Crystallization 1:
All crystallization experiments were performed using the hanging-drop
vapour diffusion method at ambient temperature. Initial
crystallization conditions for Xase were identified from the PEG/Ion
Screen (Hampton Research). Crystals suitable for X-ray diffraction
analysis (typical dimensions are 0.05 x 0.03 x 0.02 mm3; Fig. 1) were
grown from 1 μL drops containing equal volumes of protein
(1.2 mg mL-1
in 50 mM Tris pH 7.5, 100 mM sodium chloride) and reservoir solution
(25% PEG 3350, 100 mM Hepes pH 7.3, 200 mM diammonium citrate). Drops
were equilibrated against 0.5 mL reservoir solution in the wells.
Cryo-conditions for crystal 1:
Crystals were soaked in reservoir solution supplemented with 20%
glycerol for 1-2 minutes prior to flash cooling by direct immersion
into liquid nitrogen. The mounted crystal was subjected to two
annealing treatments consisting of interruption of the flow of the
cold stream with a plastic ruler for 10 seconds.
_exptl_crystal.crystal_id 1
_exptl_crystal.size_max 0.05
_exptl_crystal.size_mid 0.03
_exptl_crystal.size_min 0.02
_exptl_crystal_grow.crystal_id 1
_exptl_crystal_grow.method 'hanging-drop vapour diffusion'
_exptl_crystal_grow.temp_details 'ambient'
loop_
_pdbx_exptl_crystal_grow_sol.crystal_id
_pdbx_exptl_crystal_grow_sol.sol_id
_pdbx_exptl_crystal_grow_sol.volume
_pdbx_exptl_crystal_grow_sol.volume_units
_pdbx_exptl_crystal_grow_sol.pH
1 'protein' 0.5 'microlitre' 7.5
1 'precipitant' 0.5 'microlitre' 7.3
1 'reservoir' 0.5 'millilitre' 7.3
loop_
_pdbx_exptl_crystal_grow_comp.crystal_id
_pdbx_exptl_crystal_grow_comp.sol_id
_pdbx_exptl_crystal_grow_comp.comp_id
_pdbx_exptl_crystal_grow_comp.comp_name
_pdbx_exptl_crystal_grow_comp.conc
_pdbx_exptl_crystal_grow_comp.conc_units
1 'protein' 1 'protein' 1.2 'milligrams_per_millilitre'
1 'protein' 2 'Tris' 50. 'millimolar'
1 'protein' 3 'NaCl' 100. 'millimolar'
1 'reservoir' 1 'PEG 3350' 25. 'percent_weight_by_volume'
1 'reservoir' 2 'Hepes' 100. 'millimolar'
1 'reservoir' 3 'diammonium citrate' 200. 'millimolar'
1 'precipitant' 1 'PEG 3350' 25. 'percent_weight_by_volume'
1 'precipitant' 2 'Hepes' 100. 'millimolar'
1 'precipitant' 3 'diammonium citrate' 200. 'millimolar'
_pdbx_exptl_crystal_cryo_treatment.crystal_id 1
_pdbx_exptl_crystal_cryo_treatment.final_solution_details
; Crystals were soaked in reservoir solution supplemented with 20% glycerol
;
_pdbx_exptl_crystal_cryo_treatment.soak_details
; 1-2 min before flash-cooling
;
_pdbx_exptl_crystal_cryo_treatment.cooling_details
; direct immersion in liquid nitrogen
;
_pdbx_exptl_crystal_cryo_treatment.annealing_details
; 10 s interruption of cold stream with plastic ruler; performed twice
;
How this example will appear in the journal
| Crystallization |
| Crystallization method
|
Hanging-drop vapour diffusion
|
| Temperature
|
Ambient
|
| Crystallization solutions |
| Protein (0.5 μl)
|
Protein 1.2 mg ml-1,
Tris 50 mM,
NaCl 100 mM, pH 7.5
|
| Precipitant (0.5 μl)
|
PEG 3350 25%(w/v),
Hepes 100 mM,
diammonium citrate 200 mM, pH 7.3
|
| Reservoir (0.5 ml)
|
PEG 3350 25% (w/v),
Hepes 100 mM,
diammonium citrate 100 mM, pH 7.3
|
| Cryo treatment
|
| Final cryoprotection solution
|
Crystals were soaked in reservoir solution supplemented with 20% glycerol
|
| Soaking
|
1-2 min before flash-cooling
|
| Cooling
|
Direct immersion in liquid nitrogen
|
| Annealing
|
10 s interruption of cold stream with plastic ruler; performed twice
|
| |
|
| Crystal data |
| Crystal size (mm)
|
0.05 × 0.03 × 0.02
|
|
|
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