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- PDB-6wu0: CryoEM structure of Burkholderia pseudomallei hopanoid biosynthes... -

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Basic information

Entry
Database: PDB / ID: 6wu0
TitleCryoEM structure of Burkholderia pseudomallei hopanoid biosynthesis-associated RND transporter HpnN
ComponentsHopanoid biosynthesis associated RND transporter like protein HpnN
KeywordsPROTEIN TRANSPORT / CryoEM structure / hopanoid transporter HpnN
Function / homologyHopanoid biosynthesis associated RND transporter-like protein HpnN / Membrane transport protein MMPL domain / MMPL family / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain / membrane => GO:0016020 / plasma membrane / Hopanoid biosynthesis associated RND transporter like protein HpnN
Function and homology information
Biological speciesBurkholderia pseudomallei MSHR346 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.59 Å
AuthorsLyu, M. / Yu, E.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
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 1.0Feb 3, 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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Assembly

Deposited unit
B: Hopanoid biosynthesis associated RND transporter like protein HpnN


Theoretical massNumber of molelcules
Total (without water)92,6011
Polymers92,6011
Non-polymers00
Water0
1


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

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Components

#1: Protein Hopanoid biosynthesis associated RND transporter like protein HpnN


Mass: 92600.930 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Burkholderia pseudomallei MSHR346 (bacteria)
Gene: hpnN, GBP346_B2639 / Production host: Escherichia coli (E. coli) / References: UniProt: C4I4K0

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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: hopanoid transporter HpnN / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia Coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.15_3459: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 63910 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026467
ELECTRON MICROSCOPYf_angle_d0.6018832
ELECTRON MICROSCOPYf_dihedral_angle_d9.4423905
ELECTRON MICROSCOPYf_chiral_restr0.041088
ELECTRON MICROSCOPYf_plane_restr0.0041124

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