3OWV | pdb_00003owv

Structural insights into catalytic and substrate binding mechanisms of the strategic EndA nuclease from Streptococcus pneumoniae

X-RAY DIFFRACTION

Starting Model(s)

Initial Refinement Model(s)
Type	Source	Accession Code	Details
experimental model	Other		incomplete manually built model of EndA from a low resolution incompletely refined data set from an MBP-EndA fusion protein

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION, SITTING DROP	7	297	Crystallization solution contained 0.1M Tris pH 7, 17% PEG8000, and 0.2M MgCl2. The crystal was transferred to a hanging drop containing 0.1M Tris pH 7, 75mM MaCl, 0.2M MgCl2, and 20% PEG800, which was then placed over a vapor diffusion chamber containing a sitting drop bridge with 25% glutaraldehyde. The crystal was cross-linked for 8 minutes at room temperature, then transferred to a soaking solution containing 0.1M Tris pH 7, 75mM NaCl, 0.2M MgCl2,20% PEG8000, 11.6mM trimethyl lead acetate, and 9.63mM triethyl lead acetate for 43 hrs at room temperature. The soaked crystal was then transferred to a cryoprotectant (0.1M Tris pH 7, 75mM NaCl, 0.2M MgCl2, 20% PEG8000, 15% ethylene glycol, 11.9mM trimethyl lead acetate, and 13.75mM triethyl lead acetate) in four steps, then flash frozen in liquid nitrogen. , VAPOR DIFFUSION, SITTING DROP, temperature 297K

Crystal Properties
Matthews coefficient	Solvent content
2.19	43.72

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 67.963	α = 90
b = 75.931	β = 90
c = 90.784	γ = 90

Symmetry
Space Group	P 21 21 21

Diffraction

Diffraction Experiment
ID #	Crystal ID	Scattering Type	Data Collection Temperature	Detector	Detector Type	Details	Collection Date	Monochromator	Protocol
1	1	x-ray	93	CCD	MARMOSAIC 225 mm CCD		2009-12-10	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	APS BEAMLINE 22-ID	0.94	APS	22-ID

Data Collection

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	R Sym I (Observed)	Net I Over Average Sigma (I)	Redundancy	Number Reflections (All)	Number Reflections (Observed)	Observed Criterion Sigma (F)	Observed Criterion Sigma (I)	B (Isotropic) From Wilson Plot
1	1.75	50	96.8	0.097	26.5	13.6	46402	46402	-3	-3	17.8

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R-Sym I (Observed)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
	1.75	1.78	68.7		0.371	3.56	7.2

Refinement

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	Starting model	Resolution (High)	Resolution (Low)	Number Reflections (All)	Number Reflections (Observed)	Number Reflections (R-Free)	Percent Reflections (Observed)	R-Factor (Observed)	R-Work (Depositor)	R-Work (DCC)	R-Free (Depositor)	R-Free (DCC)	R-Free Selection Details	Mean Isotropic B
X-RAY DIFFRACTION	MOLECULAR REPLACEMENT	THROUGHOUT	incomplete manually built model of EndA from a low resolution incompletely refined data set from an MBP-EndA fusion protein	1.75	37.97	45460	45460	1962	94.7	0.195	0.195	0.2	0.217	0.2	RANDOM	23.3

Temperature Factor Modeling
Anisotropic B[1][1]	Anisotropic B[1][2]	Anisotropic B[1][3]	Anisotropic B[2][2]	Anisotropic B[2][3]	Anisotropic B[3][3]
-5.55			-2.71		8.27

RMS Deviations
Key	Refinement Restraint Deviation
c_dihedral_angle_d	25
c_scangle_it	4.68
c_scbond_it	3.83
c_mcangle_it	1.83
c_mcbond_it	1.21
c_angle_deg	1.2
c_improper_angle_d	0.68
c_bond_d	0.004
c_bond_d_na
c_bond_d_prot

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	3337
Nucleic Acid Atoms
Solvent Atoms	495
Heterogen Atoms	7

Software

Software
Software Name	Purpose
MAR345	data collection
AutoBuild	model building
CNS	refinement
HKL-2000	data reduction
HKL-2000	data scaling
AutoBuild	phasing

RCSB PDB Core Operations are funded by the U.S. National Science Foundation (DBI-2321666), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM157729.