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- PDB-6e6b: Crystal structure of the Protocadherin GammaB4 extracellular domain -

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Basic information

Entry
Database: PDB / ID: 6e6b
TitleCrystal structure of the Protocadherin GammaB4 extracellular domain
ComponentsProtocadherin gamma B4
KeywordsCELL ADHESION / Cadherin / Polymer / Neuronal self-recognition / Neuronal self-avoidance
Function / homology
Function and homology information


plasma membrane => GO:0005886 / homophilic cell adhesion via plasma membrane adhesion molecules / cell adhesion / calcium ion binding / membrane
Similarity search - Function
Cadherin, C-terminal catenin-binding domain / Cadherin C-terminal cytoplasmic tail, catenin-binding region / Cadherin, cytoplasmic C-terminal domain / Cadherin cytoplasmic C-terminal / Cadherin, N-terminal / Cadherin-like / Cadherin conserved site / Cadherin domain signature. / Cadherin repeats. / Cadherin domain ...Cadherin, C-terminal catenin-binding domain / Cadherin C-terminal cytoplasmic tail, catenin-binding region / Cadherin, cytoplasmic C-terminal domain / Cadherin cytoplasmic C-terminal / Cadherin, N-terminal / Cadherin-like / Cadherin conserved site / Cadherin domain signature. / Cadherin repeats. / Cadherin domain / Cadherins domain profile. / Cadherin-like / Cadherin-like superfamily
Similarity search - Domain/homology
alpha-D-mannopyranose / Protocadherin gamma B4
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 4.52 Å
AuthorsGoodman, K.M. / Mannepalli, S. / Bahna, F. / Honig, B. / Shapiro, L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)R01 MH114817 United States
CitationJournal: Nature / Year: 2019
Title: Visualization of clustered protocadherin neuronal self-recognition complexes.
Authors: Julia Brasch / Kerry M Goodman / Alex J Noble / Micah Rapp / Seetha Mannepalli / Fabiana Bahna / Venkata P Dandey / Tristan Bepler / Bonnie Berger / Tom Maniatis / Clinton S Potter / Bridget ...Authors: Julia Brasch / Kerry M Goodman / Alex J Noble / Micah Rapp / Seetha Mannepalli / Fabiana Bahna / Venkata P Dandey / Tristan Bepler / Bonnie Berger / Tom Maniatis / Clinton S Potter / Bridget Carragher / Barry Honig / Lawrence Shapiro /
Abstract: Neurite self-recognition and avoidance are fundamental properties of all nervous systems. These processes facilitate dendritic arborization, prevent formation of autapses and allow free interaction ...Neurite self-recognition and avoidance are fundamental properties of all nervous systems. These processes facilitate dendritic arborization, prevent formation of autapses and allow free interaction among non-self neurons. Avoidance among self neurites is mediated by stochastic cell-surface expression of combinations of about 60 isoforms of α-, β- and γ-clustered protocadherin that provide mammalian neurons with single-cell identities. Avoidance is observed between neurons that express identical protocadherin repertoires, and single-isoform differences are sufficient to prevent self-recognition. Protocadherins form isoform-promiscuous cis dimers and isoform-specific homophilic trans dimers. Although these interactions have previously been characterized in isolation, structures of full-length protocadherin ectodomains have not been determined, and how these two interfaces engage in self-recognition between neuronal surfaces remains unknown. Here we determine the molecular arrangement of full-length clustered protocadherin ectodomains in single-isoform self-recognition complexes, using X-ray crystallography and cryo-electron tomography. We determine the crystal structure of the clustered protocadherin γB4 ectodomain, which reveals a zipper-like lattice that is formed by alternating cis and trans interactions. Using cryo-electron tomography, we show that clustered protocadherin γB6 ectodomains tethered to liposomes spontaneously assemble into linear arrays at membrane contact sites, in a configuration that is consistent with the assembly observed in the crystal structure. These linear assemblies pack against each other as parallel arrays to form larger two-dimensional structures between membranes. Our results suggest that the formation of ordered linear assemblies by clustered protocadherins represents the initial self-recognition step in neuronal avoidance, and thus provide support for the isoform-mismatch chain-termination model of protocadherin-mediated self-recognition, which depends on these linear chains.
History
DepositionJul 24, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 10, 2019Provider: repository / Type: Initial release
Revision 1.1Apr 24, 2019Group: Data collection / Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2May 22, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Nov 27, 2019Group: Author supporting evidence / Data collection / Category: chem_comp / pdbx_audit_support
Item: _chem_comp.type / _pdbx_audit_support.funding_organization
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / struct_asym / struct_conn / struct_conn_type / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _chem_comp.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn_type.id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Oct 11, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn / struct_ncs_dom_lim
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI ..._chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag / _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
A: Protocadherin gamma B4
B: Protocadherin gamma B4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)146,62242
Polymers141,9842
Non-polymers4,63840
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, Sedimentation equilibrium analytical ultracentrifugation experiments show that PcdhgB4 EC1-6 is a dimer-of-dimers in solution. However in both the crystal structure and cryo- ...Evidence: microscopy, Sedimentation equilibrium analytical ultracentrifugation experiments show that PcdhgB4 EC1-6 is a dimer-of-dimers in solution. However in both the crystal structure and cryo-electron tomography of PcdhgB4 on membrane reveals a one-dimensional lattice of alternating C-terminal (cis) and N-terminal (trans) interactions, the same as those used to form the dimer-of-dimers observed in solution but generating a polymer rather than a discrete dimer-of-dimers.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6270 Å2
ΔGint-86 kcal/mol
Surface area72640 Å2
MethodPISA
Unit cell
Length a, b, c (Å)127.730, 87.580, 149.330
Angle α, β, γ (deg.)90.000, 109.940, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21

NCS domain segments:

Ens-ID: 1 / Beg auth comp-ID: GLN / Beg label comp-ID: GLN

Dom-IDComponent-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11SERSER(chain 'A' and (resid 2 through 5 or (resid 6...AA2 - 2522 - 252
12PHEPHE(chain 'A' and (resid 2 through 5 or (resid 6...AA259 - 632259 - 632
21SERSER(chain 'B' and (resid 2 through 10 or (resid 11...BB2 - 2522 - 252
22PHEPHE(chain 'B' and (resid 2 through 10 or (resid 11...BB259 - 632259 - 632
DetailsSedimentation equilibrium analytical ultracentrifugation experiments show that PcdhgB4 EC1-6 is a dimer-of-dimers in solution. However in both the crystal structure and cryo-electron tomography of PcdhgB4 on membrane reveals a one-dimensional lattice of alternating C-terminal (cis) and N-terminal (trans) interactions, the same as those used to form the dimer-of-dimers observed in solution but generating a polymer rather than a discrete dimer-of-dimers.

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Components

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Protein / Non-polymers , 2 types, 32 molecules AB

#1: Protein Protocadherin gamma B4 / Protocadherin gamma subfamily B / 4


Mass: 70991.781 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Protocadherin extracellular cadherin domains 1-6 with C-terminal octahistidine expression tag
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Pcdhgb4 / Cell line (production host): HEK-293F / Production host: Homo sapiens (human) / References: UniProt: Q91XX6
#5: Chemical...
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 30 / Source method: obtained synthetically / Formula: Ca

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Sugars , 5 types, 10 molecules

#2: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#3: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1056.964 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/4,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5][a1221m-1a_1-5]/1-1-2-3-3-4/a4-b1_a6-f1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}[(6+1)][a-L-Fucp]{}}}LINUCSPDB-CARE
#4: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta- ...2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 570.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1221m-1a_1-5]/1-1-2/a4-b1_a6-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}[(6+1)][a-L-Fucp]{}}}LINUCSPDB-CARE
#6: Sugar
ChemComp-MAN / alpha-D-mannopyranose / alpha-D-mannose / D-mannose / mannose


Type: D-saccharide, alpha linking / Mass: 180.156 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Formula: C6H12O6
IdentifierTypeProgram
DManpaCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
a-D-mannopyranoseCOMMON NAMEGMML 1.0
a-D-ManpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
ManSNFG CARBOHYDRATE SYMBOLGMML 1.0
#7: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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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: 5.55 Å3/Da / Density % sol: 77.83 %
Crystal growTemperature: 295 K / Method: batch mode / pH: 7.5
Details: 10% (w/v) PEG8000, 20% ethylene glycol, 10% Morpheus Amino Acids (Molecular Dimensions), and 0.1 M Morpheus Buffer System 2 (Hepes/MOPS buffer; Molecular Dimensions) pH 7.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.97919 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Dec 1, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97919 Å / Relative weight: 1
ReflectionResolution: 4.5→40 Å / Num. obs: 8694 / % possible obs: 93.4 % / Redundancy: 2.8 % / Biso Wilson estimate: 66.09 Å2 / CC1/2: 0.995 / Rmerge(I) obs: 0.113 / Rpim(I) all: 0.078 / Rrim(I) all: 0.138 / Net I/σ(I): 5.2
Reflection shellResolution: 4.5→5.05 Å / Redundancy: 3 % / Rmerge(I) obs: 0.173 / Mean I/σ(I) obs: 5.7 / Num. unique obs: 317 / CC1/2: 0.973 / Rpim(I) all: 0.119 / Rrim(I) all: 0.211

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Processing

Software
NameVersionClassification
PHENIX1.13_2998refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5V5X, 5T9T

5v5x

5t9t


Resolution: 4.52→38.29 Å / SU ML: 0.7857 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 34.4867
Details: Due to the severe anisotropy of the data ellipsoidal resolution limits of 6.8, 6.0, and 4.5 angstroms were applied along the three principal axes. The completeness with respect to a sphere ...Details: Due to the severe anisotropy of the data ellipsoidal resolution limits of 6.8, 6.0, and 4.5 angstroms were applied along the three principal axes. The completeness with respect to a sphere at 4.5 angstroms is therefore very low (46.9%), however the completeness within the ellipsoidal limits is 93.4%.
RfactorNum. reflection% reflection
Rfree0.2767 448 5.16 %
Rwork0.2312 --
obs0.2336 8683 93.4 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 138.7 Å2
Refinement stepCycle: LAST / Resolution: 4.52→38.29 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9489 0 253 0 9742
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00249916
X-RAY DIFFRACTIONf_angle_d0.565413567
X-RAY DIFFRACTIONf_chiral_restr0.04171629
X-RAY DIFFRACTIONf_plane_restr0.00381780
X-RAY DIFFRACTIONf_dihedral_angle_d11.57735847
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
4.52-5.170.2868200.2639444X-RAY DIFFRACTION7.6
5.17-6.510.3871400.29812393X-RAY DIFFRACTION41.51
6.51-38.290.24212880.21175398X-RAY DIFFRACTION90.74

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