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- PDB-6wtz: Cryo-EM structure of E. Coli OmpF -

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Basic information

Entry
Database: PDB / ID: 6wtz
TitleCryo-EM structure of E. Coli OmpF
ComponentsOuter membrane porin F
KeywordsMEMBRANE PROTEIN / outer membrane porin / omp / ompf
Function / homology
Function and homology information


colicin transmembrane transporter activity / monoatomic ion channel complex / porin activity / pore complex / protein homotrimerization / monoatomic ion transmembrane transport / lipopolysaccharide binding / cell outer membrane / disordered domain specific binding / monoatomic ion channel activity ...colicin transmembrane transporter activity / monoatomic ion channel complex / porin activity / pore complex / protein homotrimerization / monoatomic ion transmembrane transport / lipopolysaccharide binding / cell outer membrane / disordered domain specific binding / monoatomic ion channel activity / lipid binding / membrane / identical protein binding
Similarity search - Function
Porin, gammaproteobacterial / Porin, Gram-negative type, conserved site / General diffusion Gram-negative porins signature. / Porin domain, Gram-negative type / Gram-negative porin / Porin, Gram-negative type / Porin domain superfamily
Similarity search - Domain/homology
Outer membrane porin F
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsMorgan, C.E. / Su, C.-C. / Lyu, M. / Yu, E.W.
Funding support1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
CitationJournal: Nat Methods / Year: 2021
Title: A 'Build and Retrieve' methodology to simultaneously solve cryo-EM structures of membrane proteins.
Authors: Chih-Chia Su / Meinan Lyu / Christopher E Morgan / Jani Reddy Bolla / Carol V Robinson / Edward W Yu /
Abstract: Single-particle cryo-electron microscopy (cryo-EM) has become a powerful technique in the field of structural biology. However, the inability to reliably produce pure, homogeneous membrane protein ...Single-particle cryo-electron microscopy (cryo-EM) has become a powerful technique in the field of structural biology. However, the inability to reliably produce pure, homogeneous membrane protein samples hampers the progress of their structural determination. Here, we develop a bottom-up iterative method, Build and Retrieve (BaR), that enables the identification and determination of cryo-EM structures of a variety of inner and outer membrane proteins, including membrane protein complexes of different sizes and dimensions, from a heterogeneous, impure protein sample. We also use the BaR methodology to elucidate structural information from Escherichia coli K12 crude membrane and raw lysate. The findings demonstrate that it is possible to solve high-resolution structures of a number of relatively small (<100 kDa) and less abundant (<10%) unidentified membrane proteins within a single, heterogeneous sample. Importantly, these results highlight the potential of cryo-EM for systems structural proteomics.
History
DepositionMay 4, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 20, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: 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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Structure visualization

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Assembly

Deposited unit
A: Outer membrane porin F
B: Outer membrane porin F
C: Outer membrane porin F


Theoretical massNumber of molelcules
Total (without water)118,0953
Polymers118,0953
Non-polymers00
Water64936
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Outer membrane porin F / Outer membrane protein 1A / Outer membrane protein B / Outer membrane protein F / Outer membrane ...Outer membrane protein 1A / Outer membrane protein B / Outer membrane protein F / Outer membrane protein IA / Porin OmpF


Mass: 39365.043 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / Strain: K12 / References: UniProt: P02931
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 36 / Source method: isolated from a natural source / Formula: H2O

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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: OmpF / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameCategory
2Latitudeimage acquisition
4cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43793 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048049
ELECTRON MICROSCOPYf_angle_d0.7310890
ELECTRON MICROSCOPYf_dihedral_angle_d12.334566
ELECTRON MICROSCOPYf_chiral_restr0.0521104
ELECTRON MICROSCOPYf_plane_restr0.0051470

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