FSC curve does not fall down to zero but goes up dramatically at high resolutions

Submitted by donghua on Tue, 2014-05-13 22:54

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Frealign

Dear Niko,

I have reconstructed two maps from two subsets of data using Frealign v9.08. Then calculated the FSC directly from those two maps (reconstruction.mrc) without any masking. The FSC curve (attached) does not fall down to zero completely at high resolutions but goes up very fast. My mparameters file is as follows:

working_directory /raid2/donghua/DE-20/gset1
data_input DE-20
raw_images1 DE-20_odd_new_stack
thresh_reconst 0.95
thresh_refine 0.9
pixel_size 1.07
dstep 6.4
WGH 0.07
kV1 300.0
Cs 4.1
RO 380.0
RI 0.0
PBC 5.0
BOFF 75.0
ASYM I2
DANG 1
ITMAX 10
MODE 1
XSTD 0.0
RBFACT 0.0
FMAG F
FDEF F
FASTIG F
FPART F
dfsig 150.0
IEWALD 0
FBEAUT T
FFILT F
res_reconstruction 2.3
res_low_refinement 200.0
res_refinement 3.4

Any idea about why the FSC curve goes up fast at high resolutions (higher than 3.3Å)? Did I use any parameter(s) incorrectly? Thanks in advance!

Thanks
Donghua

niko Wed, 2014-05-14 08:01

The FSC can show a behavior like yours for a number of reasons: overfitting, inappropriate masking, rotational blurring/averaging of the reconstruction (for example, when the orientations of the particles are not well determined or random).

Your list of parameters looks like it is missing a few parameters that are needed for Frealign 9.08 (for example, the molecular weight of your particle). Please make sure that you are using the latest set of parameters by checking the Frealign log file for errors or warnings.

donghua Wed, 2014-05-14 10:17

Dear Niko,

I set MW to 0.0 and RCLAS to REFINE HIGH in the script.
No masking during FSC calculation, no warning or errors from log file.

---------
FREALIGN - Fourier REconstruction and ALIGNment

V9.08 - 05.05.14
Copyright 2013 Howard Hughes Medical Institute.
All rights reserved.
Use is subject to Janelia Farm Research Campus Software Copyright 1.1
license terms ( http://license.janelia.org/license/jfrc_copyright_1_1.html )

Parallel processing: NCPUS = 12

CFORM,IFLAG,FMAG,FDEF,FASTIG,FPART,IEWALD,
FBEAUT,FFILT,FBFACT,FMATCH,IFSC,FDUMP,IMEM,INTERP?
M 0 F F F F 0 T F F F 0 F 2 0
RO,RI,PSIZE,MW,WGH,XSTD,PBC,BOFF,DANG,ITMAX,IPMAX?
380.0 0.0 1.07 0.000 0.070 0.000 5.0 75.0 1.0 10 10
PMASK for parameter refinement (e.g. 1,1,1,1,1)
1 1 1 1 1
FIRST, LAST PARTICLES ?
1 21416
SYMMETRY CARD ?
I2
Entering GETSYMMAT with ASYM,NASYM I 2
Symmetry point group I2 redundancy 60
MAGNIFICATION, STEPSIZE, TARGET & THRESH SCORE LIMIT, CS, KV, BEAM TILT X,Y ?
1.0 6.4 0.9 0.9 4.1 300.0 0.0 0.0
NSET= 1, RELMAG= 1.000, DSTEP= 6.40
RESOLUTION RECONST., REFINE LOW/HIGH, CLASSIFY, DFSTD, RBFACT ?
2.3 200.0 3.2 3.2 100.0 0.0
PARTICLE IMAGE STACK ?