Did you run your crystals on a gel to ensure and verify that what you think you
crystallized is at least the right molecular weight ? Maybe the N-terminal part
is just chopped off and you have indeed crystallized just the 23 kDa fragment ?
Try running Xtriage or pointless over your data to verify your space group.
Try indexing e.g. with Mosflm with a 0 degree, 45 and 90 degree image and see
if your space group is still the same.
Jürgen
On Jul 16, 2011, at 4:47 AM, Appu kumar wrote:
Dear CCP4 User,
I am new to crystallography and i need your
valuable suggestion to help me out. We have crystallized full length protein
and I am solving protein structure by molecular replacement method and
molecular weight of protein is 37kDa. Template pdb i am using have only
C-terminal 24kDa protein structure. HKL2000 gave a space group C2221 and
Mathews Coefficient indicate one molecule of 37 kDa with solvent content 17%
only. When i am running phaser, it gave me a most probable molecualr weight of
protein in ASU to be 23kDa. and also i am not getting any density for
N-terminal of protein. Right now my Rwrk and Rfree are 28% and 37% but it not
decreasing further.
When i am indexing in lower symetry sapce group P1211, most probable molecular
weight ofpreotin com comes to be 46kDa in ASU. Would please anyone can help,
and suggest me what is the real situation?.
Thanks in advance
Mathews table
Data line--- CELL 57.8080 70.0520 105.7080 90.0000 90.0000 90.0000
Data line--- SYMM 'C 2 2 21'
Cell volume: 428071.531
For estimated molecular weight 36000.
Nmol/asym Matthews Coeff %solvent P(2.30) P(tot)
_____________________________________________________________
1 1.49 17.30 1.00 1.00
_____________________________________________________________
Space-Group Name: C 2 2 21
Space-Group Number: 20
Unit Cell: 57.81 70.05 105.71 90.00 90.00 90.00
MW of "average" protein to which Matthews applies: 36000
Resolution for Matthews calculation: 2.30
Z MW VM % solvent rel. freq.
1 36000 1.49 17.25 0.002 <== most probable
Z is the number of multiples of the total composition
In most cases the most probable Z value should be 1
If it is not 1, you may need to consider other compositions
Histogram of relative frequencies of VM values
----------------------------------------------
Frequency of most common VM value normalized to 1
VM values plotted in increments of 1/VM (0.02)
<--- relative frequency --->
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
| | | | | | | | | | |
10.00 -
8.33 -
7.14 -
6.25 -
5.56 -
5.00 -
4.55 -
4.17 --
3.85 ---
3.57 -----
3.33 --------
3.12 -------------
2.94 -------------------
2.78 ---------------------------
2.63 ------------------------------------
2.50 --------------------------------------------
2.38 -------------------------------------------------
2.27 --------------------------------------------------
2.17 ---------------------------------------------
2.08 -----------------------------------
2.00 -----------------------
1.92 -------------
1.85 ------
1.79 --
1.72 -
1.67 -
1.61 -
1.56 -
1.52 -
1.47 * (COMPOSITION*1)
1.43 -
1.39 -
1.35 -
1.32 -
1.28 -
1.25 -
Most probable VM for resolution = 2.30957
Most probable MW of protein in asu for resolution = 23168.4
Thanks in advance
Appu Kumar singh.
......................
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-3655
http://web.mac.com/bosch_lab/
