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- PDB-6x58: MPER-Fluc-Ec2 bound to 10E8v4 antibody -

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Basic information

Entry
Database: PDB / ID: 6x58
TitleMPER-Fluc-Ec2 bound to 10E8v4 antibody
Components
  • 10E8v4 Fab Heavy Chain
  • 10E8v4 Fab Light Chain
  • gp41 MPER peptide,Putative fluoride ion transporter CrcB
KeywordsMEMBRANE PROTEIN / antibody / chaperone / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


fluoride channel activity / cellular detoxification of fluoride / positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope ...fluoride channel activity / cellular detoxification of fluoride / positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane / virion membrane / structural molecule activity / metal ion binding / membrane / plasma membrane
Similarity search - Function
Putative fluoride ion transporter CrcB / CrcB-like protein, Camphor Resistance (CrcB) / Envelope glycoprotein Gp160 / Retroviral envelope protein / Retroviral envelope protein GP41-like / Gp120 core superfamily / Envelope glycoprotein GP120 / Human immunodeficiency virus 1, envelope glycoprotein Gp120 / Prokaryotic membrane lipoprotein lipid attachment site profile. / Immunoglobulins ...Putative fluoride ion transporter CrcB / CrcB-like protein, Camphor Resistance (CrcB) / Envelope glycoprotein Gp160 / Retroviral envelope protein / Retroviral envelope protein GP41-like / Gp120 core superfamily / Envelope glycoprotein GP120 / Human immunodeficiency virus 1, envelope glycoprotein Gp120 / Prokaryotic membrane lipoprotein lipid attachment site profile. / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
Fluoride-specific ion channel FluC / Envelope glycoprotein gp160
Similarity search - Component
Biological speciesHomo sapiens (human)
Human immunodeficiency virus
Escherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.26 Å
AuthorsMcIlwain, B.C. / Stockbridge, R.B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: J Mol Biol / Year: 2021
Title: N-terminal Transmembrane-Helix Epitope Tag for X-ray Crystallography and Electron Microscopy of Small Membrane Proteins.
Authors: Benjamin C McIlwain / Amanda L Erwin / Alexander R Davis / B Ben Koff / Louise Chang / Tatsiana Bylund / Gwo-Yu Chuang / Peter D Kwong / Melanie D Ohi / Yen-Ting Lai / Randy B Stockbridge /
Abstract: Structural studies of membrane proteins, especially small membrane proteins, are associated with well-known experimental challenges. Complexation with monoclonal antibody fragments is a common ...Structural studies of membrane proteins, especially small membrane proteins, are associated with well-known experimental challenges. Complexation with monoclonal antibody fragments is a common strategy to augment such proteins; however, generating antibody fragments that specifically bind a target protein is not trivial. Here we identify a helical epitope, from the membrane-proximal external region (MPER) of the gp41-transmembrane subunit of the HIV envelope protein, that is recognized by several well-characterized antibodies and that can be fused as a contiguous extension of the N-terminal transmembrane helix of a broad range of membrane proteins. To analyze whether this MPER-epitope tag might aid structural studies of small membrane proteins, we determined an X-ray crystal structure of a membrane protein target that does not crystallize without the aid of crystallization chaperones, the Fluc fluoride channel, fused to the MPER epitope and in complex with antibody. We also demonstrate the utility of this approach for single particle electron microscopy with Fluc and two additional small membrane proteins that represent different membrane protein folds, AdiC and GlpF. These studies show that the MPER epitope provides a structurally defined, rigid docking site for antibody fragments that is transferable among diverse membrane proteins and can be engineered without prior structural information. Antibodies that bind to the MPER epitope serve as effective crystallization chaperones and electron microscopy fiducial markers, enabling structural studies of challenging small membrane proteins.
History
DepositionMay 25, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 19, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 28, 2021Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2Oct 18, 2023Group: 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
Revision 1.3Nov 20, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: 10E8v4 Fab Heavy Chain
D: 10E8v4 Fab Light Chain
E: gp41 MPER peptide,Putative fluoride ion transporter CrcB
F: gp41 MPER peptide,Putative fluoride ion transporter CrcB
A: 10E8v4 Fab Heavy Chain
B: 10E8v4 Fab Light Chain


Theoretical massNumber of molelcules
Total (without water)126,1636
Polymers126,1636
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)99.050, 99.050, 167.600
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number145
Space group name H-MP32

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Components

#1: Antibody 10E8v4 Fab Heavy Chain


Mass: 25138.002 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK / Production host: Homo sapiens (human) / Strain (production host): Expi293
#2: Antibody 10E8v4 Fab Light Chain


Mass: 22560.049 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): HEK / Production host: Homo sapiens (human) / Strain (production host): Expi293
#3: Protein gp41 MPER peptide,Putative fluoride ion transporter CrcB


Mass: 15383.293 Da / Num. of mol.: 2 / Mutation: R25K,A51M in transporter
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus, (gene. exp.) Escherichia coli (E. coli)
Gene: env, crcB, crcB_2, flc_2, AC789_145pl00540, AKG29_01220, B6V57_25995, B7C53_24430, B9T59_30510, BANRA_05655, BANRA_05710, BEN53_26765, BIU72_24510, BK292_28205, BK334_22235, BK373_23795, BON66_ ...Gene: env, crcB, crcB_2, flc_2, AC789_145pl00540, AKG29_01220, B6V57_25995, B7C53_24430, B9T59_30510, BANRA_05655, BANRA_05710, BEN53_26765, BIU72_24510, BK292_28205, BK334_22235, BK373_23795, BON66_22385, BON69_17555, BON72_04410, BON76_23080, BON86_18360, BON94_16600, BVL39_28685, C2M16_27610, C4K41_18470, C4M78_26960, C5F73_28830, C5P01_26640, C5P44_26785, C6B13_25705, C7B08_22045, C9160_28010, C9201_28610, C9E25_24405, CDL37_21115, CSB64_25975, CWS33_26140, D1912_23150, D2184_26920, D2188_23810, D4U49_23430, D7W70_25855, D7Y10_22455, D9C99_23085, D9D33_24680, D9D94_24325, D9F87_24785, D9G11_25285, D9H70_24700, D9J44_26040, D9J61_22795, D9K02_24930, D9K48_12550, D9K54_12845, D9L99_22160, DAH18_26070, DB359_26980, DL545_01300, DLU67_24300, DLU82_24070, DN808_21420, DNI21_22025, DNQ45_14105, DNX19_24210, DP265_23725, DP277_24230, DQF72_24030, DS966_25260, DTL90_25845, DWB25_27055, E2112_25200, E2148_25435, EA239_25270, EA250_26195, EA429_25950, EA435_26495, ECONIH1_26550, ECS286_0026, EIA08_25210, EIZ93_19245, EJ366_00165, ELT22_23900, ELT58_23370, ELX56_24555, ELX56_26650, ELY05_20840, ELY05_25530, EPS76_20210, EVY14_24275, ExPECSC038_03841, EXX06_27990, EXX23_23730, EXX55_27910, EXX87_27755, EYX82_20275, F0L67_16895, F1E19_23340, F9050_24150, FORC82_p488, FQU83_00900, G3565_28150, GP698_23185, GQE58_24285, HVW93_24610, HXS78_24215, MJ49_27125, NCTC9001_00147, NCTC9077_06349, NCTC9434_05106, NCTC9969_05488, pCTXM15_EC8_00123, pO103_22, RCS79_P0115, SAMEA3472090_04449, SAMEA3752620_04806, SAMEA3753164_04868, SAMEA4370473_00090, TUM18780_48590, WP2S18E08_P10360, WP5S18E08_P10940
Production host: Escherichia coli (E. coli) / References: UniProt: Q73372, UniProt: Q6J5N4
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: 3.8 Å3/Da / Density % sol: 67.66 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / Details: 34-39% PEG 300, 0.1 M NaCl, 0.1 M MES pH 6.2-7 / PH range: 6.2-7

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-D / Wavelength: 0.976 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Jun 9, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976 Å / Relative weight: 1
ReflectionResolution: 3.26→38.18 Å / Num. obs: 28368 / % possible obs: 98.6 % / Redundancy: 10.8 % / CC1/2: 0.999 / Rmerge(I) obs: 0.135 / Rpim(I) all: 0.043 / Rrim(I) all: 0.141 / Net I/σ(I): 10.3 / Num. measured all: 306181 / Scaling rejects: 3
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
3.26-3.4611.22.4415172546010.7650.7632.558198.4
9.78-38.1810.70.0441094910190.9990.0140.04635.395.1

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Processing

Software
NameVersionClassification
REFMAC1refinement
Aimless0.7.2data scaling
PDB_EXTRACT3.25data extraction
xia2data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5A43

5a43


Resolution: 3.26→34.02 Å / SU ML: 0.68 / Cross valid method: THROUGHOUT / σ(F): 1.96 / Phase error: 39.74 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2951 1426 5.05 %RANDOM
Rwork0.251 ---
obs0.2533 28211 98.27 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 322.54 Å2 / Biso mean: 159.7412 Å2 / Biso min: 30 Å2
Refinement stepCycle: final / Resolution: 3.26→34.02 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8816 0 0 0 8816
Num. residues----1153

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