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- PDB-6cbv: Crystal structure of BRIL bound to an affinity matured synthetic ... -

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Basic information

Entry
Database: PDB / ID: 6cbv
TitleCrystal structure of BRIL bound to an affinity matured synthetic antibody.
Components
  • BRIL
  • Heavy chain, Fab fragment
  • Light Chain, Fab fragment
KeywordsIMMUNE SYSTEM / 4-helix bundle / fusion protein / synthetic antibody / Fab fragment / fusion protein in complex with Fab
Function / homology
Function and homology information


electron transport chain / electron transfer activity / periplasmic space / iron ion binding / heme binding
Similarity search - Function
Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
FORMIC ACID / Soluble cytochrome b562
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.872 Å
AuthorsMukherjee, S. / Skrobek, B. / Kossiakoff, A.A.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM117372-01 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U54GM087519-01 United States
CitationJournal: Nat Commun / Year: 2020
Title: Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins.
Authors: Somnath Mukherjee / Satchal K Erramilli / Mark Ammirati / Frances J D Alvarez / Kimberly F Fennell / Michael D Purdy / Blazej M Skrobek / Katarzyna Radziwon / John Coukos / Yanyong Kang / ...Authors: Somnath Mukherjee / Satchal K Erramilli / Mark Ammirati / Frances J D Alvarez / Kimberly F Fennell / Michael D Purdy / Blazej M Skrobek / Katarzyna Radziwon / John Coukos / Yanyong Kang / Przemysław Dutka / Xiang Gao / Xiayang Qiu / Mark Yeager / H Eric Xu / Seungil Han / Anthony A Kossiakoff /
Abstract: We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an ...We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.
History
DepositionFeb 5, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 6, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 13, 2019Group: Data collection / Structure summary / Category: struct_keywords
Item: _struct_keywords.pdbx_keywords / _struct_keywords.text
Revision 1.2Feb 20, 2019Group: Author supporting evidence / Data collection / Structure summary
Category: pdbx_audit_support / struct
Item: _pdbx_audit_support.funding_organization / _struct.title
Revision 1.3Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Aug 19, 2020Group: 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 1.5Oct 4, 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
H: Heavy chain, Fab fragment
L: Light Chain, Fab fragment
B: BRIL
hetero molecules


Theoretical massNumber of molelcules
Total (without water)59,7598
Polymers59,4603
Non-polymers2995
Water5,765320
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6190 Å2
ΔGint-44 kcal/mol
Surface area23940 Å2
MethodPISA
Unit cell
Length a, b, c (Å)87.954, 87.954, 159.107
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number170
Space group name H-MP65

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Components

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Protein , 1 types, 1 molecules B

#3: Protein BRIL / Cytochrome b-562


Mass: 11785.229 Da / Num. of mol.: 1 / Mutation: M7W, H102I, R106L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: cybC / Plasmid: pHFT2 / Details (production host): T7 promoter, Kanamycin resistant / Production host: Escherichia coli (E. coli) / References: UniProt: P0ABE7

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Antibody , 2 types, 2 molecules HL

#1: Antibody Heavy chain, Fab fragment


Mass: 24321.084 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pRH2.2 / Details (production host): IPTG inducible, Amp resistant / Production host: Escherichia coli (E. coli)
#2: Antibody Light Chain, Fab fragment /


Mass: 23353.947 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Plasmid: pRH2.2
Details (production host): IPTG inducible, Ampicillin resistant
Production host: Escherichia coli (E. coli)

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Non-polymers , 4 types, 325 molecules

#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical ChemComp-FMT / FORMIC ACID / Formic acid


Mass: 46.025 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: CH2O2
#6: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 320 / Source method: isolated from a natural source / Formula: H2O

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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: 2.97 Å3/Da / Density % sol: 58.59 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.5 / Details: 0.2 M Sodium formate, 20 % w/v PEG 3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9791 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Jun 30, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9791 Å / Relative weight: 1
ReflectionResolution: 1.87→76.171 Å / Num. obs: 55287 / % possible obs: 96.7 % / Redundancy: 4.757 % / Biso Wilson estimate: 42.15 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.041 / Rrim(I) all: 0.045 / Χ2: 1.131 / Net I/σ(I): 17.55 / Num. measured all: 263005
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsCC1/2Rrim(I) all% possible all
1.87-1.992.5380.8920.9419122923375330.4611.10681.6
1.99-2.125.0250.5652.6343227866086030.8330.6399.3
2.12-2.295.1220.3214.8941224809580490.9390.35899.4
2.29-2.515.2450.1758.6538857743574080.9810.19499.6
2.51-2.815.0830.09315.6433999671066890.9940.10499.7
2.81-3.245.2360.04929.3931344599059860.9980.05499.9
3.24-3.974.970.03344.4124771501049840.9990.03699.5
3.97-5.64.9870.02654.1319300389238700.9990.02999.4
5.6-76.1715.1550.02358.1211161217821650.9990.02699.4

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX1.12_2829refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1M6T, 3PGF

1m6t

3pgf


Resolution: 1.872→19.847 Å / SU ML: 0.23 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 24.18 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2248 2647 4.79 %
Rwork0.1938 52571 -
obs0.1952 55218 96.62 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 181.06 Å2 / Biso mean: 56.0022 Å2 / Biso min: 31.35 Å2
Refinement stepCycle: final / Resolution: 1.872→19.847 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4131 0 19 320 4470
Biso mean--52.44 52.98 -
Num. residues----543
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0044292
X-RAY DIFFRACTIONf_angle_d0.7225847
X-RAY DIFFRACTIONf_chiral_restr0.046656
X-RAY DIFFRACTIONf_plane_restr0.004747
X-RAY DIFFRACTIONf_dihedral_angle_d11.6092608
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 19

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.8721-1.90620.3105690.34111755182461
1.9062-1.94280.321390.32162433257286
1.9428-1.98240.35031460.29092737288396
1.9824-2.02550.27551670.27182820298799
2.0255-2.07250.28811460.26752815296199
2.0725-2.12430.26291460.25532827297399
2.1243-2.18170.26911620.24892837299999
2.1817-2.24580.2421590.23652856301599
2.2458-2.31820.27381520.23522848300099
2.3182-2.40090.27381660.233927892955100
2.4009-2.49690.24931360.224728853021100
2.4969-2.61030.26791480.21728412989100
2.6103-2.74750.25111030.214828952998100
2.7475-2.91920.25361330.207728542987100
2.9192-3.14380.23241350.200428953030100
3.1438-3.45860.21571330.191828552988100
3.4586-3.95560.18261290.17462866299599
3.9556-4.97070.18591390.158128743013100
4.9707-19.84780.21281390.16972889302899
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)
14.31350.7218-1.43992.64-1.37593.1427-0.15540.16140.20490.15050.19850.1455-0.00790.0156-0.05190.3644-0.003-0.02530.33060.07170.325424.4504123.453-5.4272
26.32750.4915-0.23361.19331.21134.58750.0585-0.6123-0.57160.418-0.0767-0.34640.67460.2465-0.04380.43740.0180.02050.28840.02120.284914.0163102.474420.012
37.1063-1.5821.87274.6802-0.68344.70730.2158-0.38610.03470.42570.0702-0.3465-0.01190.2131-0.28460.3731-0.02790.00180.30310.02160.330919.5278103.512119.0745
42.6437-0.40761.04488.489-5.76297.663-0.36840.3545-0.3592-0.95960.56610.58141.0027-0.3296-0.0610.884-0.1155-0.00430.5685-0.04490.410923.5506100.1681-17.1208
54.5720.2825-1.0217.4394-1.00093.576-0.28280.2984-0.3021-0.56360.0469-0.26710.98830.58380.22970.52980.04650.06730.35850.0070.282631.9497106.5221-15.0948
62.05510.2104-0.87859.7109-4.26413.5767-0.2740.2339-0.2288-0.05580.20750.17010.48820.21750.05790.5547-0.0325-0.01010.4324-0.02360.331426.8773107.9403-12.7649
72.85934.4964-3.72757.0392-5.79074.7487-0.69140.2933-0.4966-0.58080.8876-0.00671.0373-0.3021-0.1861.02350.08550.11380.40020.00040.552922.699688.9317-2.046
86.096-2.7144-4.38155.72714.21167.24920.042-0.0104-0.3368-0.1133-0.0277-0.03820.2194-0.39260.00430.46-0.0151-0.01290.25560.10140.323910.79292.21716.3062
93.7636-3.3911-2.57823.27761.38958.02270.2924-0.1630.1711-0.22890.023-0.12740.4802-0.2186-0.36880.5239-0.0124-0.0310.2760.02420.379813.173492.38688.4362
106.0323-3.33-2.68497.8151-0.1423.9423-0.4069-0.0638-0.62130.22630.14340.41790.8193-0.40480.2890.4402-0.10170.00610.30310.04130.36115.806388.216818.8523
118.7535-6.0024-1.66855.87910.36387.75150.43591.2905-0.0791-2.3493-0.06440.72060.4940.2578-0.19631.0558-0.3671-0.1721.09420.12550.542627.8047123.9742-38.5057
123.18480.31240.20847.7126-0.19275.6604-0.31990.8944-0.2143-1.38880.0925-0.73450.93730.68890.06110.868-0.05160.11690.80270.09710.390236.8163119.8238-32.176
133.7617-1.5057-0.09678.53590.66995.3026-0.09550.21640.6061-0.75620.2264-0.90950.37930.76640.07920.5389-0.1973-0.03980.75630.15550.376837.0109127.261-27.4055
148.9295-4.7575-4.27819.25032.91257.5551-0.11661.08090.8035-1.46850.1189-0.188-0.5070.4161-0.0570.7826-0.2087-0.16310.86110.2680.629231.4717133.6916-34.1841
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'H' and (resid 3 through 123 )H3 - 123
2X-RAY DIFFRACTION2chain 'H' and (resid 124 through 148 )H124 - 148
3X-RAY DIFFRACTION3chain 'H' and (resid 149 through 229 )H149 - 229
4X-RAY DIFFRACTION4chain 'L' and (resid 2 through 26 )L2 - 26
5X-RAY DIFFRACTION5chain 'L' and (resid 27 through 76 )L27 - 76
6X-RAY DIFFRACTION6chain 'L' and (resid 77 through 103 )L77 - 103
7X-RAY DIFFRACTION7chain 'L' and (resid 104 through 115 )L104 - 115
8X-RAY DIFFRACTION8chain 'L' and (resid 116 through 152 )L116 - 152
9X-RAY DIFFRACTION9chain 'L' and (resid 153 through 176 )L153 - 176
10X-RAY DIFFRACTION10chain 'L' and (resid 177 through 214 )L177 - 214
11X-RAY DIFFRACTION11chain 'B' and (resid 1 through 22 )B1 - 22
12X-RAY DIFFRACTION12chain 'B' and (resid 23 through 55 )B23 - 55
13X-RAY DIFFRACTION13chain 'B' and (resid 56 through 81 )B56 - 81
14X-RAY DIFFRACTION14chain 'B' and (resid 82 through 103 )B82 - 103

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