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Cryo-Electron MicroscopyJames ConwayUniversity of Pittsburgh School of Medicine1. Making imagesimage formationcontrast functiondetectors film & scanning, CCD, DEDenergy filtersphase plates2. Making density maps (AUTO3DEM)calculating 3D density maps from 2D projectionsestimating resolutionmodellingPenn State Med School Friday, 4-Oct-20131. How does 3D reconstruction work anyway?Images are 2D projections of the 3D structureSimple approach is to back-project into a 3D volumeMore computationally efficient is to perform the equivalent operation in Fourier (reciprocal) spaceEasier to demonstrate back-projection using1D projections from a 2D objectPenn State Med School Friday, 4-Oct-2013

Reconstructions - Central Section TheoremProjection onto 2D plane(microscopy)

Fourier

TransformFit through center of 3D volumeFourier

Transform(inverse)center of Fourier planecenter of Fourier volumeReconstruction pathwaysNeed many images at different orientations to fill Fourier volume or Real Space volumeRaw images3

ObjectBack-Projection in 2D - 1. Projection images

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ObjectBack-Projection in 2D - 2. Projecting backwards

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ObjectBack-Projection in 2D - 3. Finer sampling

1051306ObjectBack-Projection in 2D - 4. Details

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1R-weighted7ObjectBack-Projection in 2D - 5. Another Example1R-weighted

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Reconstructions - Central Section TheoremProjection onto 2D plane(microscopy)

Fourier

TransformFit through center of 3D volumeFourier

Transform(inverse)center of Fourier planecenter of Fourier volumeReconstruction pathwaysNeed many images at different orientations to fill Fourier volume or Real Space volumeRaw images9Tim BakerPurdue/UCSD2. Reconstruction Processa.Particle Pickingb.CTF estimationc.RMC initial model searchd.Auto3Deme.Resolutionf.Interpretation & modellingPenn State Med School Friday, 4-Oct-20132a. Particle PickingPenn State Med School Friday, 4-Oct-2013

50010002a. Particle PickingPenn State Med School Friday, 4-Oct-2013

2a. Particle PickingPenn State Med School Friday, 4-Oct-2013

579 capsids21 capsids

451 x 451 pixels2b. CTF EstimationPenn State Med School Friday, 4-Oct-2013micrograph

2b. CTF EstimationPenn State Med School Friday, 4-Oct-2013power spectrum

2b. CTF EstimationPenn State Med School Friday, 4-Oct-2013power spectrum

2a & b. SummaryPenn State Med School Friday, 4-Oct-2013Picked 579 biggies & 21 smallies from 1 micrograph (2134)Repeat for other micrographs:ugraphBigSmalltotal21345792160021355081752521365283055821374908498213854012552Total2645882733Mean52917546Defocus estimatesugraphdefocus21342.7721352.8521363.0821373.2021382.87Continue with analysis of biggies2c. RMC initial model searchPenn State Med School Friday, 4-Oct-2013

Iterate search with new model for 10 roundsRepeat whole procedure several times with different starting orientations look for acceptable and consistent results.2c. RMC initial model searchPenn State Med School Friday, 4-Oct-2013conway% setup_rmc------ SETUP_RMC version v4.03.1 ------

NAME setup_rmc - script for setting up random model calculation

EXAMPLE setup_rmc -ncpu 4 -seed 123 -list listfile setup_rmc -usedefaults

DESCRIPTION All input specified using the syntax -key value. Defaults values are shown in brackets following descriptions. To use all default values, use the -usedefaults flag. 'n' = integer, 'f' = float, 's' = string

-boxrad n image box size [obtained from image file] -fsc_nbins n number of bins for FSC calculation [50] -fsc_res_min f minimum resolution (A) for FSC calculation [60] -fsc_res_max f maximum resolution (A) for FSC calculation [30] -list s file or expression listing particle parameter files If not specified, then setup_rmc looks for (1) file named 'list' or (2) *00n files in rundir -multi run multiple RMCs in parallel -ncpu n number of CPUs for each RMC computation[4] -nimages n number of images to use in constructing model [150] -nmodels n number of random models [10] -noctf turn off CTF corrections -nodefile s file containing list of compute nodes -res f highest resolution used in constructing map -rmax n maximum capsid radius -rmin n minimum capsid radius -rundir s directory containing particle parameter files [dat] -seed n seed for random number generator. Use an integer for reproducible result or -seed=time for seed based on seconds since 1/1/1970 [1] -symm_code n symmetry of reconstruction [532] -trad traditional RMC calculation (best map chosen using FSC)2c. RMC initial model searchPenn State Med School Friday, 4-Oct-2013conway% setup_rmc nmodels 5--- setup_rmc starting ---

setup_rmc parameters: Default rundir = dat Default maximum map resolution = 29 list not specified - setup_rmc will first check for file 'list', then look for files dat/*00n Default ncpu = 4 Default fsc_nbins = 50 Default fsc_res_min = 60.0 Default fsc_res_max = 30.0 Default nimages = 150 User specified nmodels = 5 Default symm_code = 532 Default seed = 1

Parameter files in dat considered for model construction: all5.dat_000

Box radius from PIF/MRC file = 112

Instructions: Run RMC_run to construct starting model Run RMC_cleanup to remove temporary files after calculations complete Starting model will be found at dat/rmc.pif auto3dem input file auto-bin2_master automatically generated

--- setup_rmc done ---

conway% RMC_runNote 150 particles only2c. RMC initial model searchPenn State Med School Friday, 4-Oct-2013

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10Trial 1Iteration 02c. RMC initial model searchPenn State Med School Friday, 4-Oct-2013

Trial 1

Trial 2

Trial 3

Trial 4

Trial 5Best

Not precise, but accurate2d. Auto3DemPenn State Med School Friday, 4-Oct-2013i.Initial orientation search we have a model (RMC)but no orientationsii.Refinement improve orientationsimprove resolutionglobal refinelocal refineautomodesearchautomoderefineUses PPFTUses PORpo2rmodeglobalpo2rmodelocal

2d. Auto3DemPenn State Med School Friday, 4-Oct-2013PPFTPOR

2d. Auto3Demi. Initial orientation search (PPFT)

Penn State Med School Friday, 4-Oct-2013

vs.

I usually run 10 iterations to ensure the model is decent and particle orientations have a chance to sample their correct valuesUse coarse angle steps want a quick answer, rough but accuratee.g.ppftdelta_theta2ppftbin_factor2

Projectionfrom RMC map2d. Auto3Demi. Initial orientation search (PPFT)

automodesearch...ppftannulus_high110ppftannulus_low33ppftbin_factor2ppftctfmode3ppftdelta_theta2ppftfilter_factor_10.1ppftinput_mode2ppftpftrad_hi112ppftpftrad_lo1ppftpftrad_step1ppftresolution_high30ppftresolution_low124.2ppfttemperature_factor0Penn State Med School Friday, 4-Oct-2013

2d. Auto3Demi. Initial orientation search (PPFT)

delta map mapitr mode estres angle undamp damp time cpu nptles ntot nmg defocus--- ---- ------ ------ ------ ------ ----- --- ------ ------ --- ---------# cycle 1. Start with RMC model (5), 5 iterations 1 s(2) 18.48 2.00 15.60 13.49 35 8 2639 2645 5 2.77-3.20 2 s(2) 13.07 2.00 11.56 10.36 35 8 2638 2645 5 2.77-3.20 3 s(2) 12.58 2.00 11.17 10.05 36 8 2638 2645 5 2.77-3.20 4 s(2) 12.64 2.00 11.22 10.09 36 8 2638 2645 5 2.77-3.20 5 s(2) 13.59 2.00 11.97 10.69 36 8 2639 2645 5 2.77-3.20Penn State Med School Friday, 4-Oct-2013

RMCiter 1iter 2iter 3iter 4iter 5150p2639p2639p2638p2638p2638p2d. Auto3Demi. Initial orientation search (PPFT)

delta map mapitr mode estres angle undamp damp time cpu nptles ntot nmg defocus--- ---- ------ ------ ------ ------ ----- --- ------ ------ --- ---------# cycle 1. Start with RMC model (5), 5 iterations 1 s(2) 18.48 2.00 15.60 13.49 35 8 2639 2645 5 2.77-3.20 2 s(2) 13.07 2.00 11.56 10.36 35 8 2638 2645 5 2.77-3.20 3 s(2) 12.58 2.00 11.17 10.05 36 8 2638 2645 5 2.77-3.20 4 s(2) 12.64 2.00 11.22 10.09 36 8 2638 2645 5 2.77-3.20 5 s(2) 13.59 2.00 11.97 10.69 36 8 2639 2645 5 2.77-3.20

Penn State Med School Friday, 4-Oct-2013

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iter 1

iter 2

2d. Auto3Demi. Initial orientation search (PPFT)

delta map mapitr mode estres angle undamp damp time cpu nptles ntot nmg defocus--- ---- ------ ------ ------ ------ ----- --- ------ ------ --- ---------# cycle 1. Start with RMC model (5), 5 iterations 1 s(2) 18.48 2.00 15.60 13.49 35 8 2639 2645 5 2.77-3.20 2 s(2) 13.07 2.00 11.56 10.36 35 8 2638 2645 5 2.77-3.20 3 s(2) 12.58 2.00 11.17 10.05 36 8 2638 2645 5 2.77-3.20 4 s(2) 12.64 2.00 11.22 10.09 36 8 2638 2645 5 2.77-3.20 5 s(2) 13.59 2.00 11.97 10.69 36 8 2639 2645 5 2.77-3.20Penn State Med School Friday, 4-Oct-2013452d. Auto3Demi. Initial orientation search (PPFT)

Penn State Med School Friday, 4-Oct-2013

RMCiter 5RMCiter 5autoscore_fraction0.752d. Auto3Demii. Global orientation refine (POR mode global)

Penn State Med School Friday, 4-Oct-2013Better model, starting orientations for all particlesPOR/global generally does a better job than PPFTButvery slowAfter PPFT, do 1 iteration of POR/global, coarse stepsautomoderefine...# Iteration parametersautoiter_start6autoniter1...po2rctfmode2po2rdangle2po2rdcenter3po2rgangle3po2rmodeglobalpo2rnangle4po2rncenter4po2rres_max12.65po2rres_min124.2po2rtempfac0 delta map mapitr mode estres angle undamp damp time cpu nptles ntot nmg defocus--- ---- ------ ------ ------ ------ ----- --- ------ ------ --- ---------# cycle 1. Start with RMC model (5), 5 iterations 1 s(2) 18.48 2.00 15.60 13.49 35 8 2639 2645 5 2.77-3.20 2 s(2) 13.07 2.00 11.56 10.36 35 8 2638 2645 5 2.77-3.20 3 s(2) 12.58 2.00 11.17 10.05 36 8 2638 2645 5 2.77-3.20 4 s(2) 12.64 2.00 11.22 10.09 36 8 2638 2645 5 2.77-3.20 5 s(2) 13.59 2.00 11.97 10.69 36 8 2639 2645 5 2.77-3.20# cycle 2. Global POR, 1 iteration 6 r(g) 11.34 3.00 10.18 9.24 1766 4 1984 2645 5 2.77-3.202d. Auto3Demii. Global orientation refine (POR mode global)

Penn State Med School Friday, 4-Oct-2013 delta map mapitr mode estres angle undamp damp time cpu nptles ntot nmg defocus--- ---- ------ ------ ------ ------ ----- --- ------ ------ --- ---------# cycle 1. Start with RMC model (5), 5 iterations 1 s(2) 18.48 2.00 15.60 13.49 35 8 2639 2645 5 2.77-3.20 2 s(2) 13.07 2.00 11.56 10.36 35 8 2638 2645 5 2.77-3.20 3 s(2) 12.58 2.00 11.17 10.05 36 8 2638 2645 5 2.77-3.20 4 s(2) 12.64 2.00 11.22 10.09 36 8 2638 2645 5 2.77-3.20 5 s(2) 13.59 2.00 11.97 10.69 36 8 2639 2645 5 2.77-3.20# cycle 2. Global POR, 1 iteration 6 r(g) 11.34 3.00 10.18 9.24 1766 4 1984 2645 5 2.77-3.20

iter 52639p

iter 61766p

2d. Auto3Demii. Global orientation refine (POR mode global)

Penn State Med School Friday, 4-Oct-2013 delta map mapitr mode estres angle undamp damp time cpu nptles ntot nmg defocus--- ---- ------ ------ ------ ------ ----- --- ------ ------ --- ---------# cycle 1. Start with RMC model (5), 5 iterations 1 s(2) 18.48 2.00 15.60 13.49 35 8 2639 2645 5 2.77-3.20 2 s(2) 13.07 2.00 11.56 10.36 35 8 2638 2645 5 2.77-3.20 3 s(2) 12.58 2.00 11.17 10.05 36 8 2638 2645 5 2.77-3.20 4 s(2) 12.64 2.00 11.22 10.09 36 8 2638 2645 5 2.77-3.20 5 s(2) 13.59 2.00 11.97 10.69 36 8 2639 2645 5 2.77-3.20# cycle 2. Global POR, 1 iteration 6 r(g) 11.34 3.00 10.18 9.24 1766 4 1984 2645 5 2.77-3.20

iter 513.6iter 611.3iter 118.52d. Auto3Demii. Global orientation refine (POR mode global)

Penn State Med School Friday, 4-Oct-2013 delta map mapitr mode estres angle undamp damp time cpu nptles ntot nmg defocus--- ---- ------ ------ ------ ------ ----- --- ------ ------ --- ---------# cycle 1. Start with RMC model (5), 5 iterations 1 s(2) 18.48 2.00 15.60 13.49 35 8 2639 2645 5 2.77-3.20 2 s(2) 13.07 2.00 11.56 10.36 35 8 2638 2645 5 2.77-3.20 3 s(2) 12.58 2.00 11.17 10.05 36 8 2638 2645 5 2.77-3.20 4 s(2) 12.64 2.00 11.22 10.09 36 8 2638 2645 5 2.77-3.20 5 s(2) 13.59 2.00 11.97 10.69 36 8 2639 2645 5 2.77-3.20# cycle 2. Global POR, 1 iteration 6 r(g) 11.34 3.00 10.18 9.24 1766 4 1984 2645 5 2.77-3.20 6 r(g) 11.62 2.00 10.41 9.43 5174 4 1984 2645 5 2.77-3.20Iteration 6 repeated with a smaller angle (2 instead of 3)No significant change in resolutionTook 3 times longer to run (86 mins vs. 29 mins)Dont make your angle or center steps too small!! 2d. Auto3Demii. Local orientation refine (POR mode local)

Penn State Med School Friday, 4-Oct-2013Better model, better orientationsPOR/local is faster by inspecting a local window of orientationsDo cycles of 5 iterations, reducing angle & center stepsautomoderefine...# Iteration parametersautoiter_start7autoniter5...po2rctfmode2po2rdangle2po2rdcenter3po2rgangle3po2rmodelocalpo2rnangle4po2rncenter4po2rres_max11.34po2rres_min124.2po2rtempfac0 delta map mapitr mode estres angle undamp damp time cpu nptles ntot nmg defocus--- ---- ------ ------ ------ ------ ----- --- ------ ------ --- ---------...# cycle 2. Global POR, 1 iteration 6 r(g) 11.34 3.00 10.18 9.24 1766 4 1984 2645 5 2.77-3.20# cycle 3. Local POR, 5 iterations 7 r(l) 10.56 2.00 9.55 8.72 70 4 1984 2645 5 2.77-3.20 8 r(l) 10.63 2.00 9.61 8.77 38 4 1983 2645 5 2.77-3.20 9 r(l) 10.59 2.00 9.58 8.74 38 4 1984 2645 5 2.77-3.20 10 r(l) 10.59 2.00 9.58 8.74 37 4 1983 2645 5 2.77-3.20 11 r(l) 10.59 2.00 9.58 8.74 38 4 1983 2645 5 2.77-3.20 . . . . +8 . . . . . . . . +6 . . . . . . . . +4 . . . . . . . . +2 . . . .-8 -6 -4 -2 0 +2 +4 +6 +8 () . . . . -2 . . . . . . . . -4 . . . . . . . . -6 . . . . . . . . -8 . . . . ()2d. Auto3Demii. Local orientation refine (POR mode local)

Penn State Med School Friday, 4-Oct-2013 delta map mapitr mode estres angle undamp damp time cpu nptles ntot nmg defocus--- ---- ------ ------ ------ ------ ----- --- ------ ------ --- ---------...# cycle 2. Global POR, 1 iteration 6 r(g) 11.34 3.00 10.18 9.24 1766 4 1984 2645 5 2.77-3.20# cycle 3. Local POR, 5 iterations 7 r(l) 10.56 2.00 9.55 8.72 70 4 1984 2645 5 2.77-3.20 8 r(l) 10.63 2.00 9.61 8.77 38 4 1983 2645 5 2.77-3.20 9 r(l) 10.59 2.00 9.58 8.74 38 4 1984 2645 5 2.77-3.20 10 r(l) 10.59 2.00 9.58 8.74 37 4 1983 2645 5 2.77-3.20 11 r(l) 10.59 2.00 9.58 8.74 38 4 1983 2645 5 2.77-3.20iter 6iter 11

2d. Auto3Demii. Global orientation refine (POR mode global)

Penn State Med School Friday, 4-Oct-2013 delta map mapitr mode estres angle undamp damp time cpu nptles ntot nmg defocus--- ---- ------ ------ ------ ------ ----- --- ------ ------ --- ---------...# cycle 2. Global POR, 1 iteration 6 r(g) 11.34 3.00 10.18 9.24 1766 4 1984 2645 5 2.77-3.20# cycle 3. Local POR, 5 iterations 7 r(l) 10.56 2.00 9.55 8.72 70 4 1984 2645 5 2.77-3.20 8 r(l) 10.63 2.00 9.61 8.77 38 4 1983 2645 5 2.77-3.20 9 r(l) 10.59 2.00 9.58 8.74 38 4 1984 2645 5 2.77-3.20 10 r(l) 10.59 2.00 9.58 8.74 37 4 1983 2645 5 2.77-3.20 11 r(l) 10.59 2.00 9.58 8.74 38 4 1983 2645 5 2.77-3.20

iter 611.3iter 1110.62d. Auto3Demii. Global orientation refine (POR mode global)

Penn State Med School Friday, 4-Oct-2013 delta map mapitr mode estres angle undamp damp time cpu nptles ntot nmg defocus--- ---- ------ ------ ------ ------ ----- --- ------ ------ --- ---------... 11 r(l) 10.59 2.00 9.58 8.74 38 4 1983 2645 5 2.77-3.20# 4. Local POR, 5 iterations 16 r(l) 10.06 1.00 9.14 8.38 39 4 1983 2645 5 2.77-3.20# 5. Local POR, 5 iterations 21 r(l) 9.91 0.50 9.02 8.27 40 4 1985 2645 5 2.77-3.20# 6. Local POR, 5 iterations 26 r(l) 9.62 0.30 8.77 8.06 42 4 1983 2645 5 2.77-3.20# 7. Try some deconvolution in P3DR 31 r(l) 9.46 0.30 8.64 7.95 45 4 1983 2645 5 2.77-3.20# 8. Try some deconvolution in P3DR 36 r(l) 9.35 0.20 8.55 7.88 48 4 1983 2645 5 2.77-3.20# 9. Last round 41 r(l) 9.13 0.10 8.37 7.72 63 4 1983 2645 5 2.77-3.20

iter 611.3iter 1110.6iter 41 9.1

2d. Auto3Demii. Global orientation refine (POR mode global)

Penn State Med School Friday, 4-Oct-2013iter 11iter 41

Needs more particles:- reduce noise- increase resolution2d. Auto3Demii. Global orientation refine (POR mode global)

Penn State Med School Friday, 4-Oct-2013

100Previously bin-by-2 data (2.76/pixel)Un-binned data (1.38/pixel)FSC(0.5) = 7.0; 0.26; 11598 / 23248p 50 graphs2d. Auto3Demii. Global orientation refine (POR mode global)

Penn State Med School Friday, 4-Oct-2013

iter 611.3iter 1110.6iter 41 9.1no-bin 7.0Previously bin-by-2 data (2.76/pixel)Un-binned data (1.38/pixel)FSC(0.5) = 7.0; 0.26; 11598 / 23248p 50 graphs2d. ResolutionPenn State Med School Friday, 4-Oct-2013FSC = Fourier Shell CorrelationCorrelation between two -dataset density maps in Fourier (reciprocal) spaceMeasure of consistency vs spatial frequency (spacing)Consistency breaks down when common signal stops

Problems-dataset density maps inferior to full dataset mapCorrelation limit which? 0.5, 0.3, 0.143,Consistency may be extended by systematic error including model bias

Gold Standard (Henderson et al, Structure 20, 2012)-datasets analysed independently (RMC onwards)Correlation limit of 0.143DPRSSNR2d. ResolutionPenn State Med School Friday, 4-Oct-2013Over-fitting

Solid datasetsDashed full dataset vs xtal mapBlack gold standardGrey classic2e. Interpretation & ModellingPenn State Med School Friday, 4-Oct-2013

SummaryPenn State Med School Friday, 4-Oct-2013Recipe for RMC/Auto3DemRMC, check sectionsAuto3Dem search, 10 iterations: ppftdelta_theta2 (or 3)Auto3Dem refine (g), 1 iteration:po2rgangle2 (or 3)Auto3Dem refine (l), 5 iterations:po2rdangle1.2po2rdcenter2 5 Match dangle and dcenter to current resolutioneg: 300 radius particle, 1 1*pi/180*300 5 at edgeSo at 20, refine at1 angle steps5 center steps (change to pixels) Repeat refine (local) and adjust dangle and dcenter down to match new resolution Use a realistic fraction of particles Use phase flipping (ctfmode 2) especially in po2r Use deconvolution (ctfmode 1) with modest tempfac in p3drRun p3dr by hand to optimize data limits & tempfac