REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit polyclonal anti-GFP	Generated in-house	N/A
Mouse monoclonal anti-MIS12	Generated in-house	#QA21-74-4-3
Rabbit polyclonal anti-CENP-C	Trazzi et al., 2009	#SI410
Mouse monoclonal anti-Vinculin	Sigma-Aldrich	Cat#V9131; RRID: AB_477629
Rabbit polyclonal anti-KNL1-N	Generated in-house	#SI0787
Mouse monoclonal anti-HEC1	GeneTex	Cat#GTX70268; RRID: AB_371632
Rabbit polyclonal anti-CENP-T/W	Generated in-house	#SI0882
Mouse monoclonal anti-DSN1	Generated in-house	#RC11-3(40)
Sheep anti-mouse HRP	Amersham	Cat#NXA931-1ML; RRID: AB_772209
Donkey anti-rabbit HRP	Amersham	Cat#NXA934-1ML
Human anti-centromere (CREST)	Antibodies Inc.	Cat#15-234-0001
Goat anti-human Alex Fluor 647	Invitrogen	Cat#A-21445; RRID: AB_2535862
Chemicals, Peptides, and Recombinant Proteins
GST-Prescission (3C protease)	Musacchio Lab	N/A
Endoproteinase Asp-N	Sigma Aldrich	Cat#000000011420488001
Lysyl Endopeptidase	Wako	Cat#125-05061
Ndc80C	Petrovic et al., 2014	N/A
KNL1C	Petrovic et al., 2014	N/A
Zwint1	Musacchio Lab	N/A
Protease-inhibitor mix HP Plus	Serva	Cat#39107
PhosSTOP phosphatase inhibitors	Roche	Cat#04906845001
DNaseI	Roche	Cat#13146700
FAM-CENP-C 1-21	GenScript	N/A
FAM-GGGK	GenScript	N/A
Sortase A (S.pyogenes)	Hidde Ploegh Lab	Addgene:Cat#51139
Sortase A delta 59 (S.aureus)	Hidde Ploegh Lab	Addgene:Cat#51138
Zeocin	Invitrogen	Cat#R25001
Doxycycline	Sigma	Cat#D9891;
		CAS: 24390-14-5
Nocodazole	Sigma	Cat#M1404;
		CAS: 31430-18-9
L-glutamine	PAN Biotech	P04-80100
DAPI	Serva	Cat#18860.01
Poly-L-Lysine	Sigma Aldrich	Cat#P4832;
		CAS: 25988-63-0
Bissulfosuccinimidylsuberate (BS3)	Creative Molecules	Cat#001SS
Urea	Sigma-Aldrich	Cat#U6504;
		CAS: 57-13-6
4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)	Sigma Aldrich	Cat#74104;
		CAS: 3945-69-5
Acetonitrile	Fluka	Cat#34967;
		CAS: 75-05-8
Trypsin	Promega	Cat#V511
Ammonium Bicarbonate (Ambic)	Fluka	Cat#C990X98;
		CAS: 1066-33-7
Trifluoroacetic acid (TFA)	Sigma Aldrich	Lot#RB228879;
		CAS: 76-05-1
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl)	Pierce	Cat#20490; CAS: 51805-45-9
Iodoacetamide	Sigma Aldrich	Cat#I6125;
		CAS: 144-48-9
Critical Commercial Assays
JBS Floppy-Choppy	Jena Bioscience	Cat#CO-110
JBS Tantalum Cluster Derivitization Kit	Jena Bioscience	Cat#PK-103
JSCG Core Suites	QIAGEN	Cat#130724;130725;130726;30727
Deposited Data
MIS12/CENP-C full-length structure	This study	PDB:5LSK
MIS12Δhead2 structure	This study	PDB:5LSJ
Head2 structure	This study	PDB:5LSI
Experimental Models: Cell Lines
Trichoplusia ni:BTI-Tnao38	Garry W Blissard Lab	N/A
S.frugiperda:Sf9 cells in Sf900™ III SFM	ThermoFisher	Cat#:12659017
Human: Flp-In T-Rex HeLa	S.S. Taylor, University of Manchester	N/A
Human: Flp-In T-Rex HeLa-MIS12	This paper	N/A
Human: Flp-In T-Rex HeLa-MIS12 YFF	This paper	N/A
Human: Flp-In T-Rex HeLa-MIS12 4D/EA	This paper	N/A
Experimental Models: Organisms/Strains
E.coli:BL21-CodonPlus(DE3)-RIL strain	Agilent Technologies	#230240
E.coli:One Shot OmniMAX 2 T1R Chemically Competent Cells	ThermoFisher	#C854003
E.coli:BL21-CodonPlus(DE3)-RIPL strain	Agilent Technologies	#230280
Recombinant DNA
pGEX-2rbs	Musacchio Lab
MultiBac	Geneva Biotech	N/A
pST39	Tan et al., 2005	N/A
pST39-MIS12C	This study	N/A
pST39-MIS12C YFF	This study	N/A
pST39-MIS12C MIS12-2D/EA	This study	N/A
pST39-MIS12C MIS12-2E/DA	This study	N/A
pST39-MIS12C MIS12-4D/EA	This study	N/A
pST39-MIS12C NSL1-DED/AAA	This study	N/A
pST39-MIS12C Δ100-109	This study	N/A
pST39-MIS12C DSN1-RRAA	This study	N/A
pST39-MIS12C DSN1-RRKAAA	This study	N/A
pST39-MIS12C Δhead1	This study	N/A
pST39-MIS12C Δhead2	This study	N/A
pGEX-2rbs-Head1	This study	N/A
pGEX-2rbs-Head2	This study	N/A
pGEX-CENP-C 1-71	Screpanti et al., 2011	N/A
pCDNA 5/FRT/TO plasmid	Invitrogen	Cat#V6520-20
pcDNA 5/FRT/TO EGFP	Krenn et al., 2012	N/A
pcDNA 5/FRT/TO EGFP-MIS12	This study	N/A
pcDNA 5/FRT/TO EGFP-MIS12 YFF	This study	N/A
pcDNA 5/FRT/TO EGFP-MIS124D/EA	This study	N/A
pFL-MIS12:PMF1	This study	N/A
pUCDM-NSL1:DSN1	This study	N/A
Software and Algorithms
Origin	OriginLab	www.originlab.com
ImageJ 1.46 r	NIH	https://imagej.nih.gov/ij/
Imaris 7.3.4 32-bit	Bitplane	http://www.bitplane.com/imaris
GraphPad Prism 6.0	GraphPad software	http://www.graphpad.com
Coot	Emsley et al., 2010	http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
UCSF Chimera	Pettersen et al., 2004	https://www.cgl.ucsf.edu/chimera
Phenix	Adams et al., 2010	https://www.phenix-online.org
XDS	Kabsch, 2010	http://xds.mpimf-heidelberg.mpg.de/
PyMol	The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC	https://www.pymol.org/
UCLA Diffraction Anisotropy Server		http://services.mbi.ucla.edu/anisoscale
CCP4 package	Winn et al., 2011	www.ccp4.ac.uk
xQuest/XProphet	Walzthoeni et al., 2012	http://proteomics.ethz.ch/cgi-bin/xquest2_cgi/index.cgi
SHARP	Bricogne et al., 2003	https://www.globalphasing.com
Other
Sep-Pak C18 Vac Cartridge, 50 mg Sorbent	Waters	Cat#WAT054955
Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL	Thermo Scientific	Cat#89883
Amicon concentrators (3K/10K/30K)	Millipore	Cat#UFC900324;UFC901024;UFC903024
Nitrocellulose membrane	GE Healthcare	Cat#10600001
Mowiol mounting media	Calbiochem	Cat#475904
4-12% NuPAGE Bis-Tris gels	Life Technologies	Cat#NP0321BOX
GST-Trap FF (5 ml)	GE Healthcare	Cat#17-5130-01
His Trap FF (5ml)	GE Healthcare	Cat#17-5255-01
Superdex 75 (10/300) GL	GE Healthcare	Cat#17-5174-01
Superdex 75 (16/600) pg	GE Healthcare	Cat#28-9893-33
Superdex 200 (10/300) GL	GE Healthcare	Cat#17-5175-01
Superdex 200 Increase 5/150 GL	GE Healthcare	Cat#28-9909-45
Superdex Peptide PC 3.2/300	GE Healthcare	Cat#29-0362-31
Resource Q (6 ml)	GE Healthcare	Cat#17-1179-01
Resource S (6 ml)	GE Healthcare	Cat#17-1180-01
Glutathione Affinity Resin	Expedeon	Cat#AGS0010
Protein G-agarose beads	Amintra	Cat#APG0005
Protein A-agarose beads	Roth	Cat#1278.1
GFP-Trap_A	ChromoTek	Cat#gta-20
Corning 384 Well Low Volume Black Round Bottom Polystyrene NBS Microplate	Corning	Cat#4514
ECL Prime western blotting system	GE Healthcare	Cat#RPN 2232