REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit polyclonal GFP	ABCAM	#AB6556; RRID: AB_305564
Rabbit polyclonal anti-CENP-OP	Generated in house	N/A
Mouse monoclonal anti-CENP-P	ABCAM	#AB66058; RRID: AB_1523338
Goat polyclonal anti-CENP-QU	Generated in house	N/A
Mouse monoclonal anti-CENP-Q	ABCAM	#AB57539; RRID: AB_940733
Mouse monoclonal anti-CENP-R	ABCAM	#AB57098; RRID: AB_304751
Rabbit polyclonal anti-CENP-HK	Generated in house	#SI0930
Mouse polyclonal anti-CENP-A	Gene Tex	#GTX13939; RRID: AB_369391
Mouse monoclonal anti-CENP-A	ABCAM	#AB13939; RRID: AB_300766
Rabbit polyclonal anti-CENP-C(23-410)	Trazzi et al., 2009	#SI410
Rabbit polyclonal anti-CENP-N(1-212)	Generated in house	N/A
Rabbit polyclonal anti-CENP-E	Meraldi Lab; Meraldi et al., 2004	N/A
Human anti-centromere (CREST)	Antibodies Inc.	Cat#15-234-0001; RRID: AB_2687472
Bacterial and Virus Strains
E.coli: BL21(DE3)-RIL strain	Agilent Technologies	#230240
Chemicals, Peptides, and Recombinant Proteins
HRV 3C Prescision Protease	Generated in house	N/A
TEV Protease	Generated in house	N/A
Lambda phosphatase	Generated in house	N/A
Protease-inhibitor mix HP Plus	Serva	Cat#39107
Sortase A delta 59 (S.aureus)	Hidde Ploegh Lab	Addgene:Cat#51139
Alexa Fluor 488 C5 maleimide Protein labeling kit	ThermoFisher	#A10254
Alexa Fluor 647 C2 maleimide Protein labeling kit	ThermoFisher	#A20347
Alexa Fluor 405 Carboxylic acid, succinimidyl ester Protein labeling kit	ThermoFisher	#A30000
NHS-Rhodamine labeling kit	Thermo Scientific	#46406
FAM-LPETGG	Genscript	N/A
PhosSTOP phosphatase inhibitor	Roche	Cat#04906845001
(+)-S-Trityl-L-cysteine (STLC)	Sigma Aldrich	#164739
RO-3306	Millipore	#217699
MG-132	Calbiochem	CAS 133407-82-6
MG-132	Sigma	SML1135
Uranyl formate	SPI Supplies	CAS#16984-59-1
Fetal bovine serum (FBS)	Clonetech	#631107
Fetal bovine serum (FBS)	Sigma	Cat:f7524 Batch:111M3395
Zeocin	Invitrogen	Cat#R25001
L-glutamine	PAN Biotech	P04-80100
Nocodazole	Sigma	Cat#M1404
CENP-C(1-544)	Musacchio Lab; Screpanti et al., 2011	N/A
CENP-HIKM	Musacchio Lab, Basilico et al., 2014	N/A
CENP-LN	Musacchio Lab, Pentakota et al., 2017	N/A
CENP-CHIKM	Musacchio Lab; Klare et al. 2015	N/A
NDC80 complex	Musacchio Lab; Huis In 't Veld et al., 2016	N/A
MIS12 complex	Musacchio Lab; Petrovic et al., 2014	N/A
KNL1 complex	Musacchio Lab; Petrovic et al., 2014	N/A
CENP-A NCP	Musacchio Lab; Weir et al., 2016	N/A
Tubulin (in TIRF experiment) purified from pig brains	N/A	N/A
poly-L-lysine-poly-ethylene-glycol-biotin	SUSOS-AG	PLL (20) -g[3.5]- PEG(2)/PEGbi)
streptavidin	Sigma	S4762; CAS#: 9013-20-1
GMP-CPP	Jena Biosciences	GpCpp, NU-405S CAS#: 14997-54-7
Porcine tubulin (biotin labelled)	Cytoskeleton	Cat. # T333P-A
HiLyte 647 labeled porcine tubulin	Cytoskeleton	Cat. # TL670M-A
Paclitaxel (Taxol)	Sigma	T7402; CAS#: 33069-62-4
Glucose oxidase	Sigma	G7141; CAS#:9001-37-1
Catalase	Sigma	SRE0041 CAS#:9001-05-2
DAPI	Sigma	D9542
PenStrep	Gibco	15-140
Fugene6	Promega	E2691
Oligofectamine	Invitrogen	12252011
Critical Commercial Assays
QIAquick Kit	Qiagen	28704
Mini-prep kit	Qiagen	27104
Maxi-prep kit	Qiagen	10023
Experimental Models: Cell Lines
Trichoplusia ni:BTI-Tnao38	Garry W Blissard Lab	N/A
S.frugiperda:Sf9 cells	ThermoFisher	Cat#12659017
HeLa cells	IEO Milan	N/A
Human: Flp-IN T-Rex HeLa	S.S. Taylor, University of Manchester	N/A
Human: Flp-In T-Rex HeLa-GFP-CENP-O	This paper	N/A
Human: Flp-In T-Rex HeLa-GFP-CENP-P	This paper	N/A
Human: Flp-In T-Rex HeLa-GFP-CENP-P(F116G)	This paper	N/A
Human: Flp-In T-Rex HeLa-GFP-CENP-Q	This paper	N/A
Human: HeLa K cells	Meraldi Lab	N/A
Human: HeLa K-CENP-Q-eGFP	This paper	N/A
Human: HeLa K-CENP-Q(Ndc80(1-80))-eGFP	This paper	N/A
Human: Flp-In T-Rex HeLa-GFP-CENP-U	This paper	N/A
Human: Flp-In T-Rex HeLa-GFP-CENP-R	This paper	N/A
Experimental Models: Organisms/Strains
E.coli: BL21(DE3)-RIL strain	Agilent Technologies	#230240
E.coli: BL21CodonPlus(DE3)-RIL strain	Agilent Technologies	#230280
Oligonucleotides
CENP-O siRNA: 5’-UAGGAGACCAGACUCAUAU-3’	Dharmacon
CENP-P SMARTpool: 5’-GUGCAAGAGAGAACAACUA-3’ 5’-AGUCAUUGUUUGGAGGAUA-3’ 5’-UAUCGUAAGCGCACGUUUA-3’ 5-CCUAAGUGCUAUAUCGAUC-3’	Dharmacon
CENP-P siRNA 5’-GAACCCTGGTAGGACTGCTTGGAAT-3’	Invitrogen Stealth; Amaro et al., 2010
CENP-Q SMARTpool: 5’-GAGUUAAUGACUGGGAAUA-3’, 5’-AUGGAAAGGGCACGAGACA-3’ 5’-ACAAAGCACACUAACCUAA-3’ 5’-UGUCAGAGAAAUAAGGUUAG-3’	Dharmacon
CENP-Q siRNA: 5‘-GGUCUGGCAUUACUACAGGAAGAAA-3‘	Invitrogen Stealth; Bancroft et al., 2015
CENP-R SMARTpool: 5’-GAAGUUGGAUGGUCUGUUA-3’ 5’-UGACAGCUAUGAAUUCCUU-3’ 5’-UAAGUAGUAUACAGGCUUU-3’ 5’-GAAUUCAUGAUGUUGCUAU-3’	Dharmacon
CENP-H siRNA: 5’-CUAGUGUGCUCAUGGAUAA-3’	Dharmacon
CENP-L siRNA: 5’-UUUAUCAGCCACAAGAUUA-3’	Dharmacon
CENP-N SMARTpool: 5’-CUACCUACGUGGUGUUACUA-3’ 5’-GUUCAGCACUUGAUCCAUC-3’ 5’-AUACACCGCUUCUGGGUCA-3’ 5’-ACACAAAGCCAAACCAGUA-3’	Dharmacon
Recombinant DNA
pGEX-2rbs	Musacchio Lab	N/A
pGEX-2rbs-CENP-R	This study	N/A
pGEX-2rbs-CENP-C(1-544)	Screpanti et al., 2011	N/A
pGEX-2rbs-CENP-C(1-544)-SNAP	This study	N/A
MultiBac	Geneva Biotech	N/A
pFL-6His-CENP-P:CENP-O	This study	N/A
pFL-6His-CENP-P(F116G):CENP-O	This study	N/A
pFL-6His-CENP-Q:CENP-U	This study	N/A
pUCDM-CENP-R	This study	N/A
pLIB	Peters Lab.	Addgene #80610
pBIG1A	Peters Lab.	Addgene #80611
pLIB CENP-O	This study	N/A
pLIB CENP-P	This study	N/A
pLIB CENP-Q	This study	N/A
pLIB CENP-Q(68-C)	This study	N/A
pLIB CENP-U	This study	N/A
pLIB CENP-R	This study	N/A
pBIG1A with CENP-OPQUR (His-CENP-Q)	This study	N/A
pBIG1A with CENP-OPQ(68-C)UR (His-CENP-Q)	This study	N/A
pCDNA 5/FRT/TO	Invitrogen	Cat#V6520-20
pCDNA 5/FRT/TO EGFP	Musacchio Lab; Krenn et al., 2012	N/A
pCDNA 5/FRT/TO EGFP-CENP-O	This study	N/A
pCDNA 5/FRT/TO EGFP-CENP-P	This study	N/A
pCDNA 5/FRT/TO EGFP-CENP-P(F116G)	This study	N/A
pCDNA 5/FRT/TO EGFP-CENP-Q	This study	N/A
pCDNA 5/FRT/TO EGFP-CENP-U	This study	N/A
pCDNA 5/FRT/TO EGFP-CENP-R	This study	N/A
peGFP-C1	Clontech	#6085-1
pCENP-Q-eGFP	McAinsh Lab; Bancroft et al., 2015	N/A
pCENP-Q(NDC80(1-80))-eGFP	This study	N/A
Software and Algorithms
GraphPad Prism 6.0	GraphPad software	http://www.graphpad.com
Imaris 7.3.4 32-bit	Bitplane	http://www.bitplane.com/imaris
ImageJ 1.46 r	NIH	http://imageJ.nih.gov/ij/
SoftWorx	Applied Precision	NA
Image Lab	Bio-rad	https://www.bio-rad.com/de-de/product/image-lab-software?ID=KRE6P5E8Z
UCSF Chimera	Pettersen et al., 2004	http://www.cgl.ucsf.edu/chimera
SPHIRE suit	Moriya et al., 2017	http://www.sphire.mpg.de
SEDFIT	Schuck, 2000	http://www.analyticalultracentrifugation.com/default.htm
SEDNTERP	Laue et al., 1992	http://bitcwiki.sr.unh.edu/index.php/Main_Page
GUSSI	Chad Brautigam	http://biophysics.swmed.edu/MBR/software.html
PrDOS	Ishida and Kinoshita, 2007	http://prdos.hgc.jp/cgi-bin/top.cgi
COILS	Lupas et al., 1991	https://embnet.vital-it.ch/software/COILS_form.html
Deposited Data
Mendeley data	This paper	https://doi.org/10.17632/yv552m8s98.1