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- PDB-9g9m: Lipid III flippase WzxE with NB10 and NB7 nanobodies in outward-f... -

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Basic information

Entry
Database: PDB / ID: 9g9m
TitleLipid III flippase WzxE with NB10 and NB7 nanobodies in outward-facing conformation - crystal 1
Components
  • Lipid III flippase
  • NB10 Nanobody
  • NB7 Nanobody
KeywordsTRANSPORT PROTEIN / membrane protein / flippase / cell wall / enterobacterial common antigen / lipid III
Function / homology
Function and homology information


intramembrane lipid transporter activity / enterobacterial common antigen biosynthetic process / plasma membrane
Similarity search - Function
Polysaccharide biosynthesis protein / Lipid III flippase WzxE, Proteobacteria / Lipid III flippase WzxE / : / Polysaccharide biosynthesis protein
Similarity search - Domain/homology
PHOSPHATE ION / Lipid III flippase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Lama glama (llama)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.55 Å
AuthorsLe Bas, A. / Naismith, J.H.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: Open Biology / Year: 2025
Title: Structure of WzxE the lipid III flippase for Enterobacterial Common Antigen polysaccharide.
Authors: Le Bas, A. / Clarke, B.R. / Teelucksingh, T. / Lee, M. / El Omari, K. / Giltrap, A.M. / McMahon, S.A. / Liu, H. / Beale, J.H. / Mykhaylyk, V. / Duman, R. / Paterson, N.G. / Ward, P.N. / ...Authors: Le Bas, A. / Clarke, B.R. / Teelucksingh, T. / Lee, M. / El Omari, K. / Giltrap, A.M. / McMahon, S.A. / Liu, H. / Beale, J.H. / Mykhaylyk, V. / Duman, R. / Paterson, N.G. / Ward, P.N. / Harrison, P.J. / Weckener, M. / Pardon, E. / Steyaert, J. / Liu, H. / Quigley, A. / Davis, B.G. / Wagner, A. / Whitfield, C. / Naismith, J.H.
History
DepositionJul 25, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Lipid III flippase
B: NB10 Nanobody
C: NB7 Nanobody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)76,5824
Polymers76,4873
Non-polymers951
Water68538
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)38.855, 152.687, 287.500
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Space group name H-MC2221

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Components

#1: Protein Lipid III flippase


Mass: 46130.621 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: wzxE, wzx, yifJ, b3792, JW3766 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: P0AAA7
#2: Antibody NB10 Nanobody


Mass: 15132.753 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli) / Strain (production host): wk6 su-
#3: Antibody NB7 Nanobody


Mass: 15223.880 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli) / Strain (production host): wk6 su-
#4: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 38 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.79 Å3/Da / Density % sol: 55.87 %
Crystal growTemperature: 293 K / Method: lipidic cubic phase / pH: 5
Details: tri-sodium citrate, ammonium acetate, 1-propanol, PEG400, E. coli polar lipids, monoolein

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 0.6199 Å
DetectorType: DECTRIS EIGER2 X CdTe 9M / Detector: PIXEL / Date: Jul 7, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.6199 Å / Relative weight: 1
ReflectionResolution: 2.55→32.52 Å / Num. obs: 28454 / % possible obs: 99.2 % / Redundancy: 6.2 % / CC1/2: 1 / Net I/σ(I): 5.9
Reflection shellResolution: 2.55→2.6 Å / Redundancy: 4.1 % / Mean I/σ(I) obs: 0.6 / Num. unique obs: 1280 / CC1/2: 0.3 / % possible all: 89.4

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
DIALSdata scaling
xia2data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.55→32.52 Å / SU ML: 0.4 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 29.03 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.287 1430 5.05 %
Rwork0.2346 --
obs0.2373 28333 98.75 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.55→32.52 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5096 0 5 38 5139
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0025217
X-RAY DIFFRACTIONf_angle_d0.4037092
X-RAY DIFFRACTIONf_dihedral_angle_d3.788732
X-RAY DIFFRACTIONf_chiral_restr0.035809
X-RAY DIFFRACTIONf_plane_restr0.004879
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.55-2.640.39411440.33882403X-RAY DIFFRACTION91
2.64-2.750.29971380.28522622X-RAY DIFFRACTION97
2.75-2.870.30281240.26892671X-RAY DIFFRACTION100
2.87-3.020.3211510.26692699X-RAY DIFFRACTION100
3.02-3.210.31981530.24272675X-RAY DIFFRACTION100
3.21-3.460.29311360.24292706X-RAY DIFFRACTION100
3.46-3.810.29231540.22382704X-RAY DIFFRACTION100
3.81-4.360.2611450.21072757X-RAY DIFFRACTION100
4.36-5.490.26071400.22662754X-RAY DIFFRACTION100
5.49-32.520.27341450.21192912X-RAY DIFFRACTION100
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
11.95350.12360.9950.83420.55582.4104-0.03120.05480.09880.0284-0.03370.01070.0023-0.08160.04180.31320.01440.00450.2557-0.00340.2421-4.3142-23.3168-48.78
22.713-1.06940.31451.428-0.9172.0543-0.2412-0.3937-0.32690.35270.18390.09260.114-0.01990.05360.54980.10820.0930.30660.0570.3728-1.9887-46.8499-43.8915
33.0086-1.471-1.99095.72592.90837.07640.24230.220.06560.013-0.0175-0.1231-0.1338-0.0738-0.21330.3381-0.0234-0.0380.3564-0.00650.2607-3.4974-5.5762-13.1409
44.7075-0.53331.08034.1350.07366.25860.2051-0.08510.2156-0.1959-0.05690.0494-0.8862-0.2715-0.10150.70040.19860.04460.44880.02350.3726.8046-52.708-5.7168
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 228 )
2X-RAY DIFFRACTION2chain 'A' and (resid 229 through 415 )
3X-RAY DIFFRACTION3chain 'B' and (resid 3 through 127 )
4X-RAY DIFFRACTION4chain 'C' and (resid 3 through 124 )

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