5E7W
X-ray Structure of Human Recombinant 2Zn insulin at 0.92 Angstrom
Experimental procedure
Experimental method | SINGLE WAVELENGTH |
Source type | SYNCHROTRON |
Source details | DIAMOND BEAMLINE I02 |
Synchrotron site | Diamond |
Beamline | I02 |
Temperature [K] | 100 |
Detector technology | PIXEL |
Collection date | 2012-09-27 |
Detector | DECTRIS PILATUS 6M |
Wavelength(s) | 0.77 |
Spacegroup name | H 3 |
Unit cell lengths | 81.820, 81.820, 33.850 |
Unit cell angles | 90.00, 90.00, 120.00 |
Refinement procedure
Resolution | 10.000 - 0.952 |
Rwork | 0.111 |
R-free | 0.14440 |
Structure solution method | MOLECULAR REPLACEMENT |
Starting model (for MR) | 3w7y |
Data reduction software | XDS (Fast dp Xia 2) |
Data scaling software | Aimless (Fast dp Xia 2) |
Phasing software | PHASER |
Refinement software | SHELXL (2014/7) |
Data quality characteristics
Overall | Outer shell | |
Low resolution limit [Å] | 40.910 | 0.940 |
High resolution limit [Å] | 0.920 | 0.920 |
Rmerge | 0.034 | 0.967 |
Number of reflections | 58647 | |
<I/σ(I)> | 20.2 | 1.6 |
Completeness [%] | 100.0 | 100 |
Redundancy | 5 | 4.7 |
Crystallization Conditions
crystal ID | method | pH | temperature | details |
1 | BATCH MODE | 6.3 | 293 | The crystals were prepared by a batch method similar to that of Baker et al, 1988 [1], modified as follows: 0.01g of insulin as a fine powder was placed in a clean test tube; 0.02M HCl was added to dissolve the protein; on addition of 0.15 mL of 0.15 M zinc acetate the solution became cloudy due to precipitation of the protein; 0.3 mL of acetone and then 0.5 mL of trisodium citrate together with 0.8 mL of water were added and the solution went clear; the pH was checked and increased with NaOH to a pH between 8 and 9 for different batches, thus ensuring complete dissolution. It was then adjusted to the required value of pH 6.3. If any slight turbidity occurred, it was removed by warming the solution. The solution was then filtered using a Millipore membrane/acetate cellulose acetate filter. This removes any nuclei which will encourage precipitation or formation of masses of small crystals. The solution was then warmed to 50 deg C by surrounding the test tube with preheated water in a Dewar. This allowed the solution to cool slowly to room temperature. The test tube was lightly sealed with cling film; crystals formed within a few days and were of suitable size for X-ray diffraction within two weeks; the test tube containing crystals was kept at 4 degC prior to data collection. The crystal used for data collection was about 0.2 mm3. |