[English] 日本語
Yorodumi
- PDB-6xvi: Crystal structure of Megabody Mb-Nb207-c7HopQ_A12 -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 6xvi
TitleCrystal structure of Megabody Mb-Nb207-c7HopQ_A12
ComponentsOuter membrane protein,Outer membrane protein,Mb-Nb207-c7HopQ_A12
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 speciesHelicobacter pylori (bacteria)
Helicobacter pylori G27 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.59599994893 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 22, 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

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Outer membrane protein,Outer membrane protein,Mb-Nb207-c7HopQ_A12
B: Outer membrane protein,Outer membrane protein,Mb-Nb207-c7HopQ_A12
C: Outer membrane protein,Outer membrane protein,Mb-Nb207-c7HopQ_A12
D: Outer membrane protein,Outer membrane protein,Mb-Nb207-c7HopQ_A12


Theoretical massNumber of molelcules
Total (without water)224,8794
Polymers224,8794
Non-polymers00
Water1,36976
1
A: Outer membrane protein,Outer membrane protein,Mb-Nb207-c7HopQ_A12


Theoretical massNumber of molelcules
Total (without water)56,2201
Polymers56,2201
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Outer membrane protein,Outer membrane protein,Mb-Nb207-c7HopQ_A12


Theoretical massNumber of molelcules
Total (without water)56,2201
Polymers56,2201
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Outer membrane protein,Outer membrane protein,Mb-Nb207-c7HopQ_A12


Theoretical massNumber of molelcules
Total (without water)56,2201
Polymers56,2201
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: Outer membrane protein,Outer membrane protein,Mb-Nb207-c7HopQ_A12


Theoretical massNumber of molelcules
Total (without water)56,2201
Polymers56,2201
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)96.047, 117.376, 96.844
Angle α, β, γ (deg.)90.000, 90.719, 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

-
Components

#1: Antibody
Outer membrane protein,Outer membrane protein,Mb-Nb207-c7HopQ_A12


Mass: 56219.723 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Helicobacter pylori (strain G27) (bacteria), (gene. exp.) Helicobacter pylori G27 (bacteria)
Gene: hopQ, HPG27_1120, hopQ / Production host: Escherichia coli (E. coli) / References: UniProt: B5Z8H1
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 76 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsMegabody Mb-Nb207-c7HopQ_A12 is a chimeric protein with circular permutation of HopQ: Residues 1-12: ...Megabody Mb-Nb207-c7HopQ_A12 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 288-446, 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.

-
Experimental details

-
Experiment

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

-
Sample preparation

CrystalDensity Matthews: 2.33 Å3/Da / Density % sol: 47.2 %
Crystal growTemperature: 293 K / Method: vapor diffusion
Details: 0.2 M magnesium chloride, 0.1 M BIS-Tris pH 5.0, 21% w/v PEG3350

-
Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 0.96859 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Dec 1, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.96859 Å / Relative weight: 1
ReflectionResolution: 2.59599994893→29.6203523595 Å / Num. obs: 62815 / % possible obs: 94.6 % / Redundancy: 3.21 % / Biso Wilson estimate: 66.0814790783 Å2 / CC1/2: 0.998 / Rrim(I) all: 0.075 / Net I/σ(I): 12.86
Reflection shellResolution: 2.6→2.75 Å / Redundancy: 2.76 % / Mean I/σ(I) obs: 1.63 / Num. unique obs: 7514 / CC1/2: 0.616 / Rrim(I) all: 0.787 / % possible all: 70.7

-
Processing

Software
NameVersionClassification
PHENIX1.12_2829refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5LP2

5lp2


Resolution: 2.59599994893→29.6203523595 Å / SU ML: 0.390138560687 / Cross valid method: FREE R-VALUE / σ(F): 1.3575303206 / Phase error: 29.3401524681
RfactorNum. reflection% reflectionSelection details
Rfree0.250435429091 3140 4.99944273727 %0.05
Rwork0.223808320843 ---
obs0.225150860774 62807 94.6273334036 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 76.0978353974 Å2
Refinement stepCycle: LAST / Resolution: 2.59599994893→29.6203523595 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12939 0 0 76 13015
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01273649448213139
X-RAY DIFFRACTIONf_angle_d1.3191276236817795
X-RAY DIFFRACTIONf_chiral_restr0.07351677141992030
X-RAY DIFFRACTIONf_plane_restr0.008363645009912314
X-RAY DIFFRACTIONf_dihedral_angle_d13.57253699234779
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.596-2.63650.453668334078350.427721489267678X-RAY DIFFRACTION23.8941018767
2.6365-2.67970.3684832476141120.3339972644712114X-RAY DIFFRACTION73.8063660477
2.6797-2.72590.3596359449921480.3628355085562818X-RAY DIFFRACTION98.8996332111
2.7259-2.77540.3616661418221490.3416238408592825X-RAY DIFFRACTION99.431628218
2.7754-2.82880.3851069599911490.3179394442022839X-RAY DIFFRACTION99.26910299
2.8288-2.88650.3215327122251490.3094300060692830X-RAY DIFFRACTION99.6321070234
2.8865-2.94920.3060745133831510.2894991937512857X-RAY DIFFRACTION99.6356409407
2.9492-3.01770.3179100630821500.2945405361422867X-RAY DIFFRACTION99.6696399075
3.0177-3.09310.2949758950011510.28841321762854X-RAY DIFFRACTION99.7344839031
3.0931-3.17670.3261916990431480.2756886574282826X-RAY DIFFRACTION99.8992274101
3.1767-3.270.2473627032731500.2583380399062844X-RAY DIFFRACTION99.6007984032
3.27-3.37540.3136022209711510.2569575693772871X-RAY DIFFRACTION99.7689006273
3.3754-3.49590.2769377835911480.2401743823622806X-RAY DIFFRACTION99.3275050437
3.4959-3.63560.2735098126681500.2359674016142855X-RAY DIFFRACTION99.3060145406
3.6356-3.80080.2414879882211510.2194189534452853X-RAY DIFFRACTION99.1419141914
3.8008-4.00070.236832806241490.210440677242844X-RAY DIFFRACTION98.9748677249
4.0007-4.25070.2207708227811500.1988964479512839X-RAY DIFFRACTION99.2034517093
4.2507-4.57780.2192042050351480.1826903678382826X-RAY DIFFRACTION98.444223767
4.5778-5.03640.2115888625741510.1756537055652852X-RAY DIFFRACTION98.750411049
5.0364-5.76060.2426694306291490.1999955111242838X-RAY DIFFRACTION98.9072847682
5.7606-7.24010.245880491151510.2124945374072867X-RAY DIFFRACTION98.3382209189
7.2401-29.62035235950.190787208891500.1902134489482864X-RAY DIFFRACTION97.5404530744
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)
11.77209441889-0.28030970620.6957206248334.22133705251-0.7723101491025.0711428492-0.1638876866690.2661952124980.180435097697-0.4381581829250.03121686385920.104834439466-0.113760864223-0.4511344081920.09743466537460.4961287799220.01227328862550.04192579321460.583469660278-0.1079376673710.42121287949481.5398600161.24247635817-69.1087545194
23.96121124006-0.2310382350693.976053151870.781587706955-1.635710259097.84275568240.1546328391130.17659632558-0.293253023374-0.143731109629-0.0327528611176-0.1445195181190.4307058466430.441992842691-0.119867565690.4042079630030.06709824269620.07911600291530.48985874553-0.005191439313280.5748105285997.3901764573-6.43807034938-50.2635113117
37.82143764613-0.2485272048351.374767567627.99031599836-2.329569102526.26136693507-0.142537629805-0.0322702145667-0.4300677391410.04054663715210.6291049152870.240866017901-0.0682583124255-0.778964259446-0.4801393420650.2921920571420.03659040774730.06445318165380.6187193635520.2382496954320.530401800738124.547690471-9.47179394236-30.2336507897
42.638197762371.871665689431.645449862941.785908764271.953925596951.773180876270.604282896947-0.46445954639-0.5943138010780.571510230984-0.352562294165-0.1403061433340.666584178229-0.325986726366-0.2068763469560.681127251826-0.0520317598192-0.03888360833930.4484143870480.1008979884090.72504398886582.1664815793-22.5664850365-99.1931367886
53.670507331275.263731480692.501799885448.36231825034.064442014552.716802369540.516483001101-0.120091574797-0.4633294514641.13715513455-0.0575017796298-0.5597191234080.723800587435-0.0278036720045-0.4720028523060.592441425319-0.0225558694395-0.1146849878410.4992717591330.06824213838390.627229105061102.762676448-6.27813687791-84.1120407717
63.77438453175.820093717753.944191038939.28024148235.667913352683.50161400330.04521290257070.277739055866-0.6085342363220.8279081054830.720723113246-0.9499312230670.7089177843120.806928519226-0.7387125767180.626895371926-0.0164807829072-0.05685502885740.501357262885-0.01271760533660.794566919343112.9189998538.71283634987-79.3114057604
73.202150275592.2899690621.270020889987.241960101311.875343445644.781303748980.2828537245340.222401729531-1.332023185390.4287828465480.31272288626-0.3264862241930.757367472906-0.601546261078-0.5928680077630.60290987383-0.11556420381-0.1508545271990.4719082480650.1062028382650.51018204198586.9077007288-22.3991714865-94.1913820772
84.685952681322.001849590893.338787142741.688306685083.181018406244.427412224620.109963686306-0.0876448731528-0.2389965536450.1273675652440.115188397197-0.07910636852170.141457720571-0.256423049592-0.2323928068530.335442588036-0.0155989021325-0.04070532656270.3048332393570.08067263095040.53563679907483.1057549796-18.8466395154-102.443576088
97.22248316154-1.618465075310.5140024472798.456842598110.2552726133997.64679802137-0.166878995706-0.0296068575099-0.5442105063610.2718217497330.239392043042-0.2126914271390.4069733156110.201692561234-0.06119607724060.300565421484-0.008563614075120.01191958933780.280604203309-0.03758058371480.4289480131453.270486555-24.1099263001-115.888412329
103.29301984103-1.24388123289-1.351351359841.062049291820.5812164040461.057851895370.04687175764320.3191161065210.13074873185-0.176527322911-0.04391607579650.02127996355690.09774791244340.107577930374-0.02557165563250.456837875302-0.0429577443927-0.03492244061270.566780387760.0704195232860.377042986749140.6640269960.506149133127-58.9342591048
112.33677817469-1.8182630981-1.55871124633.991230831462.302528492513.69404155118-0.3312479857-0.052385242865-0.0005779575136430.07094313315980.623611693783-0.1940952550830.4205799316190.419867684021-0.287579664460.496068412464-0.1126051272860.05320602508470.5923707389810.1048485613570.428970351194150.088092365-7.04221475293-61.8119384255
124.47731802934-0.410311309405-3.074994537230.01471390694082.267199737493.9527499648-0.150148406960.2336389424270.05669364053240.1148516248110.1971290887060.202581887668-0.0885042326568-0.299991104409-0.0830379081980.428899575878-0.06115686185050.02665852938820.4430975620440.1962401679670.583363457102130.4457331073.97951958652-47.0910374161
136.360897569010.9053837558740.06583751180385.982714615621.186973680337.46421689740.0386299000465-0.04801840344170.605853782821-0.1376770006490.0820254972074-0.109940974274-0.5772155478840.345602527968-0.09039219688230.34572025531-0.009824078697440.04328471859410.321365232631-0.002625536333110.47770539630399.97155538928.3195583948-33.7032909315
143.054560792230.746416427734-2.530672536781.10602982631-1.091332228933.941250862090.0833650349513-0.4121478011480.1347589768490.433928727417-0.112314487588-0.0249282411035-0.3003249829650.006096471276580.01372557464050.6942292631290.0327292277266-0.05977742772350.459557004242-0.02701110974690.430741056368139.449587026-10.5607786322-85.5920720743
152.839371440222.21051068229-0.705322629925.25712816138-2.521618369125.12031253690.02265680019730.0338316439259-0.192208227461.028909648910.3638397179820.135944248377-0.235683305845-0.714998410279-0.334123518260.7992528557550.1592108066940.03177225505350.406092939185-0.04584960402580.549339383682130.936941463-16.0405645932-80.0223410036
164.240092171230.600327944349-3.562074656730.37973752838-1.724721862696.42940388819-0.01705420610860.03744849206110.275118791650.1131408793650.1338499024740.0148501619052-0.2736282008840.108090566483-0.08098730769650.4747289257070.03394734568230.0003677413991430.2703355488840.002421260023240.470430329222147.14682036-8.47402127516-100.089622145
178.33975383985-0.200719096588-0.067557344275.33867610185-1.385921736018.265710805820.07740348467660.109842284443-0.1520328228340.07472105956170.274364037951-0.3436900354220.000330494180372-0.278840581988-0.3220943518750.294215517389-0.0281098836014-0.007487465365120.3308312029170.06223711481520.473498048584173.920362479-6.69448603876-120.405872948
185.32490225026-0.7884758433173.110875978940.272171716132-0.6395473055594.419536971020.37296246240.358739483799-0.509048664184-0.405003440409-0.300064872207-0.3498529277730.8486146695270.38520880179-0.04673322059570.6499721419420.04203928347730.1134658384680.4210070492990.07262749223980.60815870806199.5553922058-10.3217636114-52.9038498096
194.9219219701-1.073731371592.921792862634.40331349219-2.87358950465.577996476280.06450236402740.7591828460170.0551427900968-0.639322812543-0.156295328345-0.2498876126750.3728962425110.03863195140120.07263912876260.6841205777630.1206224997690.08561636334620.701443730828-0.05617438925970.38163478522584.2001327807-1.01200516767-71.7629382499
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'D' and (resid 194 through 315 )
2X-RAY DIFFRACTION2chain 'D' and (resid 316 through 409 )
3X-RAY DIFFRACTION3chain 'D' and (resid 410 through 510 )
4X-RAY DIFFRACTION4chain 'A' and (resid 2 through 74 )
5X-RAY DIFFRACTION5chain 'A' and (resid 75 through 210 )
6X-RAY DIFFRACTION6chain 'A' and (resid 211 through 277 )
7X-RAY DIFFRACTION7chain 'A' and (resid 278 through 315 )
8X-RAY DIFFRACTION8chain 'A' and (resid 316 through 409 )
9X-RAY DIFFRACTION9chain 'A' and (resid 410 through 511 )
10X-RAY DIFFRACTION10chain 'B' and (resid 2 through 193 )
11X-RAY DIFFRACTION11chain 'B' and (resid 194 through 315 )
12X-RAY DIFFRACTION12chain 'B' and (resid 316 through 409 )
13X-RAY DIFFRACTION13chain 'B' and (resid 410 through 510 )
14X-RAY DIFFRACTION14chain 'C' and (resid 2 through 193 )
15X-RAY DIFFRACTION15chain 'C' and (resid 194 through 315 )
16X-RAY DIFFRACTION16chain 'C' and (resid 316 through 409 )
17X-RAY DIFFRACTION17chain 'C' and (resid 410 through 510 )
18X-RAY DIFFRACTION18chain 'D' and (resid 2 through 74 )
19X-RAY DIFFRACTION19chain 'D' and (resid 75 through 193 )

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more