crystallization of cytochrome C from horse heart

From: Danilo Belviso
Date: 26 August 2011 15:19


Dear all,

I would like to crystallize cytochrome C from horse heart, to perform a soaking. I have tried the crystallization conditions that are reported by many papers (1) but I have not obtained cytochrome C crystals until now.
Only big objects (800micrometer) that do not seem single
crystals (in attachment_1) are been obtained (conditions are in attachment_2).
Conditions were performed using cytC from horse heart of Sigma Aldrich and with protein obtained in our lab. The result was the same.

May someone give me some tricks to crystallize cyt C?

Thanks a lot.

Danilo



(1) - High res 3D structure of Horse Heart Cytochrome c J. Mol.Biol.
(1990), 214,585-595
- The low ionic strength crystal structure of horse cytochrome c at 2.1A
resolution and comparison with its high ionic strength counterpart
Structre (1995), 3(7)
- High resolution X-Ray crystallographic structure of bovine heart
cytochrome c and its application to the design of an electron transer
biosensor
Proteins (2008), 70, 83-92

Crystallization trials for CytC apo from Horse Heart

Trials with low ionic strange
Conditions	Drop 4μL	2μL ([CytC]=25mg/ml in [Na3PO4]=50mM pH7) + 2μL (PEG1000 25%w/v in [Na3PO4]=50mM pH7)	Drop: We put on cover slip 2μL of solution B and after we added 2μL of solution C
	Reservoir 700μL	PEG1000 30%w/v in [Na3PO4]=50mM pH7	The reservoir was made of: 250μL of solution A 600μL of PEG1000 50% (HR2-523 Hampton) 150μL of milliQ water
	Method	Hanging drop (Nextal Plate)
	Temperature	20°C

Solution A (buffer phosphate 200mM pH7): the solution was made of trisodic phosphate (71908 Fluka) titrated with NaOH (480507 Carlo Erba) and HCl (403912 Carlo Erba) to obtain pH 7.


Solution B (buffer phosphate 50mM pH7, PEG1000 25%w/v): the solution was made of 25μL of solution A, 50μL of PEG 1000 50%w/v (HR2-532 Hampton) and 25μL of milliQ water.


Solution C ([CytC]=25mg/ml, buffer phosphate 50mM pH7,PEG1000 25%w/v): the solution was made of 10mg of CytC (105201 Sigma Aldrich) stabilized in 100μL of solution A. The protein solution was diluted with 300μL of milliQ water