Hello Gargi
I don't think you mean Fc. Apart from anything else, there's not enough
room for 4 more domains per Fab.
I think you mean the CL:CH1 domain dimer of the Fab. Fabs have a
well-characterized variability in the "elbow angle" between the VL:VH
domain pair and the CL:CH1 domain pair. I suspect you've tried
molecular replacement without trying a range of different elbow angles.
I see +ve difference map peaks within domains and -ve difference map
peaks along polypeptide chain, which is refinement's way of saying that
it doesn't want atoms in the latter location and wants atoms in the
former location.
There's also some quite serious clashing (chain interdigitation) in
places, which can't possibly be correct.
The easiest way to do this is to re-do your molecular replacement with
VL:VH and CL:CH1 models which will inherently allow their correct
relative placement to be modeled. If you managed to get TFZ of 42 with
flawed models, this should be a pretty easy thing to pull off, but if
not please contact me off list.
Phil Jeffrey
Princeton
On 3/27/23 1:52 PM, Kher, Gargi M wrote:
Hello,
I obtained diffraction data for one of my crystallographic projects.
Data collection determined the space group to be P1 at a ~2.67Å. I
solved it using MR (it contains a Fab-Fab complex) and the Phaser
solution (TFZ of 42.0), which is close to the Matthews coefficient
prediction, placed 16 Fabs in the unit cell (8 copies of the same
Fab-Fab complex). The Matthews coefficient was predicted to be 2.44 with
9 copies in the ASU, but for 8 and 10 copies in the unit cell, the
Matthews coefficients were 2.75 and 2.20, respectively. I did set up MR
jobs searching for 9 and 10 Fab-Fab copies in the unit cell, but still,
only 8 Fabs were placed.
While most of the Fabs fit nicely in the density, 6 Fcs (chains e&f,
A&B, M&N, Q&R, U&V, and c&d) do not fit within the electron density. I
have tried re-processing my data, but P1 seems to be the “correct” space
group, and Xtriage does not indicate any red flags. There is some extra
positive density visible close to these Fcs. I believe these 6 Fcs might
be in a different orientation/position than how they’re currently being
placed, and should fit into the additional positive density I’m seeing.
However, I have been unable to place them correctly: either by using
rigid body refinement in Phenix for these 6 Fc domains or doing it
manually as Fc domains.
Does anyone have ideas as to what might be happening here and what I
could do to try and fix this? I’ve attached my PDB and MTZ file for your
reference.
Thank you,
Gargi Kher
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