TABLE 1.

Summary of data collection, phase calculation, model building and refinement

	Native	I-ATP	EMTS
Cell dimensions:
a (Å)	48.54	48.66	48.68
b (Å)	67.94	67.88	67.73
c (Å)	112.95	113.37	112.76
Data collection:
Resolution (Å)	1.8	2.5*	2.5
Rsym (%)†	6.1	17.2	14.1
Observed reflections	101,090	40,935	96,338
Unique reflections	30,582	11,951	12,726
Completeness (%)	82.4	87.4	91.1
Phasing (15.0–3.0 Å):
Rscal‡	–	14.2	21.2
Number of sites	–	1	3
Relative occupancy	–	–	1.0
			0.63
			0.40
Phasing power§		0.84	1.95
RCullis||		0.76	0.59
Mean figure of merit	0.48
Refinement (6.0–1.8Å):
Rcryst (%)¶	23.4
Rfree (%)#	30.7
Average B-factor (Å2)	26.1

The first 349 amino acids of the human kinesin gene²⁵ containing the conserved motor domain were expressed in bacteria and purified as described²⁶. Crystals were obtained using sitting drops containing 20 μl of ~5 mgml⁻¹ protein in 50 mM Na Acetate, pH 4.6, 75 mM KCl, 3.5% (w/v) PEG 4000, 2.5 mM ATP, 10 mM MgCl₂. Crystals (1,000 × 150 × 40 μm) appeared after 2–3 days at 4 °C, and were of the orthorhombic space group P2₁2₁2₁ with one molecule with bound MgADP in the asymmetric unit. All data collection was performed at −170 °C using a 30% (v/v) solution of glycerol/precipitant solution as a cryosolvent. The native data were collected at Stanford Synchrotron Laboratory Radiation beamline 7–1 (λ = 1.08 Å), and derivative data were collected using a 300-mA, 50-kV rotating anode X-ray source. The first derivative was obtained by co-crystallizing K349 with 2.5 mM 2′-iodo-ATP (I-ATP)²⁷ instead of ATP. The second derivative was obtained by soaking native crystals in the presence of 0.1 mM ethyl mercurothiosalycilate (EMTS) for ~2 h. The programs DENZO and SCALEPACK (Z. Otwinowski and W. Minor) were used to index and integrate all data sets. Heavy-atom sites were initially found by analysis of difference Patterson maps and refined using the program XTALVIEW²⁸. One I-ATP derivative site and three Hg sites (bound to Cys 174, Cys 168 and Cys 294) were found for each kinesin molecule. Heavy-atom positions were further refined, and multiple isomorphous replacement (MIR) phases were calculated using the program MLPHARE, and the initial 3.0-Å MIR map and density-modified map (solvent flattening/histogram matching using the program DM) were calculated using the CCP4 suite of programs²⁹. The K349 chain could be traced using these maps in combination with density skeletons created using the program 0 (T. A. Jones and M. Kjeldgaard). The quality of initial maps was better than expected from the phasing statistics, as indicated by visible carbonyl density in several helices. After a chain of ~160 alanines and 40 residues was placed, the chain was compared with the working model of the NCD structure. Comparison of the two independently derived chains confirmed that the chains were correctly placed and connected. An initial, four-fragment model of a ~275-amino-acid fragment of the kinesin motor was then refined in the program X-PLOR³⁰ using a least-squares protocol and, later, least-squares and simulated annealing protocols. Individual B-factors were refined isotropically. The structure satisfied the PROCHECK³¹ criteria at 1.8 Å. The final structure contains amino acids 7–325, MgADP and 30 water molecules.

^*I-ATP data were processed to 2.5 Å; diffraction was weak beyond 3.0 Å.

^†R_sym = 100 × Σ_hklΣ_i | I_i − I | /Σ_hklΣ_iI_i. All positive, non-zero reflections (O_σ intensity cut-off) were included in scaling and merging.

^‡R_scale = 100 × Σ_hkl | F_PH − F_H | /Σ_hkl | F_P |. All positive, non-zero reflections were included in this calculation.

^§Phasing power = 〈F_H〉 / 〈E〉, where 〈F_H〉 is the mean calculated heavy-atom structure factor amplitude, and 〈E〉 is the mean estimated lack of closure. All positive, non-zero reflections were included in this calculation.

^||R_cullis = 〈E〉 / 〈iso〉, where 〈E〉 is the mean estimated lack of closure and 〈iso〉 is the isomorphous difference.

^¶R_cryst = 100 × Σ_hkl | F_o − F_c | /Σ_hkl | F_o | , where F_o and F_c are observed and calculated structure factor amplitudes, respectively (for all data).

^#R_free was calculated using 10% of the data chosen randomly and omitted from the refinement.