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- PDB-7omm: Cryo-EM structure of N. gonorhoeae LptDE in complex with ProMacro... -

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Basic information

Entry
Database: PDB / ID: 7omm
TitleCryo-EM structure of N. gonorhoeae LptDE in complex with ProMacrobodies (MBPs have not been built de novo)
Components
  • LPS-assembly lipoprotein LptE
  • LPS-assembly protein LptD
  • ProMacrobody 21,Maltodextrin-binding protein
  • ProMacrobody 51,Maltodextrin-binding protein
KeywordsTRANSPORT PROTEIN / Outer membrane protein LPS transporter Bacterial transporter Neisseria gonorrhoeae Drug target ProMacrobodies Structural chaperone Cryo-electron microscopy
Function / homology
Function and homology information


lipopolysaccharide transport / Gram-negative-bacterium-type cell outer membrane assembly / carbohydrate transmembrane transporter activity / : / cell outer membrane
Similarity search - Function
LptD, C-terminal / LPS-assembly protein LptD / LPS transport system D / Lipopolysaccharide-assembly / LPS-assembly lipoprotein LptE / Pectin lyase fold/virulence factor / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein
Similarity search - Domain/homology
Maltodextrin-binding protein / LPS-assembly lipoprotein LptE / LPS-assembly protein LptD
Similarity search - Component
Biological speciesNeisseria gonorrhoeae (bacteria)
synthetic construct (others)
Methanosarcina mazei (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsBotte, M. / Ni, D. / Schenck, S. / Zimmermann, I. / Chami, M. / Bocquet, N. / Egloff, P. / Bucher, D. / Trabuco, M. / Cheng, R.K.Y. ...Botte, M. / Ni, D. / Schenck, S. / Zimmermann, I. / Chami, M. / Bocquet, N. / Egloff, P. / Bucher, D. / Trabuco, M. / Cheng, R.K.Y. / Brunner, J.D. / Seeger, M.A. / Stahlberg, H. / Hennig, M.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Innosuisse25864.1 PFLS-LS Switzerland
CitationJournal: Nat Commun / Year: 2022
Title: Cryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation.
Authors: Mathieu Botte / Dongchun Ni / Stephan Schenck / Iwan Zimmermann / Mohamed Chami / Nicolas Bocquet / Pascal Egloff / Denis Bucher / Matilde Trabuco / Robert K Y Cheng / Janine D Brunner / ...Authors: Mathieu Botte / Dongchun Ni / Stephan Schenck / Iwan Zimmermann / Mohamed Chami / Nicolas Bocquet / Pascal Egloff / Denis Bucher / Matilde Trabuco / Robert K Y Cheng / Janine D Brunner / Markus A Seeger / Henning Stahlberg / Michael Hennig /
Abstract: Lipopolysaccharides are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram- ...Lipopolysaccharides are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram-negative bacteria. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex. Detailed insights into the architecture of LptDE transporter complexes have been derived from X-ray crystallography. However, no structure of a laterally open LptD transporter, a transient state that occurs during LPS release, is available to date. Here, we report a cryo-EM structure of a partially opened LptDE transporter in complex with rigid chaperones derived from nanobodies, at 3.4 Å resolution. In addition, a subset of particles allows to model a structure of a laterally fully opened LptDE complex. Our work offers insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a highly rigid chaperone scaffold to enable structural biology of challenging protein targets.
History
DepositionMay 24, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 4, 2022Provider: repository / Type: Initial release
Revision 1.1May 11, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: LPS-assembly protein LptD
B: LPS-assembly lipoprotein LptE
C: ProMacrobody 21,Maltodextrin-binding protein
D: ProMacrobody 51,Maltodextrin-binding protein


Theoretical massNumber of molelcules
Total (without water)219,7324
Polymers219,7324
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, surface plasmon resonance
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6610 Å2
ΔGint-21 kcal/mol
Surface area80900 Å2
MethodPISA
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "D" and (resid 5 through 13 or resid 15...
d_2ens_1(chain "C" and (resid 5 through 13 or resid 15...

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1VALGLYD5 - 13
d_12ens_1VALGLND15 - 16
d_13ens_1GLYGLYD18 - 29
d_14ens_1TRPTRPD39
d_15ens_1ARGGLUD41 - 49
d_16ens_1VALALAD51 - 53
d_17ens_1THRTHRD61
d_18ens_1TYRTHRD63 - 72
d_19ens_1SERSERD74
d_110ens_1ASPALAD76 - 95
d_111ens_1TYRCYSD97 - 99
d_112ens_1GLYGLYD105
d_113ens_1TRPTRPD107
d_114ens_1ALAALAD114
d_115ens_1TYRTYRD116
d_116ens_1TYRPROD118 - 128
d_117ens_1PROTHRD1 - 361
d_21ens_1VALGLYC1 - 9
d_22ens_1VALGLNC11 - 12
d_23ens_1GLYGLYC14 - 25
d_24ens_1TRPTRPC35
d_25ens_1ARGGLUC37 - 45
d_26ens_1VALALAC47 - 49
d_27ens_1THRTHRC57
d_28ens_1TYRTHRC59 - 68
d_29ens_1SERSERC70
d_210ens_1ASPALAC72 - 91
d_211ens_1TYRCYSC93 - 95
d_212ens_1GLYGLYC101
d_213ens_1TRPALAC103 - 104
d_214ens_1TYRTYRC106
d_215ens_1TYRPROC108 - 118
d_216ens_1PROTHRD1 - 361

NCS oper: (Code: givenMatrix: (0.489826801644, -0.211509938388, 0.845773758374), (0.276182027989, -0.882513923739, -0.380647687269), (0.82691788698, 0.420038951016, -0.373863728948)Vector: 6. ...NCS oper: (Code: given
Matrix: (0.489826801644, -0.211509938388, 0.845773758374), (0.276182027989, -0.882513923739, -0.380647687269), (0.82691788698, 0.420038951016, -0.373863728948)
Vector: 6.23031577385, 348.729660034, -1.77372477356)

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Components

#1: Protein LPS-assembly protein LptD


Mass: 87656.930 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria gonorrhoeae (bacteria) / Gene: lptD, imp, ostA, NGO1715 / Production host: Escherichia coli (E. coli) / References: UniProt: Q5F651
#2: Protein LPS-assembly lipoprotein LptE


Mass: 18499.066 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria gonorrhoeae (bacteria)
Gene: lptE, F0T10_01625, F0T11_01625, F0T12_01620, WHOF_00235, WHOF_00301
Production host: Escherichia coli (E. coli) / References: UniProt: A0A5K1Q6A7
#3: Antibody ProMacrobody 21,Maltodextrin-binding protein


Mass: 56714.457 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others), (gene. exp.) Methanosarcina mazei (archaea)
Gene: malE, DU57_01695 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0F8L1I7
#4: Antibody ProMacrobody 51,Maltodextrin-binding protein


Mass: 56861.590 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others), (gene. exp.) Methanosarcina mazei (archaea)
Gene: malE, DU57_01695 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0F8L1I7

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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: Cryo-EM structure of N. gonorhoeae LptDE in complex with ProMacrobodies
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Neisseria gonorrhoeae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml / Embedding 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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 184206 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 79.99 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0114321
ELECTRON MICROSCOPYf_angle_d1.52919431
ELECTRON MICROSCOPYf_dihedral_angle_d26.2915177
ELECTRON MICROSCOPYf_chiral_restr0.1862052
ELECTRON MICROSCOPYf_plane_restr0.0072543

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