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.