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- PDB-6xux: Crystal structure of Megabody Mb-Nb207-cYgjK_NO -

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Basic information

Entry
Database: PDB / ID: 6xux
TitleCrystal structure of Megabody Mb-Nb207-cYgjK_NO
ComponentsNanobody,Glucosidase YgjK,Glucosidase YgjK,Nanobody
KeywordsSTRUCTURAL PROTEIN / Megabody / Scaffold
Function / homology
Function and homology information


glucosidase complex / trehalose catabolic process / alpha,alpha-trehalase activity / glucosidase activity / organic substance catabolic process / Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds / DNA damage response / metal ion binding
Similarity search - Function
: / Glucosidase YgjK, N-terminal / Glycoside hydrolase, family 37 / Trehalase / Six-hairpin glycosidase-like superfamily / Six-hairpin glycosidase superfamily
Similarity search - Domain/homology
Biological speciesEscherichia coli K-12 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.90000643078 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

Downloads & links

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Assembly

Deposited unit
A: Nanobody,Glucosidase YgjK,Glucosidase YgjK,Nanobody
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,6592
Polymers101,6191
Non-polymers401
Water11,710650
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area33990 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.450, 84.460, 140.320
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Antibody Nanobody,Glucosidase YgjK,Glucosidase YgjK,Nanobody


Mass: 101619.000 Da / Num. of mol.: 1 / Fragment: beta-strand A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: ygjK, b3080, JW3051 / Production host: Escherichia coli (E. coli)
References: UniProt: P42592, Hydrolases; Glycosylases; Glycosidases, i.e. enzymes that hydrolyse O- and S-glycosyl compounds
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 650 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 1.96 Å3/Da / Density % sol: 37.2 %
Crystal growTemperature: 293 K / Method: vapor diffusion
Details: 0.1 M Sodium citrate pH 7.5, 20% PEG 8000, 20% glycerol

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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 XE 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: 1.9→42.15 Å / Num. obs: 63885 / % possible obs: 100 % / Redundancy: 1.999 % / Biso Wilson estimate: 21.2241179387 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.038 / Rrim(I) all: 0.054 / Net I/σ(I): 11.1
Reflection shellResolution: 1.9→1.97 Å / Redundancy: 1.999 % / Rmerge(I) obs: 0.234 / Mean I/σ(I) obs: 3.4 / Num. unique obs: 6311 / CC1/2: 0.869 / Rrim(I) all: 0.331 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.12_2829refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3W7S

3w7s


Resolution: 1.90000643078→42.1397385191 Å / SU ML: 0.194259667698 / Cross valid method: FREE R-VALUE / σ(F): 1.34351852722 / Phase error: 25.5416642491
RfactorNum. reflection% reflectionSelection details
Rfree0.230225500153 3097 4.85058263376 %0.05
Rwork0.18882155178 ---
obs0.190855923642 63848 99.9076783451 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 26.5806085483 Å2
Refinement stepCycle: LAST / Resolution: 1.90000643078→42.1397385191 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7001 0 1 652 7654
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01375384830067214
X-RAY DIFFRACTIONf_angle_d1.487468431759808
X-RAY DIFFRACTIONf_chiral_restr0.1143876296341021
X-RAY DIFFRACTIONf_plane_restr0.007729567322881284
X-RAY DIFFRACTIONf_dihedral_angle_d30.86240240664233
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.90000643078-1.92970.2815072031471330.2575910322522739X-RAY DIFFRACTION99.7568600208
1.9297-1.96130.3041525257871400.2450379092572676X-RAY DIFFRACTION99.8581560284
1.9613-1.99520.3201749393731430.2357143442892748X-RAY DIFFRACTION100
1.9952-2.03140.2796314117921380.2349124979582724X-RAY DIFFRACTION99.8952879581
2.0314-2.07050.2500192950471300.2245675981482729X-RAY DIFFRACTION99.8602864129
2.0705-2.11280.2797412197671170.2339779478972769X-RAY DIFFRACTION99.9307479224
2.1128-2.15870.2958165253851620.2334463360162700X-RAY DIFFRACTION99.8952879581
2.1587-2.20890.2731509523221290.2307512356922736X-RAY DIFFRACTION99.7910135841
2.2089-2.26420.2878762583421440.2237871847952734X-RAY DIFFRACTION99.8612074948
2.2642-2.32540.277382652661470.2187589329672735X-RAY DIFFRACTION99.9653139091
2.3254-2.39380.2910895464531390.2054590044912734X-RAY DIFFRACTION99.9652052888
2.3938-2.4710.2705211069531210.2045172729292758X-RAY DIFFRACTION99.9305796598
2.471-2.55930.2892206067911230.2112616269272780X-RAY DIFFRACTION100
2.5593-2.66180.2658780539391490.2064618376582721X-RAY DIFFRACTION99.8608211552
2.6618-2.78290.234805019081450.1996038833082765X-RAY DIFFRACTION99.9656475438
2.7829-2.92960.2629401796971600.1957315301942764X-RAY DIFFRACTION99.9316473001
2.9296-3.11310.2192374767961540.1916402570542728X-RAY DIFFRACTION99.8613998614
3.1131-3.35340.2241191881261390.177613910182786X-RAY DIFFRACTION99.9316706525
3.3534-3.69070.1953263253121560.156633464612780X-RAY DIFFRACTION99.9659516513
3.6907-4.22430.180614016281330.1442867740872842X-RAY DIFFRACTION99.9328182734
4.2243-5.32050.1642274549771490.1472087461552820X-RAY DIFFRACTION99.8990578735
5.3205-42.13973851910.1683864931251460.1662336245372983X-RAY DIFFRACTION99.9042145594

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