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- PDB-7a4d: Crystal structure of the APH coiled-coil in complex with nanobodi... -

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Basic information

Entry
Database: PDB / ID: 7a4d
TitleCrystal structure of the APH coiled-coil in complex with nanobodies Nb28 and Nb30
Components
  • APH coiled-coil
  • Nanobody Nb28
  • Nanobody Nb30
KeywordsDE NOVO PROTEIN / coiled-coil / nanobody / antibody / protein design
Function / homologyImmunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta / ACETATE ION
Function and homology information
Biological speciesLama glama (llama)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.694 Å
AuthorsHadzi, S.
Funding supportEuropean Union, Slovenia, 3items
OrganizationGrant numberCountry
European Research Council (ERC)787115European Union
Slovenian Research AgencyP4-0176 Slovenia
Slovenian Research AgencyP1-0201 Slovenia
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2021
Title: A nanobody toolbox targeting dimeric coiled-coil modules for functionalization of designed protein origami structures.
Authors: Majerle, A. / Hadzi, S. / Aupic, J. / Satler, T. / Lapenta, F. / Strmsek, Z. / Lah, J. / Loris, R. / Jerala, R.
History
DepositionAug 19, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 5, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 11, 2021Group: Database references / Derived calculations
Category: database_2 / pdbx_struct_assembly / pdbx_struct_assembly_gen
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2May 1, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._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
Revision 1.3Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Nanobody Nb28
B: Nanobody Nb28
C: Nanobody Nb30
D: Nanobody Nb30
E: APH coiled-coil
F: APH coiled-coil
G: Nanobody Nb28
H: Nanobody Nb28
I: Nanobody Nb30
J: Nanobody Nb30
K: APH coiled-coil
L: APH coiled-coil
hetero molecules


Theoretical massNumber of molelcules
Total (without water)134,60617
Polymers134,26912
Non-polymers3375
Water1,06359
1
A: Nanobody Nb28
B: Nanobody Nb28
C: Nanobody Nb30
D: Nanobody Nb30
E: APH coiled-coil
F: APH coiled-coil
hetero molecules


Theoretical massNumber of molelcules
Total (without water)67,3519
Polymers67,1346
Non-polymers2163
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
G: Nanobody Nb28
H: Nanobody Nb28
I: Nanobody Nb30
J: Nanobody Nb30
K: APH coiled-coil
L: APH coiled-coil
hetero molecules


Theoretical massNumber of molelcules
Total (without water)67,2558
Polymers67,1346
Non-polymers1212
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)61.260, 127.450, 192.910
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 3 through 86 or (resid 87...
21(chain B and (resid 3 through 86 or (resid 87...
31(chain G and resid 3 through 118)
41(chain H and (resid 3 through 86 or (resid 87...
12(chain C and ((resid 3 and (name N or name...
22(chain D and ((resid 3 and (name N or name...
32(chain I and (resid 3 through 42 or (resid 43...
42(chain J and ((resid 3 and (name N or name...
13(chain E and ((resid 1 through 2 and (name N...
23(chain F and (resid 1 through 5 or (resid 6...
33(chain K and ((resid 1 through 2 and (name N...
43(chain L and ((resid 1 through 2 and (name N...

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
111GLNGLNLEULEU(chain A and (resid 3 through 86 or (resid 87...AA3 - 863 - 86
121LYSLYSLYSLYS(chain A and (resid 3 through 86 or (resid 87...AA8787
131GLNGLNARGARG(chain A and (resid 3 through 86 or (resid 87...AA3 - 1203 - 120
141GLNGLNARGARG(chain A and (resid 3 through 86 or (resid 87...AA3 - 1203 - 120
151GLNGLNARGARG(chain A and (resid 3 through 86 or (resid 87...AA3 - 1203 - 120
211GLNGLNLEULEU(chain B and (resid 3 through 86 or (resid 87...BB3 - 863 - 86
221LYSLYSLYSLYS(chain B and (resid 3 through 86 or (resid 87...BB8787
231VALVALSERSER(chain B and (resid 3 through 86 or (resid 87...BB2 - 1182 - 118
311GLNGLNSERSER(chain G and resid 3 through 118)GG3 - 1183 - 118
411GLNGLNLEULEU(chain H and (resid 3 through 86 or (resid 87...HH3 - 863 - 86
421LYSLYSLYSLYS(chain H and (resid 3 through 86 or (resid 87...HH8787
431GLNGLNSERSER(chain H and (resid 3 through 86 or (resid 87...HH3 - 1183 - 118
441GLNGLNSERSER(chain H and (resid 3 through 86 or (resid 87...HH3 - 1183 - 118
451GLNGLNSERSER(chain H and (resid 3 through 86 or (resid 87...HH3 - 1183 - 118
461GLNGLNSERSER(chain H and (resid 3 through 86 or (resid 87...HH3 - 1183 - 118
112GLNGLNGLNGLN(chain C and ((resid 3 and (name N or name...CC33
122GLNGLNSERSER(chain C and ((resid 3 and (name N or name...CC3 - 1153 - 115
132GLNGLNSERSER(chain C and ((resid 3 and (name N or name...CC3 - 1153 - 115
142GLNGLNSERSER(chain C and ((resid 3 and (name N or name...CC3 - 1153 - 115
152GLNGLNSERSER(chain C and ((resid 3 and (name N or name...CC3 - 1153 - 115
212GLNGLNGLNGLN(chain D and ((resid 3 and (name N or name...DD33
222GLNGLNSERSER(chain D and ((resid 3 and (name N or name...DD3 - 1143 - 114
232GLNGLNSERSER(chain D and ((resid 3 and (name N or name...DD3 - 1143 - 114
242GLNGLNSERSER(chain D and ((resid 3 and (name N or name...DD3 - 1143 - 114
252GLNGLNSERSER(chain D and ((resid 3 and (name N or name...DD3 - 1143 - 114
312GLNGLNGLYGLY(chain I and (resid 3 through 42 or (resid 43...II3 - 423 - 42
322LYSLYSLYSLYS(chain I and (resid 3 through 42 or (resid 43...II4343
332GLNGLNSERSER(chain I and (resid 3 through 42 or (resid 43...II3 - 1153 - 115
342GLNGLNSERSER(chain I and (resid 3 through 42 or (resid 43...II3 - 1153 - 115
352GLNGLNSERSER(chain I and (resid 3 through 42 or (resid 43...II3 - 1153 - 115
362GLNGLNSERSER(chain I and (resid 3 through 42 or (resid 43...II3 - 1153 - 115
412GLNGLNGLNGLN(chain J and ((resid 3 and (name N or name...JJ33
422GLNGLNSERSER(chain J and ((resid 3 and (name N or name...JJ3 - 1153 - 115
432GLNGLNSERSER(chain J and ((resid 3 and (name N or name...JJ3 - 1153 - 115
442GLNGLNSERSER(chain J and ((resid 3 and (name N or name...JJ3 - 1153 - 115
452GLNGLNSERSER(chain J and ((resid 3 and (name N or name...JJ3 - 1153 - 115
113LEULEUGLUGLU(chain E and ((resid 1 through 2 and (name N...EE1 - 22 - 3
123ACEACENH2NH2(chain E and ((resid 1 through 2 and (name N...EE0 - 411 - 42
133ACEACENH2NH2(chain E and ((resid 1 through 2 and (name N...EE0 - 411 - 42
143ACEACENH2NH2(chain E and ((resid 1 through 2 and (name N...EE0 - 411 - 42
153ACEACENH2NH2(chain E and ((resid 1 through 2 and (name N...EE0 - 411 - 42
213LEULEULEULEU(chain F and (resid 1 through 5 or (resid 6...FF1 - 52 - 6
223LYSLYSLYSLYS(chain F and (resid 1 through 5 or (resid 6...FF67
233ACEACELEULEU(chain F and (resid 1 through 5 or (resid 6...FF0 - 401 - 41
243ACEACELEULEU(chain F and (resid 1 through 5 or (resid 6...FF0 - 401 - 41
253ACEACELEULEU(chain F and (resid 1 through 5 or (resid 6...FF0 - 401 - 41
263ACEACELEULEU(chain F and (resid 1 through 5 or (resid 6...FF0 - 401 - 41
313LEULEUGLUGLU(chain K and ((resid 1 through 2 and (name N...KK1 - 22 - 3
323LEULEULEULEU(chain K and ((resid 1 through 2 and (name N...KK1 - 402 - 41
333LEULEULEULEU(chain K and ((resid 1 through 2 and (name N...KK1 - 402 - 41
343LEULEULEULEU(chain K and ((resid 1 through 2 and (name N...KK1 - 402 - 41
353LEULEULEULEU(chain K and ((resid 1 through 2 and (name N...KK1 - 402 - 41
413LEULEUGLUGLU(chain L and ((resid 1 through 2 and (name N...LL1 - 22 - 3
423LEULEULEULEU(chain L and ((resid 1 through 2 and (name N...LL1 - 402 - 41
433LEULEULEULEU(chain L and ((resid 1 through 2 and (name N...LL1 - 402 - 41
443LEULEULEULEU(chain L and ((resid 1 through 2 and (name N...LL1 - 402 - 41
453LEULEULEULEU(chain L and ((resid 1 through 2 and (name N...LL1 - 402 - 41

NCS ensembles :
ID
1
2
3

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Components

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Protein/peptide , 1 types, 4 molecules EFKL

#3: Protein/peptide
APH coiled-coil


Mass: 4835.549 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Antibody , 2 types, 8 molecules ABGHCDIJ

#1: Antibody
Nanobody Nb28


Mass: 14426.892 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
#2: Antibody
Nanobody Nb30


Mass: 14304.715 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Non-polymers , 4 types, 64 molecules

#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H6O2
#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#6: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H3O2
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 59 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
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: 2.8 Å3/Da / Density % sol: 56.14 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 0.2 M sodium chloride, 2.0 M ammonium sulfate, 0.1 M Sodium cacodylate 6.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.97857 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 18, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97857 Å / Relative weight: 1
ReflectionResolution: 2.694→48.227 Å / Num. obs: 42539 / % possible obs: 99.48 % / Redundancy: 11.82 % / Biso Wilson estimate: 69.55 Å2 / CC1/2: 0.998 / Rrim(I) all: 0.144 / Net I/σ(I): 15.52
Reflection shellResolution: 2.694→2.791 Å / Num. unique obs: 4077 / CC1/2: 0.654 / Rrim(I) all: 1.516 / % possible all: 95.74

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
PHENIX1.12_2829refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Modeler

Resolution: 2.694→48.227 Å / SU ML: 0.35 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 24.49 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2227 2121 4.99 %
Rwork0.1765 40343 -
obs0.1789 42464 99.44 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 170.66 Å2 / Biso mean: 69.58 Å2 / Biso min: 30 Å2
Refinement stepCycle: final / Resolution: 2.694→48.227 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8303 0 51 59 8413
Biso mean--105.26 63.56 -
Num. residues----1085
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0098507
X-RAY DIFFRACTIONf_angle_d1.12811524
X-RAY DIFFRACTIONf_chiral_restr0.0591221
X-RAY DIFFRACTIONf_plane_restr0.0071505
X-RAY DIFFRACTIONf_dihedral_angle_d16.4565047
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2131X-RAY DIFFRACTION14.006TORSIONAL
12B2131X-RAY DIFFRACTION14.006TORSIONAL
13G2131X-RAY DIFFRACTION14.006TORSIONAL
14H2131X-RAY DIFFRACTION14.006TORSIONAL
21C2022X-RAY DIFFRACTION14.006TORSIONAL
22D2022X-RAY DIFFRACTION14.006TORSIONAL
23I2022X-RAY DIFFRACTION14.006TORSIONAL
24J2022X-RAY DIFFRACTION14.006TORSIONAL
31E782X-RAY DIFFRACTION14.006TORSIONAL
32F782X-RAY DIFFRACTION14.006TORSIONAL
33K782X-RAY DIFFRACTION14.006TORSIONAL
34L782X-RAY DIFFRACTION14.006TORSIONAL
LS refinement shell

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

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.694-2.75620.33551280.3137247193
2.7562-2.82510.33191390.2632644100
2.8251-2.90150.31621410.26522682100
2.9015-2.98690.30081390.25522636100
2.9869-3.08330.32861420.25252684100
3.0833-3.19350.27371400.22352663100
3.1935-3.32130.29981410.21822683100
3.3213-3.47240.2521400.1912671100
3.4724-3.65540.26391420.18482696100
3.6554-3.88440.2441410.1652684100
3.8844-4.18410.19361420.15022696100
4.1841-4.60490.15221440.12462738100
4.6049-5.27050.17131430.13092724100
5.2705-6.63750.19181460.16842777100
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)
13.75-0.6913-0.40898.29223.03064.08980.14680.14980.3620.17620.0561-0.5458-0.12130.1626-0.18750.457-0.01830.03930.35890.02180.49174.4783-0.0507-9.054
24.9424-1.22851.01898.7414-0.18934.2578-0.11010.0179-0.40731.14450.127-0.23360.96310.11130.04870.8240.0683-0.0640.3666-0.09970.513979.2951-32.469-9.9887
35.20331.6623-0.55642.2108-0.58823.11470.27080.6095-0.1364-0.0902-0.34610.2095-0.4782-1.33640.08450.69640.22210.02781.0525-0.20280.479353.124-17.909-33.2027
44.0803-0.1711-1.01643.7874-0.50227.74890.24380.1348-0.0419-0.4182-0.0832-0.2460.05490.6857-0.17990.56590.18940.03110.5543-0.06220.416476.8296-17.4502-42.9157
53.7314-3.1445-3.67723.56662.27387.12330.55340.41380.6056-0.1586-0.3595-0.2236-0.5560.3142-0.09260.45950.0164-0.09540.4678-0.02230.340771.9886-12.5328-22.1712
63.7033-1.2617-4.8792.15940.71736.36580.53410.5085-1.2354-0.3002-0.51810.2074-0.0512-0.1196-0.04240.56470.0699-0.03830.4169-0.0910.333671.1195-21.1457-21.6044
73.4868-1.4622-0.38967.00633.19117.3878-0.1528-0.0039-0.34880.14540.0622-0.1490.6540.1860.04670.54110.00010.05440.28720.00420.342746.79190.11375.3835
82.5893-0.04940.23518.1619-0.48944.3068-0.12750.06960.5256-0.0486-0.0970.0848-0.0355-0.06030.23760.37590.014-0.0710.2862-0.01080.476747.002432.65886.5353
96.6193-2.3948-2.54172.55731.32045.7416-0.3568-0.0378-0.227-0.04480.21330.30430.3151-0.21480.1390.84640.018-0.07540.4437-0.04110.457724.317413.807330.7779
105.95110.31220.62642.5568-0.66486.61150.2587-1.5169-0.05910.8704-0.0326-0.43330.32041.0856-0.18040.90070.0214-0.13771.0299-0.03360.467647.938216.789139.4639
113.86114.39590.43647.50434.79625.52820.4297-0.0998-0.7050.5939-0.2541-0.23010.78370.2379-0.18530.6310.05590.05280.37360.01440.398642.773311.731718.747
126.96637.54344.92196.98973.31274.81730.3161-0.79790.93840.3797-0.45560.39540.0698-0.36930.07810.65250.0533-0.02440.3625-0.00740.418941.086420.038718.3874
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1(chain 'A' and resid 3 through 120)A3 - 120
2X-RAY DIFFRACTION2(chain 'B' and resid 2 through 118)B2 - 118
3X-RAY DIFFRACTION3(chain 'C' and resid 3 through 115)C3 - 115
4X-RAY DIFFRACTION4(chain 'D' and resid 3 through 114)D3 - 114
5X-RAY DIFFRACTION5(chain 'E' and resid 0 through 41)E0 - 41
6X-RAY DIFFRACTION6(chain 'F' and resid 0 through 40)F0 - 40
7X-RAY DIFFRACTION7(chain 'G' and resid 1 through 120)G1 - 120
8X-RAY DIFFRACTION8(chain 'H' and resid 3 through 118)H3 - 118
9X-RAY DIFFRACTION9(chain 'I' and resid 3 through 115)I3 - 115
10X-RAY DIFFRACTION10(chain 'J' and resid 3 through 115)J3 - 115
11X-RAY DIFFRACTION11(chain 'K' and resid 1 through 40)K1 - 40
12X-RAY DIFFRACTION12(chain 'L' and resid 1 through 40)L1 - 40

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