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- PDB-7bm5: Crystal structure of Fab1, the Fab fragment of the anti-BamA mono... -

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Basic information

Entry
Database: PDB / ID: 7bm5
TitleCrystal structure of Fab1, the Fab fragment of the anti-BamA monoclonal antibody MAB1
Components
  • Fab1 heavy chain
  • Fab1 light chain
KeywordsIMMUNE SYSTEM / Antibody / antibiotic / Fab / BamA
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.95 Å
AuthorsWhite, P. / Storek, K.M. / Rutherford, S.T. / Radford, S.E.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MR/P018491/1 United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding.
Authors: Paul White / Samuel F Haysom / Matthew G Iadanza / Anna J Higgins / Jonathan M Machin / James M Whitehouse / Jim E Horne / Bob Schiffrin / Charlotte Carpenter-Platt / Antonio N Calabrese / ...Authors: Paul White / Samuel F Haysom / Matthew G Iadanza / Anna J Higgins / Jonathan M Machin / James M Whitehouse / Jim E Horne / Bob Schiffrin / Charlotte Carpenter-Platt / Antonio N Calabrese / Kelly M Storek / Steven T Rutherford / David J Brockwell / Neil A Ranson / Sheena E Radford /
Abstract: The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit ...The folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.
History
DepositionJan 19, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 21, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jan 31, 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 / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
L: Fab1 light chain
H: Fab1 heavy chain
A: Fab1 light chain
C: Fab1 light chain
E: Fab1 light chain
J: Fab1 light chain
G: Fab1 light chain
B: Fab1 heavy chain
D: Fab1 heavy chain
F: Fab1 heavy chain
K: Fab1 heavy chain
I: Fab1 heavy chain


Theoretical massNumber of molelcules
Total (without water)288,86312
Polymers288,86312
Non-polymers00
Water00
1
L: Fab1 light chain
H: Fab1 heavy chain


Theoretical massNumber of molelcules
Total (without water)48,1442
Polymers48,1442
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3680 Å2
ΔGint-18 kcal/mol
Surface area19550 Å2
MethodPISA
2
A: Fab1 light chain
B: Fab1 heavy chain


Theoretical massNumber of molelcules
Total (without water)48,1442
Polymers48,1442
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3310 Å2
ΔGint-22 kcal/mol
Surface area19730 Å2
MethodPISA
3
C: Fab1 light chain
D: Fab1 heavy chain


Theoretical massNumber of molelcules
Total (without water)48,1442
Polymers48,1442
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3310 Å2
ΔGint-22 kcal/mol
Surface area19450 Å2
MethodPISA
4
E: Fab1 light chain
F: Fab1 heavy chain


Theoretical massNumber of molelcules
Total (without water)48,1442
Polymers48,1442
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2810 Å2
ΔGint-21 kcal/mol
Surface area19550 Å2
MethodPISA
5
J: Fab1 light chain
K: Fab1 heavy chain


Theoretical massNumber of molelcules
Total (without water)48,1442
Polymers48,1442
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3340 Å2
ΔGint-22 kcal/mol
Surface area19280 Å2
MethodPISA
6
G: Fab1 light chain
I: Fab1 heavy chain


Theoretical massNumber of molelcules
Total (without water)48,1442
Polymers48,1442
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2820 Å2
ΔGint-22 kcal/mol
Surface area18490 Å2
MethodPISA
Unit cell
Length a, b, c (Å)92.014, 130.144, 138.921
Angle α, β, γ (deg.)90.000, 106.060, 90.000
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A
21chain C
31chain E
41chain G
51chain J
61chain L
12chain B
22chain D
32chain F
42chain H
52chain K

NCS domain segments:

Component-ID: 1

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ASPASPARGARGchain AAC1 - 2121 - 212
21GLNGLNLYSLYSchain CCD3 - 2083 - 208
31ASPASPCYSCYSchain EEE1 - 2151 - 215
41GLNGLNASNASNchain GGG3 - 2113 - 211
51ASPASPARGARGchain JJF1 - 2121 - 212
61ASPASPARGARGchain LLA1 - 2121 - 212
12GLUGLUSERSERchain BBH1 - 2221 - 222
22GLUGLUGLUGLUchain DDI1 - 2191 - 219
32VALVALPROPROchain FFJ2 - 2202 - 220
42VALVALSERSERchain HHB2 - 2222 - 222
52GLUGLULYSLYSchain KKK1 - 2211 - 221

NCS ensembles :
ID
1
2

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Components

#1: Antibody
Fab1 light chain


Mass: 23642.215 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#2: Antibody
Fab1 heavy chain


Mass: 24501.666 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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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: 2.77 Å3/Da / Density % sol: 55.55 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 6
Details: 0.16 M lithium chloride, 22% (w/v) PEG6000, 0.1 M MES pH 6.0

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Feb 3, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 2.95→93.189 Å / Num. obs: 30473 / % possible obs: 92.1 % / Redundancy: 6.8 % / Biso Wilson estimate: 46 Å2 / CC1/2: 0.978 / Rmerge(I) obs: 0.422 / Rpim(I) all: 0.173 / Rrim(I) all: 0.457 / Net I/σ(I): 3.6
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
2.955-3.42661.058909915250.640.4591.1571.668.8
10.513-93.1896.70.0841023615220.9960.0350.0918.899.6

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
PHENIX1.9_1692refinement
XDSdata reduction
Aimless0.7.4data scaling
PHASERphasing
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1ZAN

1zan


Resolution: 2.95→88.423 Å / SU ML: 0.44 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 28.19 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2897 1494 4.91 %
Rwork0.2569 28952 -
obs0.2585 30446 46.08 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 117.11 Å2 / Biso mean: 41.5631 Å2 / Biso min: 12.67 Å2
Refinement stepCycle: final / Resolution: 2.95→88.423 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms18443 0 0 0 18443
Num. residues----2443
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00518890
X-RAY DIFFRACTIONf_angle_d0.99325659
X-RAY DIFFRACTIONf_chiral_restr0.0432919
X-RAY DIFFRACTIONf_plane_restr0.0053278
X-RAY DIFFRACTIONf_dihedral_angle_d10.3316650
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A5686X-RAY DIFFRACTION15.986TORSIONAL
12C5686X-RAY DIFFRACTION15.986TORSIONAL
13E5686X-RAY DIFFRACTION15.986TORSIONAL
14G5686X-RAY DIFFRACTION15.986TORSIONAL
15J5686X-RAY DIFFRACTION15.986TORSIONAL
16L5686X-RAY DIFFRACTION15.986TORSIONAL
21B4720X-RAY DIFFRACTION15.986TORSIONAL
22D4720X-RAY DIFFRACTION15.986TORSIONAL
23F4720X-RAY DIFFRACTION15.986TORSIONAL
24H4720X-RAY DIFFRACTION15.986TORSIONAL
25K4720X-RAY DIFFRACTION15.986TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.9532-3.04850.295390.4463671
3.0485-3.15750.5119110.37762184
3.1575-3.28390.4527240.36514638
3.2839-3.43330.4452350.34973913
3.4333-3.61440.3911640.3168126322
3.6144-3.84080.3592820.285171430
3.8408-4.13740.33621880.2842332558
4.1374-4.55370.27752100.2391438076
4.5537-5.21260.26922630.2359524892
5.2126-6.5670.28212890.26755756100
6.567-88.4230.25653190.23975779100

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