9YPM

MboA with Leu-Ala-Arg peptide substrate and two Fe(II) ions bound

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	ALS BEAMLINE 8.3.1
Synchrotron site	ALS
Beamline	8.3.1
Temperature [K]	90
Detector technology	PIXEL
Collection date	2025-05-07
Detector	DECTRIS PILATUS3 S 6M
Wavelength(s)	1.11
Spacegroup name	P 43 21 2
Unit cell lengths	157.094, 157.094, 157.423
Unit cell angles	90.00, 90.00, 90.00

Refinement procedure

Resolution	90.760 - 2.450
R-factor	0.1642
Rwork	0.163
R-free	0.19510
Structure solution method	MOLECULAR REPLACEMENT
RMSD bond length	0.007
RMSD bond angle	0.540
Data reduction software	XDS (Jun 1, 2017)
Data scaling software	Aimless (0.8.2)
Phasing software	PHASER (2.8.3)
Refinement software	PHENIX (2.0_5761)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	90.760	2.500
High resolution limit [Å]	2.450	2.450
Rmerge	0.192	3.043
Rmeas	0.199	3.158
Rpim	0.053	0.842
Number of reflections	72777	4437
<I/σ(I)>	18.1	1.4
Completeness [%]	100.0	100
Redundancy	26.38	27.05
CC(1/2)	0.999	0.571

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	VAPOR DIFFUSION, HANGING DROP	4.6	298	MboA crystals were prepared via the hanging drop vapor diffusion method in an Coy anaerobic chamber with an atmosphere of < 5 ppm of O2. Hanging drops were prepared by combining equal volumes of MboA (9 mg/mL) protein solution, reservoir solution (0.6 M lithium sulfate and 0.1 M sodium acetate, pH 4.6), and 0.2 uL of a 1:1000 dilution microseed master stock generated from apo crystals for a total drop volume of 2.2 uL. The Fe(II) + substrate bound structure was prepared by incubating apo crystals with 0.4 uL of 100 mM LAR peptide and 100 mM Fe2+ in 0.1 M sodium acetate, pH 4.6 for two hours prior to looping. Crystals were cryoprotected with the addition of 0.75 uL of 40% glycerol in 0.1 M sodium acetate, pH 4.6 and flash frozen in LN2