EMDB-6717: Contactin-2 reconstructed by Individual-Particle Electron Tomography

Basic information

• Entry: Database: EMDB / ID: EMD-6717
• Title: Contactin-2 reconstructed by Individual-Particle Electron Tomography
• Map data: Contactin-2 conformation 1

Sample

• Complex: Contactin-2

• Biological species: Homo sapiens (human)
• Method: electron tomography / negative staining / Resolution: 20.0 Å
• Authors: Lu Z / Liu JK / Ren G / Rudenko G

Funding support

United States, China, 6 items

Organization	Grant number	Country
National Institutes of Health/National Institute of Mental Health	R01MH077303	United States
Department of Energy (United States)	DE-AC02-05CH11231	United States
National Institutes of Health/National Institute of Mental Health	R01MH070860	United States
China Fundamental Research Funds for the Central Universities	08143008	China
China Scholarship Council	201206280093	China
China Fundamental Research Funds for the Central Universities	08143101	China

• Citation: Journal: J Biol Chem / Year: 2016; Title: Molecular Architecture of Contactin-associated Protein-like 2 (CNTNAP2) and Its Interaction with Contactin 2 (CNTN2).; Authors: Zhuoyang Lu / M V V V Sekhar Reddy / Jianfang Liu / Ana Kalichava / Jiankang Liu / Lei Zhang / Fang Chen / Yun Wang / Luis Marcelo F Holthauzen / Mark A White / Suchithra Seshadrinathan / Xiaoying Zhong / Gang Ren / Gabby Rudenko / ; Abstract: Contactin-associated protein-like 2 (CNTNAP2) is a large multidomain neuronal adhesion molecule implicated in a number of neurological disorders, including epilepsy, schizophrenia, autism spectrum disorder, intellectual disability, and language delay. We reveal here by electron microscopy that the architecture of CNTNAP2 is composed of a large, medium, and small lobe that flex with respect to each other. Using epitope labeling and fragments, we assign the F58C, L1, and L2 domains to the large lobe, the FBG and L3 domains to the middle lobe, and the L4 domain to the small lobe of the CNTNAP2 molecular envelope. Our data reveal that CNTNAP2 has a very different architecture compared with neurexin 1α, a fellow member of the neurexin superfamily and a prototype, suggesting that CNTNAP2 uses a different strategy to integrate into the synaptic protein network. We show that the ectodomains of CNTNAP2 and contactin 2 (CNTN2) bind directly and specifically, with low nanomolar affinity. We show further that mutations in CNTNAP2 implicated in autism spectrum disorder are not segregated but are distributed over the whole ectodomain. The molecular shape and dimensions of CNTNAP2 place constraints on how CNTNAP2 integrates in the cleft of axo-glial and neuronal contact sites and how it functions as an organizing and adhesive molecule.

History

Deposition	Mar 21, 2017	-
Header (metadata) release	Apr 12, 2017	-
Map release	Sep 26, 2018	-
Update	Nov 6, 2019	-
Current status	Nov 6, 2019	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_6717.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Contactin-2 conformation 1
• Voxel size: X=Y=Z: 2.96 Å

Density

Contour Level	Movie #1: 0.12
Minimum - Maximum	-0.20281947 - 1.0123029
Average (Standard dev.)	-0.00012699359 (±0.020009542)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 65 0 Dimensions 192 192 192 Spacing 192 192 192
Cell	A=B=C: 568.32 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	2.96	2.96	2.96
M x/y/z	192	192	192
origin x/y/z	0.000	0.000	0.000
length x/y/z	568.320	568.320	568.320
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	65	0	0
NC/NR/NS	192	192	192
D min/max/mean	-0.203	1.012	-0.000

Supplemental data

Additional map: Contactin-2 conformation 2

• File: emd_6717_additional_1.map
• Annotation: Contactin-2 conformation 2

Additional map: Contactin-2 conformation 11

• File: emd_6717_additional_10.map
• Annotation: Contactin-2 conformation 11

Additional map: Contactin-2 conformation 12

• File: emd_6717_additional_11.map
• Annotation: Contactin-2 conformation 12

Additional map: Contactin-2 conformation 13

• File: emd_6717_additional_12.map
• Annotation: Contactin-2 conformation 13

Additional map: Contactin-2 conformation 14

• File: emd_6717_additional_13.map
• Annotation: Contactin-2 conformation 14

Additional map: Contactin-2 conformation 15

• File: emd_6717_additional_14.map
• Annotation: Contactin-2 conformation 15

Additional map: Contactin-2 conformation 16

• File: emd_6717_additional_15.map
• Annotation: Contactin-2 conformation 16

Additional map: Contactin-2 conformation 17

• File: emd_6717_additional_16.map
• Annotation: Contactin-2 conformation 17

Additional map: Contactin-2 conformation 18

• File: emd_6717_additional_17.map
• Annotation: Contactin-2 conformation 18

Additional map: Contactin-2 conformation 19

• File: emd_6717_additional_18.map
• Annotation: Contactin-2 conformation 19

Additional map: Contactin-2 conformation 20

• File: emd_6717_additional_19.map
• Annotation: Contactin-2 conformation 20

Additional map: Contactin-2 conformation 3

• File: emd_6717_additional_2.map
• Annotation: Contactin-2 conformation 3

Additional map: Contactin-2 conformation 21

• File: emd_6717_additional_20.map
• Annotation: Contactin-2 conformation 21

Additional map: Contactin-2 conformation 22

• File: emd_6717_additional_21.map
• Annotation: Contactin-2 conformation 22

Additional map: Contactin-2 conformation 23

• File: emd_6717_additional_22.map
• Annotation: Contactin-2 conformation 23

Additional map: Contactin-2 conformation 24

• File: emd_6717_additional_23.map
• Annotation: Contactin-2 conformation 24

Additional map: Contactin-2 conformation 25

• File: emd_6717_additional_24.map
• Annotation: Contactin-2 conformation 25

Additional map: Contactin-2 conformation 26

• File: emd_6717_additional_25.map
• Annotation: Contactin-2 conformation 26

Additional map: Contactin-2 conformation 27

• File: emd_6717_additional_26.map
• Annotation: Contactin-2 conformation 27

Additional map: Contactin-2 conformation 28

• File: emd_6717_additional_27.map
• Annotation: Contactin-2 conformation 28

Additional map: Contactin-2 conformation 29

• File: emd_6717_additional_28.map
• Annotation: Contactin-2 conformation 29

Additional map: Contactin-2 conformation 30

• File: emd_6717_additional_29.map
• Annotation: Contactin-2 conformation 30

Additional map: Contactin-2 conformation 4

• File: emd_6717_additional_3.map
• Annotation: Contactin-2 conformation 4

Additional map: Contactin-2 conformation 31

• File: emd_6717_additional_30.map
• Annotation: Contactin-2 conformation 31

Additional map: Contactin-2 conformation 32

• File: emd_6717_additional_31.map
• Annotation: Contactin-2 conformation 32

Additional map: Contactin-2 conformation 33

• File: emd_6717_additional_32.map
• Annotation: Contactin-2 conformation 33

Additional map: Contactin-2 conformation 34

• File: emd_6717_additional_33.map
• Annotation: Contactin-2 conformation 34

Additional map: Contactin-2 conformation 35

• File: emd_6717_additional_34.map
• Annotation: Contactin-2 conformation 35

Additional map: Contactin-2 conformation 36

• File: emd_6717_additional_35.map
• Annotation: Contactin-2 conformation 36

Additional map: Contactin-2 conformation 37

• File: emd_6717_additional_36.map
• Annotation: Contactin-2 conformation 37

Additional map: Contactin-2 conformation 38

• File: emd_6717_additional_37.map
• Annotation: Contactin-2 conformation 38

Additional map: Contactin-2 conformation 39

• File: emd_6717_additional_38.map
• Annotation: Contactin-2 conformation 39

Additional map: Contactin-2 conformation 40

• File: emd_6717_additional_39.map
• Annotation: Contactin-2 conformation 40

Additional map: Contactin-2 conformation 5

• File: emd_6717_additional_4.map
• Annotation: Contactin-2 conformation 5

Additional map: Contactin-2 conformation 41

• File: emd_6717_additional_40.map
• Annotation: Contactin-2 conformation 41

Additional map: Contactin-2 conformation 42

• File: emd_6717_additional_41.map
• Annotation: Contactin-2 conformation 42

Additional map: Contactin-2 conformation 43

• File: emd_6717_additional_42.map
• Annotation: Contactin-2 conformation 43

Additional map: Contactin-2 conformation 44

• File: emd_6717_additional_43.map
• Annotation: Contactin-2 conformation 44

Additional map: Contactin-2 conformation 45

• File: emd_6717_additional_44.map
• Annotation: Contactin-2 conformation 45

Additional map: Contactin-2 conformation 46

• File: emd_6717_additional_45.map
• Annotation: Contactin-2 conformation 46

Additional map: Contactin-2 conformation 47

• File: emd_6717_additional_46.map
• Annotation: Contactin-2 conformation 47

Additional map: Contactin-2 conformation 48

• File: emd_6717_additional_47.map
• Annotation: Contactin-2 conformation 48

Additional map: Contactin-2 conformation 49

• File: emd_6717_additional_48.map
• Annotation: Contactin-2 conformation 49

Additional map: Contactin-2 conformation 50

• File: emd_6717_additional_49.map
• Annotation: Contactin-2 conformation 50

Additional map: Contactin-2 conformation 6

• File: emd_6717_additional_5.map
• Annotation: Contactin-2 conformation 6

Additional map: Contactin-2 conformation 51

• File: emd_6717_additional_50.map
• Annotation: Contactin-2 conformation 51

Additional map: Contactin-2 conformation 52

• File: emd_6717_additional_51.map
• Annotation: Contactin-2 conformation 52

Additional map: Contactin-2 conformation 53

• File: emd_6717_additional_52.map
• Annotation: Contactin-2 conformation 53

Additional map: Contactin-2 conformation 54

• File: emd_6717_additional_53.map
• Annotation: Contactin-2 conformation 54

Additional map: Contactin-2 conformation 55

• File: emd_6717_additional_54.map
• Annotation: Contactin-2 conformation 55

Additional map: Contactin-2 conformation 56

• File: emd_6717_additional_55.map
• Annotation: Contactin-2 conformation 56

Additional map: Contactin-2 conformation 57

• File: emd_6717_additional_56.map
• Annotation: Contactin-2 conformation 57

Additional map: Contactin-2 conformation 58

• File: emd_6717_additional_57.map
• Annotation: Contactin-2 conformation 58

Additional map: Contactin-2 conformation 59

• File: emd_6717_additional_58.map
• Annotation: Contactin-2 conformation 59

Additional map: Contactin-2 conformation 7

• File: emd_6717_additional_6.map
• Annotation: Contactin-2 conformation 7

Additional map: Contactin-2 conformation 8

• File: emd_6717_additional_7.map
• Annotation: Contactin-2 conformation 8

Additional map: Contactin-2 conformation 9

• File: emd_6717_additional_8.map
• Annotation: Contactin-2 conformation 9

Additional map: Contactin-2 conformation 10

• File: emd_6717_additional_9.map
• Annotation: Contactin-2 conformation 10

Sample components

Entire : Contactin-2

• Entire: Name: Contactin-2

Components

• Complex: Contactin-2

Supramolecule #1: Contactin-2

• Supramolecule: Name: Contactin-2 / type: complex / ID: 1 / Parent: 0
• Source (natural): Organism: Homo sapiens (human)

Experimental details

Structure determination

• Method: negative staining
• Processing: electron tomography
• Aggregation state: particle

Sample preparation

• Concentration: 0.0025 mg/mL

Buffer

pH: 8
Component:

Concentration	Formula	Name
25.0 mM	C4H11NO3	Tris
100.0 mM	NaClSodium chloride	sodium chloride
3.0 mM	CaCl2	calcium chloride

• Staining: Type: NEGATIVE / Material: uranium formate / Details: 1% w/v uranyl formate
• Grid: Model: EMSCF, 200-Cu / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 5.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
• Details: This sample was monodisperse.
• Sectioning: Other: NO SECTIONING

Electron microscopy

• Microscope: ZEISS LIBRA120PLUS
• Electron beam: Acceleration voltage: 120 kV / Electron source: LAB6
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 0.6 µm / Nominal defocus min: 0.1 µm / Nominal magnification: 80000
• Sample stage: Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
• Image recording: Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 3.0 e/Ų

Image processing

• CTF correction: Software - Name: TOMOCTF
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IPET / Number images used: 81