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). |