CryoEM processing

workshop
PNCC center, July 20-24 2020

1
 July20: cryoSPARC
9 am -11 am cryoSPARC overview – presented by Cryosparc CEO and
co-founder (RECORDED session)
11:00 am -1 pm cryoSPARC setup, overview, gain references(ZOOM-
LIVE)
July21: cryoSPARC
9 am-1 pm cryoSPARC training on the dataset #1 (ZOOM-LIVE)
July22: cryoSPARC/relion

Schedule
9 am-11 am cryoSPARC training on the dataset #2 (ZOOM-LIVE)
11 am-1 pm relion setup and training on the dataset #1 start (ZOOM-
LIVE)
July23: relion
9am -1 pm relion training on the dataset #1 continue Follow up on
the cryoSPARC processing of dataset #2 (ZOOM-LIVE)

July24: relion/wrapping up/masks

9am -1 pm relion training on the dataset #1 continue. Discussing the
results, wrapping up. Masks (ZOOM-LIVE)

2
 • Location on cascade
/dtemp/emslpXXXXX/workshop_package/

• ApoFe (36 images)

Pixel size – 0.821 A; total dose – 30 e/A^2; 40
frames; voltage – 300 kV; Cs – 2.7
Particle diameter – 120 A; symmetry – O PDB ID: 2W0O
(octahedral)
K3 camera (correlated double samplings
Training CDS mode)

datasets 0.4 bin=2 => pixel size 0.8

\
• Connexin (535 images + 1 gain image)
Pixel size – 0.665 A; total dose – 72 e/A^2; 40
frames; voltage – 300 kV; Cs – 2.7
Particle diameter (in the largest dimension) – 150
A; symmetry – D6
K2 camera

https://www.nature.com/articles/s41586-018-0786-7
3
 To login:
ssh userID@cascade.emsl.pnl.gov -X -Y

Useful commands:

Cascade gbalance # to check the number of available

compute hours awarded on your project.
login and showq -u userID # to check the status of your
jobs

other squeue -p pncc # to check all jobs running on

pncc nodes by all users

useful scancel -u userID # to cancel all your jobs

scancel JOBNUMBER # to cancel a specific job
commands screen –S name # keeps the session active in
case connection fails. Very useful when copy
large chunks of data.
https://gist.github.com/jctosta/af918e1618682638aa82

4
 Single Particle
2D classification
Data Processing
(SIMPLIFIED)
Import Movies

Ab initio reconstruction

Motion correction

CTF estimation 3D refinement

Particle picking

Post-processing

5
 Cryosparc 2.15

RELION3.1
Available on cascade
RELION3.0

Available cisTEM
software
Other platforms: EMAN2, SPHIRE; Scipion; Focus

6
 1) Your cryosparc password is saved in your
/home/userID/ directory in .cryosparc file. Your
username is your home institution email.
vi .cryosparc

2) To launch Cryosparc in your web browser,

paste the following line in your new
terminal window (not logged in).
Cryosparc ssh -L 8443:cryosparc-pncc.emsl.pnl.gov:443
userID@cascade.emsl.pnl.gov
access
Enter your PASSCODE and cascade password.
3) Then you can point your web browser
to https://localhost:8443

4) Enter your username (home institution email)

and cryosparc password. You are IN!!!

7
 • Let’s DIVE IN
In your terminal, type:

cd /dtemp/emslpXXXXX/

Cryosparc ### where XXXXX is your 5-digit PNCC project

number!!!

STEP1 ll
cd workshop_package/

8
 Cryosparc
Organization/Recommendations
STEP2

Reserve for each distinct protein

sample or protein structure you want
Projects to determine.
Files and jobs cannot be connected
between the projects
Recommend to use one workspace for
each dataset.
Workspaces Allows exploring different processing
workflows.
Allows logical separation of jobs.
Jobs can be linked, and files can be
#####Please remember that these are recommendations, exchanged.
and it is up to you how you want to organize you space
9
 Cryosparc
Organization/Recommendations
STEP2
+ Add project

+ Add workspace.

10
Import
STEP3

Select Job Builder

Select Import Movies

Guide to Jobs and Common Parameters:

https://cryosparc.com/docs/reference/jobs
11
Import
STEP3

12
Submission lanes
STEP3

For Admins

For automated proc.

Pick your 5-digit

PNCC project number!

13
Motion Correction From the “Job Builder” panel
STEP4 a)
2-in-1: Full-frame + Local motion
(multi) parallelized over multiple GPUs
No need for particle locations unlike
“Local Motion Correction” job

b)

DRAG

c) Up 2 GPUs can be requested

14
CTF estimation From the “Job Builder” panel
STEP5 a)

Fast, auto-tuning patch-based local

CTF estimation.

b)

DRAG

c) Up 2 GPUs can be requested

15
Exposure curation (OPTIONAL)
STEP6

Save the selection Press

once
done

The goal is to identify and remove outliers (low-quality micrographs). Two

most common metrics for filtering bad data: CTF Fit resolution and
Total full-frame motion distance.
Detailed tutorial: https://cryosparc.com/docs/tutorials/exposure-curation 16
 Particle picking
STEP7
ROUTE 1:
Template picker
• The best way to • Template-free
familiarize yourself
with the particles!!!
• Manual (interactive
mode) picking
followed by template
generation and
template-based
picking
Both will be tried using ApoFe dataset
ROUTE 2: ROUTE 3:
Blob picker Deep picker
Topaz
• Best for challenging
samples (asymmetric,
automatic picking,
non-globular or
detecting regions
unusually shaped
which differ in
proteins).
properties (such as
• Retrieves many more
brightness) in
real particles; low false-
comparison to
positive rates.
surrounding regions.
• Low or no need for post-
processing (such as 2D
classification and
filtering)
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 Particle picking Box size = 2 x (particle diameter). Please note

STEP7 –TEMPLATE PICKING (ROUTE 1) that it is in pix. ### For elongated particles 1.5
values are often used instead of 2.
For ApoFe, box size =2x120(A)/0.821(A/pix)=292
• Select “Manual picker” from the JobBuilder pix
For Connexin, box size
=2x150(A)/0.665(A/pix)=451 pix. 512

• For ApoFe dataset, aim for 100 particles picked across several micrographs
• Once done, click “Done Pocking! Extract Particles”

18
 Particle picking
STEP7 –TEMPLATE PICKING (ROUTE 1)
• Select “2D classification” from the JobBuilder; drag particles from the previous job;
set number of classes to 10 and press Queue.
• Once the previous job is complete, press “Select 2D” from the JobBuilder, drag
particles and class_averages and press Queue.

Ideally, select a few templates with different

projection views. For ApoFe, it is globular and
highly symmetric object => 1 view

• Press “Done” after selection.

• Select “Template Picker” from the JobBuilder; drag template_selected from the
previous job and exposures_accepted from the ”Curate Exposure” job; set particle
diameter and press Queue.

19
 Particle picking
STEP7 –TEMPLATE PICKING (ROUTE 1)
• Select “Inspect Picks” from the JobBuilder. Drag particles and micrographs from the
previous job and press Queue. Goal is to eliminate false positives. True particles
generally have higher NCC and Power scores.

NCC stands for Normalized cross

correlation (agreement with the
provided template)
Power threshold indicates the signal
strength.
Change the box size to
small number, so you
can see the particles
clearly. The box size is
not important here.

Adjust the NCC and Power parameters

here to include only the desired
particles. Once complete, press “Done
Picking”.

• Select “Extract from Micrographs” from the JobBuilder. Drag particles and micrographs
from the previous job, specify up to 2 GPUs and the box size and press Queue.
• Select “2D classification” from the JobBuilder. Drag particles from the previous job, specify
20
up to 2 GPUs and the box size and press Queue.
 Particle picking
STEP7 –BLOB PICKING (ROUTE 2)
• Let’s create a new workspace with a new name “blob picking route” to keep
ourselves organized. Link “Curate Exposures” job from the other workspace to your
new workspace.
• In your new workspace, select “Blob picker” from the JobBuilder. Specify minimum
and maximum for the particle diameter and press Queue. (I used 100A and 150A
for ApoFe)
• Perform “Inspect Picks”, “ Extract from micrographs” and “2D classification” as
specified in the slide 21 but using a current blob-selected particle pool.

21
 Particle picking
STEP7 -TOPAZ

105 kDa Toll receptor

(asymmetric and non-globular)

Topaz picks

0.5% false positive rate

1 round of 2D class=> 1,006K particles from 1,010K pool
Aggregation
4 rounds of 2D class=> 627K particles from 1,265K pool

4 rounds of 2D class=> 770K particles from 1,599K pool

Adapted from https://www.nature.com/articles/s41592-019-0575-8.pdf

Path to Topaz executable on Cascade:

/home/svc-pncc/singularity/topaz_latest.sif
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 2D classification
STEP8

DRAG

DRAG
Should look like 2D
projections of your
Press “Select 2D” from the JobBuilder. Select good classes, Press Done.
atomic model. Should
see internal features.
The quality of 2D
classes defines the
quality of your 3D
map.

23
2D classification
STEP8
The most common questions:

• What number of 2D classes to specify?

• How many rounds of 2D classification are needed?

There is no one defined answer. This will depend on your protein sample (such as protein shape, symmetry and
heterogeneity) and the quality and size of your image datasets.

Generally, start with a minimum of 2 rounds of 2D classification for highly-symmetric molecules such as ApoFe. For
complex proteins, at least 3 to 4 rounds of 2D classifications are needed.

The same trend applies for the number of classes: 50 classes are sufficient for more globular, highly symmetric
molecules. For more complex samples and large datasets, 150—200 classes are often chosen. We don’t find it
necessary to run more then >250 classes, it becomes computationally exhausting and does not provide any additional
data curation benefits.

Compare 2D classification results from template-picking and blob-picking

24
 Ab initio reconstruction/Homogenous
refinement
STEP10-11
• Select “Ab-initio reconstruction” from the JobBuilder. Drag particles and press
Queue.
######### Note that you can pick >1 “ Number of Ab initio classes” to find multiple
conformations or distinct particles.
• Once a previous job is complete, select “Homogenous Refinement (NEW)” (for the
ApoFe dataset) from the JobBuilder. Drag particles and a volume, specify
”Refinement box size” and “ Symmetry” and press Queue.

A single homogenous structure is expected.

Refine multiple conformers or sort small differences within

a single particle pool

Best for regions of a structure, which display disorder or

high flexibility. Prevents over-fitting of these regions.
Recommended for membrane proteins.

Focus on a specific region of a structure when dealing with

flexible proteins.
https://cryosparc.com/blog/local-refinement
25
Homogenous refinement
STEP11

Your unsharpened
volume

Do not use this

sharpened volume for
publication. It is not
optimal. Run a separate
sharpening job.

26
 Sharpening
STEP12
From the “Homogenous refinement (NEW)” job:

Make sure that B-actor is entered

as a negative value!!!!!

27
 Other useful information

Create an automatic workflow: you don’t have to wait until the job is finished!!!

Card View and Tree view

28
 Please contact Irina at:
irina.novikova@pnnl.gov
For
questions/
If the problem is regarding a specific job, copy
and paste a job link along with your question.

errors in
cryosparc

29
 • Create a destination folder where all your data will
reside. New dataset/protein structure to solve = new
folder.
• The folder organization is critical!!!!
In your terminal, type:
cd /dtemp/emslpXXXXX/
mkdir relion/
cd relion/
mkdir dataset_name/
cd dataset_name/

Relion3.1 For each dataset:

1) Create a Movies/folder

STEP1 2) Transfer raw files by copying or generate symbolic links

or

3) copy relion31_launch.csh; sbatch_relion3_1.csh scripts and

modular transfer function curve for the camera used
(https://www.gatan.com/techniques/cryo-em)
Final project setup (project directory) should look like this:

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Relion 3.1
Launching GUI
STEP2
Do not launch the GUI on the general login node (glogin). The glogin is shared between many
supercomputer users and is generally used to browse files , do simple operations and submit
slurm jobs. It is not designed to handle visualization of large images, which cause the problems for
other users.

To launch the GUI, you will have to reserve a node just for you by running the following command.
## -W 180 stands for 180 min session

You will see a new terminal window appear (give it a minute), and gXXXX will be displayed
instead. -r pncc_workshop

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 Relion 3.1
Launching GUI
STEP2

REMEMBER to always launch the

GUI from the project directory!

JOBS

JOB SETTINGS OUTPUT

ERRORS
32
 Relion 3.1
Import
STEP3
For ApoFe:

MTF curves for Gatan cameras (K2

and K3):
https://www.gatan.com/techniques
/cryo-em

Submit to queue: No and

Press RUN
You can also check the status and the output of
every job on the terminal:

33
vi movies.star
 Relion 3.1
Motion Correction STEP4
Once the job is submitted via a
slurm scheduler (sbatch), the
GUI can be closed and opened
I/O tab

as desired. You can monitor the

job on the terminal as well.

use larger values for super-resolution movies

Motion tab

2 is often used for super-resolution movies

if available

if available
preferred, allows Bayesian polishing at the post-processing stage
Running tab

for workshop, we will use 3. Otherwise, up to 12 MPIs can be requested

we have 16 CPUs per MPI. Thus, up to 16 threads can be requested
Specify your project number

Press RUN ~13 min for ApoFe

34
 Relion 3.1
Motion Correction STEP4

Using these settings, the job takes around 13 min. The output of the job can
be visualized by clicking on MotionCor/job002/ and then on Display: out:
corrected_micrographs.star and logfile.pdf.

Note: to visualize a specific subset of images, use ‘Subset selection” job first.
This is particularly important when you have hundreds of images, relion will
try to read all of them which takes a significant amount of time. 35
 Relion 3.1
I/O tab
CTF estimation STEP5

Preferred, as it allows reading in the movie-averaged power spectra calculation.

CTFFIND-4.1

Specify Yes

Make sure it is a No.

Gctf

Press RUN
36
 Relion 3.1
CTF estimation STEP5

Using these settings, the job takes around 35 sec. The output of the job can
be visualized by clicking on Ctffind/job003/ and then on Display: out:
micrographs_ctf.star.

Note: to discard unsatisfactory micrographs, use ‘Subset selection” job.

37
 Relion3.1
Particle picking
STEP6
ROUTE 2:
ROUTE 1: Template-free
Template picker picking
(called as LoG-
based)

• The best way to • Template-free

familiarize yourself automatic picking,
with the particles!!! detecting regions
• Manual picking which differ in
followed by template properties (such as
generation and brightness) in
template-based comparison to
picking surrounding regions.
• used in on-the-fly
processing

38
 Relion3.1
Particle picking
STEP6 –MANUAL PICKING

Specify the particle diameter

Will depend on your screen type

Helpful to play with if the images are noisy Make sure that you
Specify the pixel size save the coordinates!

Note: you can always come back to the

same job and continue picking. Click first
Press RUN on Manual/job004/ and then press
Continue.
Need ~500-1000 particles to calculate initial 2D
templates.
For ApoFe,~250 particles are sufficient. (##6-8) 39
Relion3.1
Particle picking
STEP6 –LoG PICKING
Due to time limits, we will perform LoG picking only on a few micrographs.
However, first we have to select those. So go to “Subset selection” tab,

Press RUN
Start to use
alias

Go to ‘’File”, press “Invert selection”,

pick a few micrographs and then press
“Save selection”.
40
 Relion3.1
Particle picking
STEP6 –LoG PICKING
Go to “Auto-picking” tab.

Specify the output of the previous job

I/O tab

YES
Laplacian tab

Depends on the contrast of the images. For low contrast micrographs, values of 1.5 may be reasonable

See next slide…

41
 Relion3.1
Particle picking
STEP6 –LoG PICKING
autopicking tab

These values will be ignored

LoG-based picking is not GPU-accelerated

Running tab

The job takes around 1 min. The output of

the job can be visualized by clicking on
Autopick/job00X/ and then on Display:
out: cords_suffix_autopick.star.

Press RUN
42
 Relion3.1
Particle extraction
STEP7
I/O tab

For ApoFe, box size = 2x120(A)/0.821(A/pix)=292 pix

extract tab

The job takes around 2 min. The output of

YES
the job can be visualized by clicking on
Extract/job00X/ and then on Display: out:
particles.star.

Press RUN, no queue

43
 Relion3.1
Making templates for auto-picking
STEP8 Go to 2D classification job:
I/O
Optimization CTF

Always min 50 is recommended

For large datasets, always specify YES

It should be > than the particle diameter, but keep the mask tight to remove solvent noise and neigbouring particl
Compute

10-30 value is recommended for GPUs

YES The job takes around 6 min. The output of

KEEP EMPTY
the job can be visualized by clicking on
Class2D/job00X/ and then on Display: out:
run_it025_model.star.
Running

Press RUN 44
 Relion3.1
Making templates for auto-picking
STEP8

Go to “Subset selection” job.

I/O
options

YES
Class

Press RUN

45
 Relion3.1
Auto-picking optimization
STEP9
Before the use of generated 2D templates in the auto-picking procedure, 4 parameters
require optimization:
Picking threshold (how restrictive the particle picking is)
Minimum inter-particle distance (min is 50% of the longest dimension)
Maximum stddev noise
Minimum avg noise
To save time, only a few micrographs will be used for this.
Go to “Auto-picking” job.

Select/afewmic/micrographs_selected.star

Select the job output from previous slide

I/O

Make sure it is a NO

Ignore the“Laplacian” tab.

References

REMEMBER that we downscaled the

particles from 292 pix to 74 pix. This gives is
the factor of 3.946. So the pixels size in See next slide…
references will be 3.946*0.821=3.24

46
 Relion3.1
Auto-picking optimization
STEP9
autopicking

Use lower values to pick more particles (or possibly junk)

We see from the images that ApoFe particle pack tightly

Specify YES

Specify YES
KEEP EMPTY

Press RUN, no queue The job takes around 6 min. The output of
the job can be visualized by clicking on
AutoPick/job00X/ and then on Display:
out: coords_suffix_autopick.star.

Display window will take the

parameters from the last “Manual
picking” job. To change that, go to
“Manual picking” job and navigate to
“Colors” tab:

47
 Relion3.1
Auto-picking optimization
STEP9
Let’s continue to play with the optimization parameters. Click on your previous
Autopick job (in Finished jobs) and go to “autopicking” tab.
autopicking

Let’s try 0.05

Specify NO
Specify YES

Press CONTINUE
Play around with parameters until optimal values are found. Once ready:

Select all micrographs!!!!!!!!!

I/O

See next slide…

48
 Relion3.1
Auto-picking + extraction
STEP10
autopicking

Your optimal values!!!!

Specify NO
Specify NO

Press RUN, no queue.

The job takes around 6 min. For large
datasets with many images, submit to
queue. Check the results by clicking on
AutoPick/job00X/ and then on Display:
out: coords_suffix_autopick.star.

Re-run “Particle Extraction” job with new

“Input coordinates” !!!! Submit to queue
to speed it up (158K particles final).

49
 Relion3.1
2D classification
STEP11
The main goal is to remove bad particles in order to clean up your data!!!
Optimization I/O

Specify YES, we have more particles now

Submit to queue and press RUN

The job takes around 55 min. Launch
“Subset selection” job using the output
of this job (run_it25_model.star file).
Select the best classes and save the
selection by “Save selected classes”.

51K particles after 1st round.

Perform a 2nd round => 27 min
and 48K particles final
50
 Relion3.1
3D initial model
I/O
Optimization STEP12

Occasionally, more than 1 class is specified to ‘sink’ suboptimal particles

In general, keep this tab

SGD

unchanged with 50, 200 and 50

numbers for SGD iterations.
However, to speed things up in
this tutorial, use 25, 100 and 25.
Compute

Specify NO, GPUs will only slow down this job

See next slide…
Submit to queue: YES and press RUN 51
Relion3.1
3D initial model
STEP12
The job takes around 7 min. Check the output 3D volume (run_it150_class001.mrc) by
Chimera. Chimera is available on cascade and you can source it by the command below:

/home/scicons/cascade/apps/chimera/bin/chimera run_it150_class001.mrc

However, we would not advise to use it. The mouse movements for this image
visualization software are quite delayed when traveling over the network. It is the best
just to copy the output file for examination by Chimera on your local computer.

Open a new terminal window (do not login to cascade!!!) and copy the file to your local
computer:

[WE37474:~/Desktop] novi385% scp

userID@cascade.emsl.pnl.gov:/full/path/to/the/file/run_it150_class001.mrc ./

52
 Relion3.1
3D classification
The goal is to identify a
STEP13 homogenous subset.
I/O
Reference

Always YES, if an initial reference was generated in relion

Even though we know the symmetry, it is the best to specify C1 for the first round
CTF
Optimization

Using more classes is useful for heterogenous datasets. For ApoFe, use just 1
Compute

See next slide…

Submit to queue: YES and press RUN 53

Relion3.1
3D classification
STEP13
This job takes ~ 11 min. Copy your new
output file (run_it150_class001.mrc) to your
local computer and examine with Chimera

Also useful to look at your structure in slices view: click on Class3D/jobXXX/ and then on
Display: out: run_it025_class001.mrc

54
 Relion3.1
High-resolution 3D refinement
STEP14
The goal is to refine a selected homogenous subset to high-resolution.
However, all our particles were previously downscaled. Let’s re-extract
the particles with less or no downscaling.
I/O
extract

The job takes 2 min.

For ApoFe, keeping original 0.821 will result in maximum achievable resolution of 1.64 A

Submit to queue: YES and press RUN

We also need to re-scale the best 3D map obtained so far:
$ /dtemp/emslc50414/kschmidt/relion31_cuda/bin/relion_image_handler --i
Class3D/job022/run_it025_class001.mrc --angpix 3.23962 --rescale_angpix 0.821 --new_box 292 --o
Class3D/job022/run_it025_class001_box292.mrc 55
 Relion3.1
High-resolution 3D refinement
STEP14
Go to 3D auto-refine tab:

New re-extracted particles with no or less downscaling

I/O

Re-scaled reference, type manually!!!!!

Reference

NO, re-scaled reference is no longer on a correct greyscale

Specify the symmetry

The rest of the parameters are the same as in 3D classification job!!!!

The job takes ~1 hour.

Submit to queue: YES and press RUN Upon convergence, a single
run_model.star and
run_class001.mrc will be
written. Look at the files in
chimera and in slices view.

56
Relion3.1
High-resolution 3D refinement
STEP14

FSC curve estimate on unmasked maps

57
Relion3.1
Post-processing: sharpening and calculating masked FSC
curves
STEP15
Create a mask:

1) Find an initial binarization threshold for mask creation.

• Low-pass filter one of the unfiltered half-maps (run_half1_class001_unfil.mrc):

$ /dtemp/emslc50414/kschmidt/relion31_cuda/bin/relion_image_handler --i
run_half1_class001_unfil.mrc --lowpass 15 --angpix 0.821 --o
run_half1_class001_unfil_lowpass.mrc

• Look at the resulting file (run_half1_class001_unfil_lowpass.mrc) in Chimera

and determine the lowest threshold that gives no noise peaks outside the
reconstruction.

58
 Relion3.1
Post-processing: sharpening and calculating masked FSC
curves
STEP15
I/O
Mask

Determined in Chimera
Add more pixels to make the mask less tight
Try different values

Use 16 threads, submit to queue: NO and press RUN

Check the resulting file (mask.mrc) in Chimera to make sure it encapsulates the entire
structure but has minimal solvent area !!!!

59
 Relion3.1
Post-processing: sharpening and calculating masked FSC
curves
STEP15
Go to Post-processing job:
I/O

Submit to queue: NO and press RUN

Check the resulting output (logfile.pdf for FSC curves) and the final map in chimera
(postprocess.mrc)

60
Relion3.1
Post-processing: sharpening and calculating masked FSC
curves
STEP15
Make sure that the FSC of the phase-randomized maps (red curve) is
close to 0 at the estimated resolution of the map. If it is not, the mask is
too sharp. Re-do the mask by using a stronger low-pass filter or softer
mask parameters.

61
 Relion3.1
CTF refinement (OPTIONAL)
STEP16
This job can lead to further improvements in resolution (correct for
aberrations. Go to ”CTF refinement” job.
I/O

Correct for beamtilt first:

Specify NO initially
Specify NO initially
Fit

Submit to queue: YES

and press RUN
Takes ~1 min. Check the resulting output
(logfile.pdf). Some blue-red trends present 62
 Relion3.1
CTF refinement
STEP16
Correct for anisotropic magnification:

Use the output from previous job!!!

I/O

Specify YES
Fit

Submit to queue: YES and press RUN

Takes ~ 1 min. Check the resulting output

(logfile.pdf).

63
 Relion3.1
CTF refinement
STEP16
Re-estimate the defocus values for each particle:

Use the output from previous job!!!

I/O

Specify NO
Specify YES
Fit

Specify NO

Specify NO

Submit to queue: YES and press RUN

Takes ~ 1h10min. Check the output (logfile.pdf). Can you

see a tilted ice layer?

64
 Relion3.1
Bayesian polishing(OPTIONAL)
STEP17
It is per-particle, reference-based beam-induced motion correction. IT
TAKES TIME and WILL NOT BE PERFORMED IN WORKSHOP.
Run first in Training mode, followed by Polishing mode on entire dataset.

Training mode:
I/O

Specify YES
Train

Use minimum 4,000

Submit to queue: YES
(make sure that you use
only 1 MPI and 16
threads; it is not MPI-
Specify NO
parallelized) and press
Polish

RUN

Takes ~ 12 hours. See

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the next slide ..
 Relion3.1
Bayesian polishing
STEP17
Go to “Bayesian polishing” job and specify the following to run it in the polishing
mode now:

Specify NO Submit to queue: YES

(you can use as many
Train

MPIs and 16 threads as

needed; it is MPI-
parallelized) and press
RUN
Polish

Takes ~ 1.5 hours (using 3 MPIs). The

main output files are shiny.star and
logfile.pdf.

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 Relion3.1
Re-do high-resolution 3D refinement using polished
particles
STEP18
Go to 3D auto-refine tab:

Use shiny.star
I/O

Use the same mask that was used in post-processing

Optimization

Specify YES
The job takes ~1 hour 40 min.
Output files are
run_model.star and
run_class001.mrc will be
Submit to queue: YES and press RUN written. The resolution got
improved from 2.42 to 2.15!!!
Re-do postprocess job with
shiny particles!!!

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 Relion3.1
Local resolution estimation
STEP19
Go to “Local Resolution” job:

Use the same mask that was used in post-processing

I/O

Specify NO
ResMap

The job takes ~22 min.

Specify YES
Use B-factor from the post-process job Output file is
Relion

relion_locres.mrc.

Submit to queue: YES and press RUN 68

 Relion3.1
Visualize in Chimera
STEP20
Go to Chimera: Tools -> Volume Data -> Surface Color

Check also the handedness of the structure (50% chance of being wrong). To flip:
$ /dtemp/emslc50414/kschmidt/relion31_cuda/bin/relion_image_handler --i
PostProcess/jobXXX/postprocess.mrc --o PostProcess/jobXXX/postprocess_invert.mrc
–invert_hand
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 Relion3.1
Heterogeneity
All datasets are heterogenous!!!!

A good read: Processing of

Structurally Heterogenous Cryo-EM
Data in RELION by S.H.W. Scheres in
Methods in Enzymology (2016).

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 Cryosparc
Useful info
3D variability analysis: https://www.youtube.com/watch?v=0O781Od1z_E

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