Computational Imaging System for Transmission Electron ... › presentations ›...
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Computational Imaging System for Transmission Electron Microscopy
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Nikolaus GrigorieffAlexis Rohou
Computational Imaging System for Transmission Electron Microscopy
Tim Grant
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Tools for Single Particle Cryo-EM
Process Tool
Mag. distortion correction mag_distortion
CTF determination CTFFind4
Movie processing Unblur
Exposure filtering Unblur
3D refine. & reconstr. Frealign
B-factor sharpening bfactor
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Complete Pipeline
Decompression & gain correction
Decompression & gain correction
Mag. distortion correction
Mag. distortion correction
Movie processingMovie processing
2D classification2D classification Particle pickingParticle picking CTF determinationCTF determination
Ab initio 3D reconstructionAb initio 3D
reconstruction
Auto/manual refinement & classification
Auto/manual refinement & classification
B-factor sharpeningB-factor
sharpening
Unblur mag_distortion_correct
CTFFind4New! (Sigworth, 2013)
New ML Based
New! See later Frealign bfactor / new
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Robust Architecture
-Parallelized for CPUs, no GPUs -Written entirely in C++ (Open-Source)
-SQLite database stores all meta data
-Custom socket-based parallelization
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cisTEM GUI
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GUI Design
-No command line / editing of text files (and command line only)-Single window-Easy presentation / sorting of results-Easy selection of good results-Live feedback while jobs are running-Only enter information once-Easy Import / Export to other packages
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GUI Movie
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cisTEM is fast!
-Optimized code (especially MKL FFT)-On the fly binning-On the fly adjustment of particle no. / resolution -Only 3D classification is ML, not refinement
Abeyrathne et al, 2016
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Benchmark: β-galactosidase
Processing Step Details Time (hours)
Movie Processing 1539 movies, 38 frames, super-resolution 1.1
CTF Determination On Images 0.1
Particle Picking 131,298 particles 0.1
2D Classification 50 classes, 28 selected with 119,523 particles 0.8
Ab-inito 3D 40 iterations 0.8
Auto refinement 8 iterations, final resolution 2.2 A 1.4
Manual refinement 1 iteration (incl. defocus), final resolution 2.2 A 0.4
Total 4.7
Processing on single workstation
2 x Xeon (44 cpu cores)
512 GB Memory
16TB SSD Scratch
Sits under my desk
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Bartesaghi et al. 2015, reprocessed
0
0.2
0.4
0.6
0.8
1
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
FS
CResolution [1/Å]
2.2 Å
Trp, Asp… Tyr, Asn, carbonyl, H2O Pro, Met…
Phe, Ile, α-helix Trp, carbonyl, β-sheet (no masking)
Benchmark: β-galactosidase
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Web Page: cistem.org
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cisTEM Ab-inito
Angular searchAngular search 3D reconstruction3D reconstruction
Ideal case, ab-inito is just a refinement
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cisTEM Ab-inito
Angular searchAngular search 3D reconstruction3D reconstruction
Ideal case, ab-inito is just an interative refinement
How to prevent getting stuck in local minima?
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cisTEM Ab-inito
cisTEM does 40 rounds of global refinement
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cisTEM Ab-inito
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cisTEM Ab-inito
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cisTEM Ab-inito
“Heuristic” approach?
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CisTEM Ab-inito
1. Start at low resolution2. Random subsets of particles3. Sloppy alignment4. Use best 1/3rd particles5. Good automasking6. C1 with symmetry alignment
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Low Resolution to High
Round 1 (20 Å) Round 40 (8 Å)
-Smoother search space at low resolution
-Some high resolution probably required for distinguishing right / wrong (protein dependent)
-Jumping back eliminates high-res bias.
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Random Subsets of Particles
-Faster!
-Also improves convergence / reduces overfitting.
N1 = 2500, N40 = 10000
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Best 3rd of particles
Taking the best 3rd of scores appears to improve convergence.
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“Sloppy” alignment
Elmlund et al, 2013
cisTEM alignment is “deterministic”
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“Sloppy” alignment
Gatsogiannis et al, EMPIAR-10089
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Randomly select from all global search results whose score is in the top 15% of the difference between the best and worst score.
Actually Faster than normal cisTEM algorithm.
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“Sloppy” alignment
Gatsogiannis et al, EMPIAR-10089
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Auto masking
Apply, then threshold
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Symmetry Alignment
Best result from starting in C1 then finishing with symmetry.
Need to align to symmetry axes.
Needs to be fast.
Gatsogiannis et al, EMPIAR-10089
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Symmetry Alignment
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Symmetry Alignment
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Symmetry Alignment
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Symmetry Alignment
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Symmetry Alignment
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Symmetry Alignment
Brute Force search over angles
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Symmetry Alignment
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Seems robust...
Start Cycle 9 Cycle 27 Cycle 40 1.0 h
Start Cycle 9 Cycle 27 Cycle 40 4.2 h
Start Cycle 9 Cycle 27 Cycle 40 0.3 h
D2460 kDa
C1240 kDa
O440 kDa
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Thanks!
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Web Page: cistem.org
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Auto masking
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Auto masking
Threshold to average value at edges.
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Auto masking
Filter to 50 Å.
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Web Page: cistem.org
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Auto masking
Binarise.
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Auto masking
Remove disconnected density.
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Ab-Initio 3D Procedure
Angular searchAngular search 3D reconstruction3D reconstruction
40 cyclesResolution incremented from 20 Å to 8 Å
3N particlesN1 = 2500, N40 = 10000
N particles with highest scores
Repeat if needed
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Custom Parallelization
GUI
WorkstationWorkstation
Job controller
Slave jobs
GUI
WorkstationWorkstation
Job controller
Slave jobs
Cluster HeadCluster Head
Cluster NodesCluster Nodes
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Run Profiles
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Ab-Initio 3D Procedure
Angular searchAngular search 3D reconstruction3D reconstruction
40 cyclesResolution incremented from 20 Å to 8 Å
3N particlesN1 = 2500, N40 = 10000
N particles with highest scores
Repeat if needed