3ZIA

The structure of F1-ATPase from Saccharomyces cerevisiae inhibited by its regulatory protein IF1

Experimental procedure

Experimental method	SINGLE WAVELENGTH
Source type	SYNCHROTRON
Source details	DIAMOND BEAMLINE I24
Synchrotron site	Diamond
Beamline	I24
Temperature [K]	100
Detector technology	CCD
Collection date	2009-10-15
Detector	MARRESEARCH
Spacegroup name	P 1 21 1
Unit cell lengths	118.224, 187.846, 181.812
Unit cell angles	90.00, 89.96, 90.00

Refinement procedure

Resolution	181.810 - 2.500
R-factor	0.22618
Rwork	0.224
R-free	0.26194
Structure solution method	MOLECULAR REPLACEMENT
Starting model (for MR)	2hld
RMSD bond length	0.009
RMSD bond angle	1.175
Data reduction software	iMOSFLM
Data scaling software	SCALA
Phasing software	PHASER
Refinement software	REFMAC (5.5.0110)

Data quality characteristics

	Overall	Outer shell
Low resolution limit [Å]	43.840	2.640
High resolution limit [Å]	2.500	2.500
Rmerge	0.110	0.750
Number of reflections	268620
<I/σ(I)>	8.7	2
Completeness [%]	98.4	97.6
Redundancy	3.9	3.9

Crystallization Conditions

crystal ID	method	pH	temperature	details
1	MICROBATCH	7.5	296	ACTIVE F1-ATPASE (12 MG/ML) WAS EXCHANGED ON A DESALTING COLUMN INTO CRYSTALLISATION BUFFER, PREPARED IN D2O CONSISTING OF 100 MM BIS-TRIS PROPANE, PH 7.5, 100 MM SUCROSE, 1 MM ADP AND 10 MM MAGNESIUM SULPHATE. THEN THE ENZYME WAS INHIBITED AT 23C WITH A 4-FOLD MOLAR EXCESS OF YI1-53 (MUTATION E21A) IN THE PRESENCE OF 1 MM ATP AND 2 MM MAGNESIUM SULPHATE. FURTHER PORTIONS (5 UL OF A NEUTRALISED STOCK SOLUTION CONTAINING 200 MM ATP AND 400 MM MAGNESIUM SULPHATE/ML PROTEIN SOLUTION) WERE ADDED AFTER 5 AND 10 MIN. MORE THAN 95% OF THE ATP HYDROLYSIS ACTIVITY OF THE ENZYME WAS INHIBITED. SODIUM-POTASSIUM TARTRATE WAS ADDED TO 100 MM, AND THE CONCENTRATION OF THE PROTEIN SOLUTION WAS ADJUSTED TO 10 MG/ML WITH CRYSTALLISATION BUFFER. CRYSTALS WERE GROWN AT 23C IN 72 WELL MICRO-BATCH PLATES UNDER FILTERED PARAFFIN OIL. THE CRYSTALLISATION DROPS (4 UL) CONTAINED A 1:1 MIXTURE OF PROTEIN SOLUTION AND PRECIPITANT SOLUTION (20%-26% POLYETHYLENE GLYCOL 3000 AND 600 MM NACL PREPARED IN D2O).