REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
α Tubulin Antibody (B-7) Alexa Fluor®647	Santa Cruz Biotechnology	sc-5286 AF647
gamma Tubulin Polyclonal Antibody, Cyanine3	Thermo Fisher	PA1–28043
Bacterial and Virus Strains
E. coli Rosetta (DE3)	Millipore	70954
Bac-to-Bac Baculovirus Expression System	Thermo Fischer Scientific	10359016
Chemicals, Peptides, and Recombinant Proteins
TEV Protease	This Paper	N/A
HyCloneTM CCM3 SFM	GE Life Sciences	SH30065.02
Thrombin Protease	GE Life Sciences	27084601
PreScission Protease	This Paper	N/A
isopropylthiogalactoside	Sigma Aldrich	I675
TWEEN® 20	Sigma Aldrich	P1379
IGEPAL® CA-630	Sigma Aldrich	I8896
ATP	Jena Bioscience	NU-1010
ADP	Sigma Aldrich	A2754
ATPyS	Jena Bioscience	NU-406
AMPPNP	Jena Bioscience	NU-407
GMPCPP	Jena Bioscience	NU-405
GTP	Sigma Aldrich	G8877
Beryllium Sulfate	Sigma Aldrich	202789
Sodium Fluoride	Sigma Aldrich	201154
Pierce™ Bond-Breaker™ TCEP Solution	Thermo Fischer Scientific	PI-77720
Tubulin	PurSolutions	32005
Tubulin	Cytoskeleton	T240
Tubulin X-Rhodamine Labeled	Cytoskeleton	TL620M
Tubulin HiLyte647 Labeled	Cytoskeleton	TL670M
Biotin-PEG-SVA	Laysan Bio	#Biotin-PEG-SVA-5000
mPEG-Succinimidyl Valerate	Laysan Bio	#NH2-PEG-VA-5K
1,4-piperazinediethanesulfonic acid	Sigma Aldrich	P6757
Ethylene glycol tetraacetic acid	Sigma Aldrich	3777
Methyl cellulose	Sigma Aldrich	M0512
Paclitaxel	Sigma Aldrich	T7402
2-Mercaptoethanol	Sigma Aldrich	M-6250
Neutravidin	Thermo Fischer Scientific	A2666
GelCodeTM Blue Stain	Thermo Fischer Scientific	PI24592
SAG	Fischer Scientific	56–666
ProLong™ Diamond Antifade Mountant	Thermo Fischer Scientific	P36961
[a-32P] ATP	PerkinElmer	BLU003H250UC
Cellulose PEI TLC	Millipore	105579
1.2/1.3 400-mesh grids	Electron Microscopy Services	CF413
Catalase	Sigma Aldrich	C40
Glucose Oxidase	Sigma Aldrich	G2133
6His-TEV-Kif7DM1–543-GFP	This paper	N/A
6His-TEV-Kif7DM1–543	This paper	N/A
6His-TEV-Kif7DM-L124K-GFP	This paper	N/A
6His-TEV-Kif7DM-L123A-GFP	This paper	N/A
6His-TEV-Kif7DM-L8KHC-GFP	This paper	N/A
6His-TEV-Kif7DM-L10KHC-GFP	This paper	N/A
6His-TEV-Kif7MM1–386	This paper	N/A
6His-TEV-Kif7MM1–386-GFP	This paper	N/A
6His-TEV-Kif7MM-L10	This paper	N/A
Strep-PreScission-Clasp1(654–1455)-GFP-TEV-6His	This paper	N/A
6His-Thrombin-EB1-GFP	Laboratory of Mike Blower	N/A
Critical Commercial Assays
Q5® Site-Directed Mutagenesis Kit	NEB	E0554S
EnzChek™ Phosphate Assay Kit	Invitrogen	E6646
Anti-Penta-HIS biosensor Chip	ForteBio	His1K
Amine Reactive Second-Generation Biosensors	ForteBio	AR2G
Deposited Data
ADP-bound Kif7 atomic model	Protein Data Bank Archive	4A14
Taxol stabilized Sus scrofa tubulin atomic model	Protein Data Bank Archive	3J6G
Taxol stabilized Sus scrofa tubulin electron density	Electron Microscopy Data Bank	EMD-5897
GmpCpp stabilized Sus scrofa tubulin electron density	Electron Microscopy Data Bank	EMD-5895
ATP analog-bound kinesin-1 H. Sapiens on tubulin atomic model	Protein Data Bank Archive	3J8Y
ATP analog-bound kinesin-1 H. Sapiens on tubulin electron density	Electron Microscopy Data Bank	EMD-6188
ADP-Mg-AlFx-bound kinesin motor domain in complex with tubulin atomic model	Protein Data Bank Archive	4HNA
HsMCAK motor domain complex atomic model	Protein Data Bank Archive	4UBF
Hs Kinesin-1 motor domain atomic model	Protein Data Bank Archive	1BG2
ADP-bound kinesin-3 on microtubules atomic model	Protein Data Bank Archive	4UXS
ADP-bound kinesin-3 on microtubules electron density	Electron Microscopy Data Bank	EMD-2768
AMPPNP-bound kinesin-3 on microtubules atomic model	Protein Data Bank Archive	4UXP
AMPPNP-bound kinesin-3 on microtubules electron density	Electron Microscopy Data Bank	EMD-2766
AMPPNP-bound Hs Kif7 motor domain on GDP-taxol microtubules atomic model	This work	6MLR
AMPPNP-bound Hs Kif7 motor domain on GDP-taxol microtubules electron density	This work	EMD-9141
ADP-bound Hs Kif7 motor domain on GDP-taxol microtubules atomic model	This work	6MLQ
ADP-bound Hs Kif7 motor domain on GDP-taxol microtubules electron density	This work	EMD-9140
Experimental Models: Cell Lines
C3H10T1/2	Lab of Stephane Angers
Recombinant DNA
pI-SUMOstar-6His-TEV-Kif7DM1–543-GFP	This paper	RS177 (Subramanian Lab Collection)
pI-SUMOstar-6His-TEV-Kif7DM1–543	This paper	RS168 (Subramanian Lab Collection)
pI-SUMOstar-6His-TEV-Kif7DM-L124K-GFP	This paper	SJ034 (Subramanian Lab Collection)
pI-SUMOstar-6His-TEV-Kif7DM-L123A-GFP	This paper	SJ035 (Subramanian Lab Collection)
pI-SUMOstar-6His-TEV-Kif7DM-L8KHC-GFP	This paper	SJ045 (Subramanian Lab Collection)
pI-SUMOstar-6His-TEV-Kif7DM-L10KHC-GFP	This paper	SJ042 (Subramanian Lab Collection)
pET28a-Kif7MM1–386	This paper	SJ029 (Subramanian Lab Collection)
pET30a-Kif7MM1–386-GFP	This paper	SJ051 (Subramanian Lab Collection)
pET28a-Kif7MM-L10	This paper	SJ055 (Subramanian Lab Collection)
pFastBac-Strep-PreScission-Clasp1(654–1455)-GFP-TEV-6His	This paper	NM503 (Subramanian Lab Collection)
pET30a-EB1-GFP	Laboratory of Mike Blower	pMB283 (Blower Lab Collection)
CMV-Kif7-GFP	This paper	pk13 (Subramanian Lab Collection)
CMV-Kif7–3A-GFP	This paper	pk28 (Subramanian Lab Collection)
CMV-Kif7–4K-GFP	This paper	pk13 (Subramanian Lab Collection)
CMV-Kif7-L10-GFP	This paper	pk13 (Subramanian Lab Collection)
Software and Algorithms
Matlab	Mathworks	https://www.mathworks.com
ImageJ	NIH	https://imagej.nih.gov/ij/
NIS Elements	Nikon	https://www.nikoninstruments.com/Products/Software
Octet Analysis	ForteBio	http://www.fortebio.com/octet-platform.html
UCSF Chimera	RBVI	https://www.cgl.ucsf.edu/chimera/
Appion	Lander et al., 2009	http://emg.nysbc.org/redmine/ projects/appion/wiki/Appion_Home
Leginon	Suloway et al., 2005	http://emg.nysbc.org/redmine/ projects/leginon/wiki/Leginon_Homepage
MotionCor	Li et al., 2013, Hirschi et al., 2017	http://msg.ucsf.edu/em/software/motioncor2.html
CTFFIND4	Rohou and Grigorieff, 2015	http://grigoriefflab.janelia.org/ctffind4
EMAN2	Tang et al., 2007	https://blake.bcm.edu/emanwiki/EMAN2
FREALIGN	Grigorieff, 2007
Image Studio Lite	LI-COR	https://www.licor.com/bio/products/ software/image_studio_lite/
Octet Data Analysis	ForteBio	https://www.fortebio.com/octet-software.html
phenix.real_space_refine	Afonine et al., 2013	https://www.phenix-online.org/documentation/reference/ real_space_refine.html
Prism	GraphPad	https://www.graphpad.com/scientific-software/prism/
Coot	Emsley and Cowtan, 2004	https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
Contact	Winn et al., 2011	http://www.ccp4.ac.uk/html/contact.html
PDBSUM	de Beer et al., 2014	www.ebi.ac.uk/pdbsum
APBS	Jurrus et al., 2018	http://nbcr-222.ucsd.edu/pdb2pqr_2.1.1/
Clustal Omega	Sievers et al., 2011	https://www.ebi.ac.uk/Tools/msa/clustalo/
Curvature Analysis Algorithm	Bechstedt et al., 2014	Lab of Gary Brouhard