frames were weighted according to electron dose and particle movement during Relion bayesian polishing procedure.
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 3710 / Height: 3838 / Movie frames/image: 56 / Used frames/image: 1-56

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategoryDetails
1RELION3particle selectionautopicking after 2D classification on a subset
2SerialEMimage acquisition
4Gctf1.06CTF correction
5RELION3CTF correction
8UCSF Chimera1.15model fitting
10PHENIX1.14model refinementphenix_real_space_refinement
11RELION3initial Euler assignment
12RELION3final Euler assignment
13RELION3classification
14RELION33D reconstruction
Image processingDetails: images were processed with Motioncor2
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1291382
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 157111 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 55.3 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Details: the ExbB sequence from S. marcescens was modeled by homology from the 5SV0 monomer from E. coli using Phyre software and the pentamer was generated using the 5SV0 symmetry. real space ...Details: the ExbB sequence from S. marcescens was modeled by homology from the 5SV0 monomer from E. coli using Phyre software and the pentamer was generated using the 5SV0 symmetry. real space refinement was carried out with rigid body, simulated annealing and morphing steps.
Atomic model buildingPDB-ID: 5SV0

5sv0


Pdb chain-ID: A / Accession code: 5SV0 / Pdb chain residue range: 10-234 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 45.97 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.02629270
ELECTRON MICROSCOPYf_angle_d1.555812515
ELECTRON MICROSCOPYf_chiral_restr0.10231450
ELECTRON MICROSCOPYf_plane_restr0.011610
ELECTRON MICROSCOPYf_dihedral_angle_d23.06273395

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