Structure of the endosomal Commander complex linked to Ritscher-Schinzel syndrome

. 2023 May 11;186(10):2219–2237.e29. doi: 10.1016/j.cell.2023.04.003

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

CCDC22	Proteintech	Cat# 16636-1-AP
CCDC93	LS Bio	Cat# C336997
CCDC93	Proteintech	Cat# 20861-1-AP
EEA1	BD Biosciences	Cat# 610457
FAM21	Gift from Dan Billadeau	N/A
GFP	Roche	Cat# 11814460001
Integrin α5	BD Biosciences	Cat# 555651
Integrin α5	Abcam	Cat# EPR7854
Integrin β1	BD Biosciences	Cat# 610467
LAMP1	Developmental Studies Hybridoma Bank	Cat# H4A3
VPS26A	Abcam	Cat# ab137447
VPS26C	Merck Millipore	Cat# ABN87
VPS29	Abcam	Cat# ab98929
VPS29	Santa Cruz	Cat# 398874
VPS35	Abcam	Cat# ab157220
VPS35	Abcam	Cat# ab97545
VPS35	Abcam	Cat# ab10099
VPS35L	Abcam	Cat# ab97889
VPS35L	Pierce	Cat# PA5-28553
WASH1	Gift from Dan Billadeau	N/A
TBC1D5	Abcam	Cat# 203896
ANKRD27	Abcam	Cat# ab108216
GFP	Roche	Cat# 11814460001
N-cadherin	Cell Signaling	Cat# 14215
N-cadherin	Cell Signaling	Cat# 13116
β actin	Sigma	Cat# A1978
COMMD1	Proteintech	Cat# 11938-1-AP
COMMD1	Sigma	Cat# WH0150684M1
COMMD2	Millipore Sigma	Cat# HPA044190
COMMD3	Abcam	Cat# ab176583
COMMD4	Abcam	Cat# ab115169
COMMD5	Proteintech	Cat# 67043-1-Ig
COMMD7	Abcam	Cat# ab96091
COMMD7	GeneTex	Cat# GTX112076
COMMD8	Proteintech	Cat# 25237-1-AP
COMMD8	Santa Cruz	Cat# sc-3973869
COMMD9	Abcam	Cat# Ab121303
COMMD10	Santa Cruz	Cat# sc-398798
COMMD10	GeneTex	Cat# GTX121488
HA-tag	Insight Biotechnology	Cat# A02040
Strep-tag	GeneTex	Cat# GTX128061
6x His-tag	Abcam	Cat# Ab18184
Myc-tag	GeneTex	Cat# GTX115046
FLAG tag	Sigma	Cat# F3165
SNAP-tag	GeneTex	Cat# GTX54523
Alexa Fluor 680 anti-mouse IgG	Life Technologies	Cat# A10038
Alexa Fluor 800 anti-rabbit IgG	Life Technologies	Cat# W10824
Goat anti-rabbit IgG	Life Technologies	Cat# AB2533967
Alexa Fluor 488 anti-mouse IgG	Life Technologies	Cat# A21202
Alexa Fluor 568 anti-mouse IgG	Life Technologies	Cat# A10037
Alexa Fluor 647 anti-mouse IgG	Life Technologies	Cat# A31571
Alexa Fluor 488 anti-rabbit IgG	Life Technologies	Cat# A21206
Alexa Fluor 568 anti-rabbit IgG	Life Technologies	Cat# A10042
Alexa Fluor 647 anti-rabbit IgG	Life Technologies	Cat# A31573
Alexa Fluor 488 anti-goat IgG	Life Technologies	Cat# A11055
Alexa Fluor 568 anti-goat IgG	Life Technologies	Cat# A11057
Alexa Fluor 647 anti-goat IgG	Life Technologies	Cat# A21447

Bacterial and virus strains

E. coli DH5α	Invitrogen	Cat# 18265017
BL21 (DE3)	New England Biolabs	Cat# C2527H
DH10EMBacY	ThermoFisher	Cat# 10361012
NEB 5-alpha	New England Biolabs	Cat# C2987H

Experimental models: Cell lines

HeLa	ATCC	Cat# CCL-2
HEK293T	ATCC	Cat# CRL-3216
RPE1	ATCC	Cat# CRL-4000
Spodoptera frugiperda Sf21	ThermoFisher	Cat# 11497013
eHap	Horizon Discovery	Cat# C669
VPS35 knock-out HeLa cells	⁹⁷	N/A
VPS29 knock-out HeLa cells	This study	N/A
VPS35L knock-out HeLa cells	This study	N/A
COMMD1 knock-out eHAP cells	This study	N/A
COMMD2 knock-out eHAP cells	This study	N/A
COMMD3 knock-out eHAP cells	This study	N/A
COMMD4 knock-out eHAP cells	This study	N/A
COMMD5 knock-out eHAP cells	This study	N/A
COMMD6 knock-out eHAP cells	This study	N/A
COMMD7 knock-out eHAP cells	This study	N/A
COMMD8 knock-out eHAP cells	This study	N/A
COMMD9 knock-out eHAP cells	This study	N/A
COMMD10 knock-out eHAP cells	This study	N/A

Chemicals and peptides

Benzamidine hydrochloride hydrate	Sigma Aldrich	Cat# B6506
Deoxyribonuclease I (DNase I)	Sigma Aldrich	Cat# DN25
Talon® resin	Clontech	Cat# 635503
Glutathione Sepharose 4B	GE Healthcare	Cat# GEHE17-0756-0
Isopropyl β-D-1-thiogalactopyranoside	Bioline	Cat# BIO-37036
Triton X-100	Sigma Aldrich	Cat# X100-500ML
VPS35L (16-38)	Genscript	N/A
VPS35L (27-38)	Genscript	N/A
Sf-900 II SFM media	ThermoFisher Scientific	Cat# 10902088
X-tremeGENE HP DNA Transfection Reagent	Sigma-Aldrich	Cat# 6366244001
Graphene oxide	Sigma-Aldrich	Cat# 763705
DMEM	Sigma-Aldrich	Cat# D5796
25 kDa PEI	Polysciences	Cat# 23966-2
Sulfo-NHS-SS Biotin	ThermoFisher	Cat# 21217
Fluoromount-G	Invitrogen	Cat# 004958-02

Critical commercial assays

QIAprep Spin Miniprep Kit	QIAGEN	Cat# 27106
NEBuilder HiFi DNA Assembly System (NEB)	New England Biolabs	Cat# E5520S
TMT seven-plex	ThermoFisher	Cat# 90061

Oligonucleotides

Primers used in this study, see Table S3	This paper	N/A
gRNA targeting VPS29 exon 2 GGCAAACTGTTGCACCGGTG	This paper	N/A
gRNA targeting VPS29 exon 3 GGACATCAAGTTATTCCATG	This paper	N/A
gRNA targeting VPS35L exon 1 GCAGACGCCATGCTTGTGAG	This paper	N/A
gRNA targeting COMMD1 CGCTGGAATAGCGGGCTTCGGGG	This paper	N/A
gRNA targeting COMMD2 GCGGCGCCTTCGTAGATTTTTGG	This paper	N/A
gRNA targeting COMMD3 GCCGAGCGGCTTACCTAACACGG	This paper	N/A
gRNA targeting COMMD4 AGCGGGATACTCACCAAACTTGG	This paper	N/A
gRNA targeting COMMD5 AGCAATGGCCCGGCTACTAGGGG	This paper	N/A
gRNA targeting COMMD6 CGTTGCAGTGATGCTAAAAGTGG	This paper	N/A
gRNA targeting COMMD7 ACCCTTGCTCGATGGGCCATAGG	This paper	N/A
gRNA targeting COMMD8 TACCTTATTCGCTGCTTCCAAGG	This paper	N/A
gRNA targeting COMMD9 CACGCCTGGTCGATCTGGACTGG	This paper	N/A
gRNA targeting COMMD10 AGAATCCGAGTGAGCAACCGTGG	This paper	N/A

Recombinant DNA

pACEBAC1	NovoPro	Cat# V011691#
MultiBAC pKL vector	Gift from Prof. Imre Berger	N/A
pbiG1a	Gift from Dr. Andrew Carter, MRC-LMB, UK.⁹⁸	N/A
pbiG2b	Gift from Dr. Andrew Carter, MRC-LMB, UK.⁹⁸	N/A
pbiG2d	Gift from Dr. Andrew Carter, MRC-LMB, UK.⁹⁸	N/A
pbiG2e	Gift from Dr. Andrew Carter, MRC-LMB, UK.⁹⁸	N/A
pbiG1a Strep-VPS26C, VPS35L, VPS29-His (Retriever)	This study	N/A
pbiG2bde COMMD1, myc-COMMD2, SNAP-COMMD3, His-COMMD4, HA-COMMD5, HA-COMMD6, V5-COMMD7, COMMD8, FLAG-COMMD9, COMMD10, CCDC22, CCDC93-Strep (CCC complex)	This study	N/A
pbiG1A VPS35, Strep-VPS26A, VPS29-His (Retromer)	This study	N/A
pACEBac1 His-TBC1D5	This study	N/A
pST39 Commd1-FLAG, Commd2, Commd6, Commd4, Commd7, Commd8, Commd9, Commd3, Commd5-Strep, Commd10-His.	This study	N/A
pST39 Commd1-His, Commd2, Commd6, Commd4, Commd7, Commd8, Commd9, Commd3, Commd5, Commd10.	This study	N/A
pST39 Commd1, Commd2-His, Commd6, Commd4, Commd7, Commd8, Commd9, Commd3, Commd5, Commd10.	This study	N/A
pST39 Commd1, Commd2, Commd6, Commd4, Commd7, Commd8, Commd9, Commd3, Commd5-His, Commd10.	This study	N/A
pRSF-Duet-1 COMMD1-His, COMMD6, COMMD4, COMMD8	This study	N/A
pRSF-Duet-1 COMMD2-His, COMMD3, COMMD4, COMMD8	This study	N/A
pST39 COMMD7, COMMD9, COMMD5, COMMD10-His	This study	N/A
pSpCas9(BB)-2A-GFP	Addgene	PX458
pUMVC3	Addgene	8449
pCMV-VSV-G	Addgene	8454
pEGFP-N1 VPS35L	This study	N/A
pLVX VPS35L	This study	N/A
pGEX4T-2 VPS29	⁴⁴	N/A
pGEX6P-1 DENND10	This study	N/A
pRSF CCDC22 (325–485) + CCDC93 (310–488) CC1 and CC2	This study	N/A
DENND10-GFP	This study	N/A
GFP-CCDC22	McNally et al.⁶	N/A
GFP-CCDC93	McNally et al.⁶	N/A
mCherry RidL	Gift from Da Jia.⁴⁷	N/A
mCherry RidL(1–200)	Gift from Da Jia.⁴⁷	N/A

Deposited data

Commd5-10-7-9 complex (crystal structure)	RCSB Protein DataBank (this study)	PDB: 8ESD
VPS29-VPS35L peptide complex (crystal structure)	RCSB Protein DataBank (this study)	PDB: 8ESE
CCC complex (cryoEM structure; RELION map)	RCSB Protein DataBank (this study)	PDB: 8F2R
CCC complex (cryoEM structure; CryoSPARC map)	RCSB Protein DataBank (this study)	PDB: 8F2U
CCC complex (cryoEM structure; RELION map)	Electron Microscopy DataBank (this study)	EMDB ID: EMD-28827
CCC complex (cryoEM structure; CryoSPARC map)	Electron Microscopy DataBank (this study)	EMDB ID: EMD-28825
Commd9 COMM domain complex (crystal structure)	RCSB Protein DataBank⁴²	PDB: 6BP6
Commd9 HN domain (crystal structure)	RCSB Protein DataBank⁴²	PDB: 4OE9
Commd10 HN domain (crystal structure)	This study	N/A
Commd1 human protein sequence	NCBI	Q8N668
Commd2 human protein sequence	NCBI	Q86X83
Commd3 human protein sequence	NCBI	Q9UBI1
Commd4 human protein sequence	NCBI	Q9H0A8
Commd5 human protein sequence	NCBI	Q9GZQ3
Commd6 human protein sequence	NCBI	Q7Z4G1
Commd7 human protein sequence	NCBI	Q86VX2
Commd8 human protein sequence	NCBI	Q9NX08
Commd9 human protein sequence	NCBI	Q9P000
Commd10 human protein sequence	NCBI	Q9Y6G5
CCDC22 human protein sequence	NCBI	O60826
CCDC93 human protein sequence	NCBI	Q567U6
VPS35L human protein sequence	NCBI	Q7Z3J2
VPS26C human protein sequence	NCBI	O14972
VPS29 human protein sequence	NCBI	Q9UBQ0
DENND10/FAM45A human protein sequence	NCBI	Q8TCE6
COMMD1-10+ CCDC22 (1–223) + CCDC93 (1–300) (AlphaFold2 Multimer prediction)	Model Archive (https://www.modelarchive.org)	ma-iplv4
CCDC22+ CCDC93 (AlphaFold2 Multimer prediction)	Model Archive (https://www.modelarchive.org)	ma-9nv72
VPS35L + VPS26C+ VPS29 (AlphaFold2 Multimer prediction)	Model Archive (https://www.modelarchive.org)	ma-3cag5
VPS35L + VPS26C+ VPS29+ CCDC22 (1–115) + CCDC22 (386–627) + CCDC93 (378–631) (AlphaFold2 Multimer prediction)	Model Archive (https://www.modelarchive.org)	ma-4097m
Combined models 1 + 2 + 3 (AlphaFold2 Multimer prediction)	Model Archive (https://www.modelarchive.org)	ma-ri7tb
COMMD1-10 Danio rerio (AlphaFold2 Multimer prediction)	Model Archive (https://www.modelarchive.org)	ma-99f82
COMMD1-10 Salpingoeca rosetta (AlphaFold2 Multimer prediction)	Model Archive (https://www.modelarchive.org)	ma-xfc84
DENND1B in complex with Rab35 (crystal structure)	RCSB Protein DataBank⁷⁰	PDB: 3TW8

Software and algorithms

XDS	⁹⁹	http://xds.mpimf-heidelberg.mpg.de/
AIMLESS	¹⁰⁰	http://www.ccp4.ac.uk/html/aimless.html
Phaser	¹⁰¹	http://www.phaser.cimr.cam.ac.uk/index.php/Phaser_Crystallographic_Software
Phenix	¹⁰²	https://www.phenix-online.org/
Coot	¹⁰³^,¹⁰⁴	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
Molprobity	¹⁰⁵	http://molprobity.biochem.duke.edu
Pymol	Schrodinger, USA.	https://pymol.org/2/
ChimeraX	¹⁰⁶	https://www.rbvi.ucsf.edu/chimerax/
iSOLDE	¹⁰⁷	https://isolde.cimr.cam.ac.uk
RELION 4.0	¹⁰⁸	https://relion.readthedocs.io/en/release-4.0/
Topaz	¹⁰⁹	https://cb.csail.mit.edu/cb/topaz/
CryoSPARC V3.3.1	¹¹⁰	https://cryosparc.com
AlphaFold2 Multimer	⁵⁴^,⁵⁵	https://github.com/deepmind/alphafold
ColabFold	⁵⁶	https://github.com/sokrypton/ColabFold
MAFFT L-INS-i (v7.505)	¹¹¹	https://mafft.cbrc.jp/alignment/software/
IQTree2.1.3	¹¹²	http://www.iqtree.org
Consurf	¹¹³	https://consurf.tau.ac.il/consurf_index.php

Other

Superose6 Increase10/300 GL column	Cytiva	Cat# 29091596
HiLoad™ Superdex75 PG column	Cytiva	Cat# 28989333
Mono Q 5/50 GL column	Cytiva	Cat# 17516601
C-Flat 1.2/1.3 grids	C-Flat	N/A
Quantifoil 1.2/1.3 Copper 300 mesh grids	Quantifoil	N/A
Anti-FLAG M2 affinity gel	Sigma Aldrich	Cat# A2220
GFP-trap beads	Chromotek	Cat# gta-20
mCherry-trap beads	Chromotek	Cat# rta-20
Streptavidin Sepharose beads	Cytiva	Cat# 90100484