Appion

Hi Anchi, Can I get the voltage for this micrograph, I want to include it in a test dataset.

200 kV, Cs = 2.0 mm

Hmm, I see that in the new plot it fit the curve just fine, but the generated CTF signal did not match. I will download the image and test it, probably Friday.

Hi Neil,
I thin this needs to be fixed ASAP or everyone will just not use it anymore and that would be sad. It is too slow and not giving good results now and so the users will vote with their feet and move on to something else. In my experience it is very hard to get them back after they leave!
Let us know if you want to chat.
Bridget

I have not changed anything to it in months, so what happened? There is a slight difference in the defocus values and at that close to focus it is often enough to shift the CTF oscillations off at high resolution, I see 0.786/0.808 um in the new and 0.784/0.812 um in the old.

I understand the need to be quick, but the CTF fitting is very touchy, so I do not like to make a modification that works with one image and then breaks the rest. I just got the kids to bed, so I will do some initial tests now, but unfortunately, my time is mostly booked for the next 24 hours. I have a quiz and 5 hours of lecture to prepare for tomorrow.

OK, get those kids bedded down happily and don't fret too much! I am a big fan of your method and want to protect its use but it is not an emergency so do not lose any sleep over it or neglect those kids or that quiz. Let's just keep it on the high priority list and see what we can do to restore confidence....
B

Ignore this comment

never mind, I messed up the pixel size when I uploaded it

Something is wrong. According to the CTFFIND4 2d images it easily fits 7 Thon rings. Looking at my 2d images, it shows green for only 4 Thon rings, but the 2D image fits the 7 Thon rings despite them being red. When we look at the 1D graph it shows a different story.

The 1D graph and 2D graph are not in agreement. I am looking into this now.

I just tested the current code on Joachim Frank's simulated images from the CTF challenge and the resolution went out to 2 Angstroms (max) for all of the images.

Are you sure that the pixel size and spherical aberration is correct for the image you uploaded? Should I try this on some similar images. Based on the fit it appears the things are getting out of sync once the spherical aberration term kicks in, which leads me to question the correctness of the pixel size and spherical aberration.

Pixel Size is correct. Your CTF estimation is way off. For this, the nominal defocus was 0.8 um. resmax I used was 3 Å, and resmin was also lowered to 30 Å, At such low defocus, defstep of 1000 is also not reasonable, so I was using 200. This is the typical setting for Phase plate data collection even though this one does not use phase plate, so I am not making it estimate phase shift. Low defocus, high resolution estimation is common now.

By the way, the CTFFind4 I used was 4.1.5

What was the CTFFIND 4 resolution it was reporting. I ran several run at low defsteps.

4.5 Å

Here is CTFFind4 output captured by Appion

Input image file name                              : 17apr12a_019en.mrc
Output diagnostic image file name                  : 17apr12a_019en-pow.mrc
Pixel size                                         : 0.963
Acceleration voltage                               : 120.0
Spherical aberration                               : 2.2
Amplitude contrast                                 : 0.15
Size of amplitude spectrum to compute              : 1024
Minimum resolution                                 : 30.0
Maximum resolution                                 : 4.27
Minimum defocus                                    : 2844.7
Maximum defocus                                    : 12844.7
Defocus search step                                : 200.0
Do you know what astigmatism is present?           : no
Slower, more exhaustive search?                    : no
Use a restraint on astigmatism?                    : yes
Expected (tolerated) astigmatism                   : 273.8
Find additional phase shift?                       : no
Do you want to set expert options?                 : no

Summary information for file 17apr12a_019en.mrc
Number of columns, rows, sections: 4096, 4096, 1
MRC data mode: 2
Bit depth: 32
Pixel size: 0.963 0.963 0.963
Bytes in symmetry header: 0

Working on micrograph 1 of 1
Estimated defocus values        : 8082.36 , 7862.55 Angstroms
Estimated azimuth of astigmatism: -53.71 degrees
Score                           : 0.14389
Pixel size for fitting          : 1.401 Angstroms
Thon rings with good fit up to  : 4.5 Angstroms
Did not detect CTF aliasing

and my appion script

ctffind4.py --ampcontrast=0.07 --fieldsize=1024 --resmin=30 --resmax=3 --defstep=0.02 --numstep=25 --dast=0.05 --min_phase_shift=10 --max_phase_shift=170 --phase_search_step=10 --runname=ctffind4run3 --rundir=/data/appiondata/17apr12a/ctf/ctffind4run3 --preset=en --commit --projectid=18 --session=17apr12a --best-images --no-wait --continue --bestdb --expid=9311 --jobtype=ctfestimate