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- PDB-6xv8: Crystal structure of Megabody Mb-Nb207-c7HopQ_G10 -

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Basic information

Entry
Database: PDB / ID: 6xv8
TitleCrystal structure of Megabody Mb-Nb207-c7HopQ_G10
ComponentsOuter membrane proteinVirulence-related outer membrane protein family
KeywordsSTRUCTURAL PROTEIN / Scaffold / Megabody
Function / homologySabA, N-terminal extracellular adhesion domain / SabA N-terminal extracellular adhesion domain / Outer membrane protein, Helicobacter / Helicobacter outer membrane protein / Outer membrane protein
Function and homology information
Biological speciesLama glama (llama)
Helicobacter pylori (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.15 Å
AuthorsSteyaert, J. / Uchanski, T. / Fischer, B.
Funding support Belgium, 1items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO) Belgium
CitationJournal: Nat Methods / Year: 2021
Title: Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM.
Authors: Tomasz Uchański / Simonas Masiulis / Baptiste Fischer / Valentina Kalichuk / Uriel López-Sánchez / Eleftherios Zarkadas / Miriam Weckener / Andrija Sente / Philip Ward / Alexandre ...Authors: Tomasz Uchański / Simonas Masiulis / Baptiste Fischer / Valentina Kalichuk / Uriel López-Sánchez / Eleftherios Zarkadas / Miriam Weckener / Andrija Sente / Philip Ward / Alexandre Wohlkönig / Thomas Zögg / Han Remaut / James H Naismith / Hugues Nury / Wim Vranken / A Radu Aricescu / Els Pardon / Jan Steyaert /
Abstract: Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their ...Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.
History
DepositionJan 21, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 13, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 20, 2021Group: Database references / Category: citation
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Outer membrane protein
B: Outer membrane protein
C: Outer membrane protein
D: Outer membrane protein


Theoretical massNumber of molelcules
Total (without water)224,6634
Polymers224,6634
Non-polymers00
Water0
1
A: Outer membrane protein


Theoretical massNumber of molelcules
Total (without water)56,1661
Polymers56,1661
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Outer membrane protein


Theoretical massNumber of molelcules
Total (without water)56,1661
Polymers56,1661
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Outer membrane protein


Theoretical massNumber of molelcules
Total (without water)56,1661
Polymers56,1661
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: Outer membrane protein


Theoretical massNumber of molelcules
Total (without water)56,1661
Polymers56,1661
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)79.241, 155.049, 89.754
Angle α, β, γ (deg.)90.000, 91.726, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Antibody
Outer membrane protein / Virulence-related outer membrane protein family / Mb-Nb207-c7HopQ_G10


Mass: 56165.656 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama), (gene. exp.) Helicobacter pylori (strain G27) (bacteria)
Gene: Nb207, hopQ, HPG27_1120 / Strain: G27 / Production host: Escherichia coli (E. coli) / References: UniProt: B5Z8H1
Sequence detailsMegabody Mb-Nb207-c7HopQ_G10 is a chimeric protein with circular permutation of HopQ: Residues 1-12: ...Megabody Mb-Nb207-c7HopQ_G10 is a chimeric protein with circular permutation of HopQ: Residues 1-12: a part of a β-strand A of the Nanobody fold. Residue 13: one amino acid linker. Residues 14-232: C-terminal part of HopQ (residues 228-446, UniProtKB B5Z8H1). Residues 233-400: N-terminal part of HopQ (residues 53-220, UniProtKB B5Z8H1). Residue 401: one amino acid linker. Residues 402-511: a part of the Nanobody fold. Residues 512-517: the His6 tag. Residues 518-521: the EPEA tag.

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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: 2.35 Å3/Da / Density % sol: 47.7 %
Crystal growTemperature: 293 K / Method: vapor diffusion / pH: 7.5 / Details: 0.1 M MgCl2, 0.1 M HEPES pH 7.5, 19% PEG 4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9763 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: May 20, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 3.15→79.23 Å / Num. obs: 36041 / % possible obs: 96.1 % / Redundancy: 1.99 % / Biso Wilson estimate: 98.93 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.04 / Net I/σ(I): 7.9
Reflection shellResolution: 3.15→3.26 Å / Redundancy: 1.99 % / Rmerge(I) obs: 0.514 / Mean I/σ(I) obs: 1.3 / Num. unique obs: 3735 / CC1/2: 0.631 / Rrim(I) all: 0.726 / % possible all: 99.8

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5LP2

5lp2


Resolution: 3.15→79.21 Å / SU ML: 0.4924 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 36.3019
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflectionSelection details
Rfree0.2966 1684 4.68 %0.05
Rwork0.2546 34322 --
obs0.2566 36006 96.01 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 114.68 Å2
Refinement stepCycle: LAST / Resolution: 3.15→79.21 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12431 0 0 0 12431
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.009612599
X-RAY DIFFRACTIONf_angle_d1.338917035
X-RAY DIFFRACTIONf_chiral_restr0.13751946
X-RAY DIFFRACTIONf_plane_restr0.00942214
X-RAY DIFFRACTIONf_dihedral_angle_d14.7914531
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.15-3.240.37011470.36092944X-RAY DIFFRACTION99.74
3.24-3.350.34861380.31432986X-RAY DIFFRACTION99.9
3.35-3.470.36711850.30592944X-RAY DIFFRACTION99.9
3.47-3.580.36971410.35252429X-RAY DIFFRACTION99.15
3.61-3.770.545950.48722154X-RAY DIFFRACTION74.32
3.77-3.970.33911590.27462936X-RAY DIFFRACTION99.87
3.97-4.220.29221030.23753005X-RAY DIFFRACTION99.78
4.22-4.540.25061190.21152989X-RAY DIFFRACTION99.78
4.54-50.27881510.20352979X-RAY DIFFRACTION99.9
5-5.720.2691430.232977X-RAY DIFFRACTION99.9
5.72-7.210.33871490.26762993X-RAY DIFFRACTION99.46
7.21-79.210.22791540.21732986X-RAY DIFFRACTION99.05

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