Practical N°1 The random conical tilt series, using
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1. their orientation in space With all these informations we can compute the 3D reconstruction of our particle Convention des angles Eul riens par rapport aux axes A Y Z dans le SPIDER Pnnempe de la reconstruction conique al atoire i e88booes lt ob SS8880 SAPP SHS CAE SE SE SE SE BE JE 1 pi a 4 5 6 T i p phi rotation autour de Z YESS m i theta rotation autour de Y S A E a 5 3 x e i rotation autour de Z The above figure gives the Euler angular conventions with respect to axes X Y and Z within spider and it shows the basic principle of the random conical tilt series 3D reconctruction approach As shown in this figure the limitation of the tilt that one can impose to the grid in the microscope induces a direct limitation of the Euler angle theta Hence within Fourier space where each 2D projection corresponds to a central slice orthogonal to the projection axis some areas remain empty This phenomenon is responsible for the missing cone artifact as the empty regions correspond to two opposite cones pointing along the Z axis To prevent this missing cone artifact we will use the symmetries of the particle and we will merge the different series of images to compute a global or merged 3D reconstruction volume 4 1 Creation of the symmetry document file To take into account the symmetries of the molecule one must create two types of docu2. also save the image by clicking the central button of the mouse or stop the computation of surface representations and come back to the main menu by clicking on the right button of the mouse see below BUTTON left Recalls Menu center Save the current image right Stop surfacing 4 3 determination of the respective positions of the three volumes in real space A method to understand the orientation of the volumes in real space is to project them on a plan to create a pseudo cryoEM image This can be done using operation PJ 3 OPERATION PJ 3 THREED FILE r3d vcla001 PROJECTION SAMPLE DIM 100 OUTPUT FILE scr001 AZIMUTH ANGLE PHI 0 0 TILT ANGLE THETA 0 0 Here the azimuth angle corresponds to a rotation of the object around the Z axis within the plan of the specimen grid and the tilt angle corresponds to the real tilt angle imposed on the specimen grid within the microscope In our example phi and theta are set to zero which corresponds to a projection of our volume along the Z axis in its original orientation as if it were resting on the electron microscope grid If you compute similar projections with the other volumes vcla003 hbl and vcla004 hbl you will obtain this type of images which are directly comparable to the average maps of our automatic clustering on the untilted specimen images section 3 2 To rotate a volume in space we will use the operation RT 3D OPERATION RT 3D INPUT FILE vela0
3. by alpha 2 Solution N 1 AVSO002 rotated by 90 alpha 2 Solution N 2 MALIGNO001 hbl The first row of this montage shows the output file and its mirror inverted copy The second raw of the montage shows their respective autocorrelation functions Finally the last two images correspond to the two solutions of our alignment alpha 2 and alpha 2 90 At this stage we can decide which of the two solutions we want to apply here it is solution 2 and move to the next step of the alignment Hence we have to edit batch bO5 fed and if necessary modify the values of parameters indicating the number of cycles produced in the previous steps apply solilion Nowl or 2 tO all original files b05 fed hbf after reference free alignment and visual checking EE EEEE E E E E PARAMETERS Xol 2 ending reference free alignment cycle number X10 2 gt solution choosed No l or No 2 for final orientation of particles Here x61 2 means that AP SR operation produced 2 cycles of alignment Once more if you got 3 4 or 5 cycles modify parameter x61 accordingly More important here is the value you want to give to x10 Depending on which angle you choose X10 1 for alpha 2 or X10 2 for alpha 2 90 Then save your new version of the batch b0O5 fed with your text editor and run it with spider The 76 images resulting from this final alignment step are kept under the name images cenu 00001 00076 hbl and the
4. i ol 3 S i E i pei a e Ha F i Now click on the central button of the mouse to look at the main menu of the interactive selection Key number G offset 0 Fit angles Shift image Enhance left Enhance right Backgrounding Erase notations Show particle locations Show particle numbers TOP CANCEL ACCEPT Click on the first option Fit angles The program will ask which of your selected particle will be used as the origin for calculs Key number for origin type 1 for instance A new menu appears X origin 284 000000 origin 144 000000 Phi 0 000000 Gamma 0 000000 Theta 0 000000 Tiltangle area 5000 00000 Determine theta Fit angles Draw fitted locations Save angles __ CANCEL Click successively on options Determine theta Fit angles and Save angles The new Theta value appears in the menu normally Theta is close to 48 and phi amp gamma are close to 0 see below X origin 284 000000 Y origin 144 000000 Phi 0 333951 Gamma 0 237597 Theta 48 954857 F Tiltangle area 5000 000000 Determine theta Ss E E Fit angles Draw fitted locations save angles CANCEL Click once on CANCEL to shut the menu Fit angles Then in the main menu click on ACCEPT to resume the interactive particle picking From now on the program knows the first estimation of respective o
5. the correspondence between Fourier units and real space distances The spider software uses Fourier units which are independant of the size of the image As some operations will ask you some radius for filtering in Fourier space this little paragraph reminds you how to deal with Fourier units m in reciprocal distances m in absolute Fourier units 0 lt Rf lt 0 5 with Nyquist Frequency 0 5 n or in pixel numbers Lets take the image r2d avgu001 hbl used to create the MSA mask Knowing that this images has a size of NSAM 100 pixels and a pixel size of 3 5 A You can excecute a low pass filtering of this image at 1 35 A 1 resolution and a high pass filtering at 1 25 A 1 resolution NSAM 100 Gmage of 100 x 100 pixels PS 3 5 A pixel size 3 5 A Rx Radius in Fourier space with r2d avgu001 hbl Always remind these conversion rules in spider f NSAM 100 pixels and PS Pixels Size of 3 5A Ps Ra Ps Rf RT Rf x NSAM Ra Radius in reciprocal Angstroems 1 A Rf Radius in Absolute Fourier units 0 lt Rf lt 0 5 Rp Radius in pixels Ra Ps x NSAM Ra Ps x NSAM Rp Rp Rp NSAM In these experimental conditions Ra 35A Rp 3 5 x 100 35 10 pixels Rf 3 5 35 0 10 Fourier units Ra 25A Rp 3 5 x 100 25 14 pixels Rf 3 5 25 0 14 Fourier units OPERATION FQ NP INPUT FILE r2d avgu001 OUTPUT FILE scr00
6. 0 and put it on screen In our example we use this mean with operation UD to read back the values contained in line No 1 of file dcu001 hbl The values are stored in consecutive order in registers X11 to X16 for example the coordinates of the particles in X and Y on the micrograph mic002 hbl are respectively X12 568 and X13 288 OPERATION 18 1 1H 1 OPERATION 18 21H 1 HAE OPERATION ud ud 1H x11 DOCUMENT FILE OPERATION 12 iz with 144 4 SOFERT LOM S Yu 16 1 HAE OPERATION Now for the windowing the operation WI is used and need answers to the following questions see figure below In this example if one wants to extract an image from mic002 hbl in a smaller image unt00001 hbl 100 pixels in X and 100 pixels in Y for locating the exact position of the image the programs asks for coordinates of the upper left corner of the images therefore we must subtract half of 100 pixles to the coordinates stored in the original document files hence 568 50 518 and 288 50 238 OPERATION wi Th INPUT FILE _ micrographs mi cha micrographs mi cha micragraphs mi chaz Rh 3 life Yee CREATED 15 Feb 1941 AT 15 44 11 O HEADER BYTES 44484 DUE UT FILE images untheae images untae ENTER X amp Y DIMENSIONS NSAM NROW 1668 160 LHH LHH images untBAad R3 188 188 CREATED 82 SEP 2884 AT 18 44 53 N HEADER BYTES 12488 SO Ler EO BOC TE c ys asia 518 2 Sta A FERATION
7. 01 OUTPUT FILE VOUTOO1 Phi Theta 90 90 by convention phi rotation around Z then theta rotation around Y Psi 90 and finally psi rotation around Z again This example corresponds to a 90 rotation around the X axis If you turn the two first volumes of 90 around the X axis and reproject them along the Z axis as previously Azimuth phi and tilt theta 0 you will produce this new type of projections In this series the first and third volumes are in the same orientations To align the second volume in a similar orientation you must apply an additional 30 rotation around the Z axis The batch b13 fed computes these series of rotations and the corresponding montages of 2D projections shown above mpro 001 003 hbl If you are completely lost in your alignment the spider software can help you to find the best matching orientation This is done stochastically from an original orientation and the result found is not always the optimal orientation but corresponds to a local minimum searched by random jumps Therefore you should run this orientation search several times and always using different starting positions The correlation coefficient printed at the end of the search provides an evaluation of the orientation search e g if the correlation is around 0 3 it is really a bad orientation but if it is larger than 0 85 it is rather good OPERATION OR 3Q X11 X12 X13 X14 Reference 3D FILE r3d vcla0
8. 04 second FILE r3d vcla001 Radius of the mask 41 Phi Theta 90 90 lt Starting position for the orientation search Psi 90 Iteration 1 Distance 0 5315237 r 0 4684763064118211 FUNIQ new parameters 90 0000 90 0000 90 0000 lt iterations V V Iteration 182 Distance 0 1053701 r 0 8946299150353252 The Euler angles found by minimization procedure Phi theta psi and function value 90 0001 89 9993 90 0005 0 8946275 lt these values are stored in registers X11 X12 X13 et X14 The batch b14 fed starts several orientation searches and the resulting values in document file doc dalv001 hbl 4 4 Merging of the three volume Rather than adding the three reconstruction volumes aligned in real space a more elegant solution is to compute a new global volume after modifying the Euler angles assigned to the images to take into account the results of the orientation search As it is a delicate step it 1s safer to use the batch mode to keep track of what you do The batch b15 fed execute the following treatments for each image series 1 a modification of Euler angles 2 a copy of the images under a new name and with consecutive numbers images cent 10001 10072 hbl 3 a copy of the new Euler angles with consecutive numbers in a new angular document file and 4 computation of the global or merged volume f vtot001 hbl f gt I7 A daea FAN eoj I USI JUL Finally we hope this r
9. 1 1 LOW PASS 3 GAUSS LOW PASS HIGH PASS GAUSS HIGH PASS Z 4 5 FERMI LOW PASS 6 FERMI HIGH PASS 8 7 BUTER LOW PASS FILTER TYPE l s 5 FILTER RADIUS 0 1 lt TEMPERATURE 0 CUTOFF 0 02 BUIER HIGH PASS EEEE EE EER Radius in Rf OPERATION FQ NP INPUT FILE r2d avgu001 OUTPUT FILE ser003 L LOW PASS 2 HIGH PASS 3 GAUSS LOW PASS 4 GAUSS HIGH PASS 5 FERMI LOW PASS 6 FERMI HIGH PASS 7 BUTER LOW PASS 8 BUTER HIGH PASS s PITLTER TYPE 1 8 2 6 FILTER RADIUS 0 14 lt Radius in Rf TEMPERATURE O CUTOFF 0 02 Original image Low_pass filtered at 1 35 A 1 High pass filtered at 1 25 A 1 r2d avgu001 hbl r2d scr001 hbl r2d scr003 hbl Running correspondence analysis The multivariate statistical analysis is now done by a single operation CA S It will ask you for a prefix name for instance coranl and it creates four output files corresponding to the sequential file SEQ the image coordinates file IMC the pixel coordinate file PIX and the eigen values file EIG The output file names will then be formed by a conbinaison of the prefix and of the suffix as shown below coranl_SEQ hbl coranl IMC hbl coranl PIX hbl coranl EIG hbl cd r2d spider hbl OPERATION CAS ENTER IMAGE FILE TEMPLATE images cenu ENTER FILE NUMBERS OR SLECTION DOC FILE 1 76 MASK FILE r2d mask01 N
10. 2 S YMBOL A SSIGN SYMBOL C LASS D OC DD S ENTER 1 CHAR SYMBOL FOR ACTIVE IMAGES o PREPARE POSTSCRIPT MAP Y N Y NUMBER OF SD OR lt CR gt 2 3 3 1 FLIP 1 2 FLIP 2 3 FLIP 1 2 lt CR gt NO FLIP lt CR gt POSTSCRIPT OUTPUT FILE r2d map12 ps TEXT SIZE FOR AXIS AND DATA 12 12 ENTER X AXIS OFFSET lt CR gt ENTER NEW LOWER UPPER AXIS BOUND or lt CR gt lt CR gt ENTER NEW AXIS LABEL UNITS LABEL or lt CR gt lt CR gt ENTER LABEL NO ete or lt CR gt TO CONTINUE lt CR gt ENTER Y AXIS OFFSET lt CR gt ENTER NEW LOWER UPPER AXIS BOUND or lt CR gt lt CR gt ENTER NEW AXIS LABEL UNITS LABEL or lt CR gt lt CR gt ENTER LABEL NO ete or lt CR gt TO CONTINUE lt CR gt OPERATION en Remark All these steps that you just carried out in interactive mode are summerized 1n the batch b08 fed In this batch the projection map is created as a postscript file called r2d map ps If you can open it on your machine you should see a map similar as the one below map ps 1140 awe Fo bbe Ue e4 ra ng 113 r 198 112 iii 3986 1442 edt To understand which trends correspond to factorial axes 1 and 2 we keep track of the extreme values on each axis e g Axis 1 0 2045 0 2424 and Axis 2 0 11 0 12 and we can use operations CA SRD and CA SRA to create importance and reconstitution images for factorial axes and 2 imp121 rec121 recl22 The importance i
11. D EVEN A O A AVERAGE FILE r2d clavg003 VARIANCE FILE r2d clvar003 OPERATION AS DC INPUT FILE images cenu DOCUMENT WITH FILE NUMBERS FILE doc dcla004 ALL ODD EVEN A O A AVERAGE FILE r2d clavg004 VARIANCE FILE r2d clvar004 One can see below the average and variance maps of our four classes In this situation we can see that classes 1 and 3 correspond to two types of rectangular side views while class 4 corresponds to the hexagonal top view and the small class 2 only 4 images to an intermediate undefined orientation This last small class will not be used for the rest of the project CLVAROOL CLYAROQO2 CLYAROOS CLVAROO4 Remark All these classification steps can be carried out automatically using the batch b10 fed However the new Web software allows now a fast computation of the truncated dendrogram and of the corresponding average images Therefore the exploration of automatic classifications is now done almost instantly To try this option you need to overcome a small bug in Web which currently seems to open by default only images with a DAT suffix The batch b16 fed will help you to rename the files necessary for trying the Web interactive clustering program Then you only need to start the program web Web hbl amp Then choose Command Dendrogram The program will ask for the dendrogram doc file gt doc dhac001 hbl A new window will open asking you to pick a Threshold
12. ILE doc dhac001 DOCUMENT FILE doc dcount001 OPENED DOC FILE doc dcount001 hbl 18 2 4 3 18 4 36 Total number of objects 76 Number of classes 4 Classe dasse j classe 4 liimajes 18 images 36 images Classe 2 04 imayes phe In our case the resulting truncated dendrogram postscript file r2d psdndplot ps looks as shown above We are now going to keep in document files the list of images corresponding to each class OPERATION x11 3 203 lt same threshold value OPERATION cl he Threshold x11 Dendrogram document FILE doc dhac001 Enter template for selection doc doc dcla OPENED DOC FILE doc dcla001 hbl Group number Number of elements l 18 OPENED DOC FILE doc dcla002 hbl 2 4 OPENED DOC FILE doc dcla003 hbl 3 18 OPENED DOC FILE doc dcla004 hbl 4 36 Total number of objects 76 Number of classes 4 Finally we will compute the average and variance maps of each class as shown below OPERATION AS DC INPUT FILE images cenu DOCUMENT WITH FILE NUMBERS FILE doc dcla001 ALL ODD EVEN A O A AVERAGE FILE r2d clavg001 VARIANCE FILE r2d clvar001 OPERATION AS DC INPUT FILE images cenu DOCUMENT WITH FILE NUMBERS FILE doc dcla002 ALL ODD EVEN A O A AVERAGE FILE r2d clavg002 VARIANCE FILE r2d clvar002 OPERATION AS DC INPUT FILE images cenu DOCUMENT WITH FILE NUMBERS FILE doc dcla003 ALL OD
13. MAND Surface computes a surface rendering of the volume corresponding to a specific threshold value and in a specific orientation The user manual of web can be checked to better understand how these operations work but we will see here a quick preview Start the web software typing web hbl amp Within the menu Commands select option Surface The program will ask for the name of the name of the volume Click on vela001 hbl then on OK A new window then pops up see below Chose the following options XYZ Scale 4 0 You can also change the threshold value for the surface rendering Threshold This value must remain between the actual minimum and maximum densities of the volume reminded in the line range at the top of the page here minimum 0 009736 amp maximum 0 047027 Viewed along rows of volume nrow x nslice plane Range 0 009736 0 047027 MOVIE M xyz Threshold 0 016645 Scale I Number of colors 50 Reflection 2 Attenuation jam 100 1 1 ROWS sda 100 1 SLICES mE o a 0 ay Y aa HEA 0 may CANCEL ACCEPT To change the orientation of the volume change the values of X Y and Z at the bottom of the screen Remark here X 90 Y 90 amp Z 0 will put the observer on top of the volume and looking down along the Z axis Once the surface rendering is computed you can start again by clicking on the left button of the mouse You can
14. Practical N 1 The random conical tilt series using a simulated tilted pair of micrographs of Lumbricus terrestris giant hemoglobin PDE copy Practical01 pdf amp annexe01 pdf IMPMC CNRS UMR 7590 Nicolas Boisset Campus Boucicaut Universit Paris 6 and Ricardo Aramayo 140 Rue de Lourmel 75015 Paris Some informations For questions on this practical email to Nicolas Boisset and to Ricardo Aramayo 1 To use the Web software you need to start an 8 bytes session The JavaWed never incorporated the Random conical tilt particle picking and lacks most of the functionnalities required for a normal usage of display or interactive clustering commonly used in the RCT reconstruction scheme 2 To get information about SPIDER operations open this Web link http www wadsworth org spider_doc spider docs operations_doc html and keep it open during the practical 3 You will need ghostview or an equivalent program to display postcript files on your computers You will also need gnuplot to draw resolution curves 3 For news about the SPIRE reconstruction engine contact Bill Baxter 4 If you forgot it check the paragraph Reminder about Fourier units Introduction For this practical session we will need some files kept in TP 1 zip click with left button of your mouse to download it or send me an email if you have trouble getting it and we will need to define a working directory and several subdirectories l Create the f
15. Remark we could also have used the values stored in the registers X12 and X13 by typing x12 x12 50 x13 x13 50 wi micrographs mic002 images unt00001 100 100 x12 x13 Now we can do these operations automatically to the rest of the images using a command or batch file See for instance the batches bOl fed and b02 fed which will be used for the windowing of the two micrographs These batches read the document files dcu001 hbl dct001 hbl of the interactive particle selection and create images of 100 x 100 pixels corresponding to all the particles in subdirectory TP1 images The tilted specimen images are termed tilt 00001 00076 hbI and the untilted specimen images are called unt 00001 00076 hbl Warning if you edit these batches you will see that most of the time except for bOO fed I took care to write some lines of explanation or descriptive note always preceded by a semicolon 3 bO1l fed hbl batch for boxing images from the 45 degree tilted specimen Then I declared my numerical PARAMETERS INPUT file names and OUTPUT file names following a certain format and using operations FR L or FR G to declare file names I recommand that you follow the same format because 1 it is a good habit to get right from the start and 2 because this format for editing your batches will allow you to use a new metaprogram called SPIRE and currently under the developpement of Bill Baxter This metaprogram will help you t
16. UMBER OF POINTS IN MASK 5785 lt remark here the mask contains 5785 pixels NUMBER OF FACTORS 7 CORAN PCA OR SKIP ANALYSIS C P S C ADDITIVE CONSTANT 0 2 OUTPUT FILE PREFIX r2d coran1 Here one could imagine that within a multi dimensional space with 5785 dimensions each image can be represented by a single point whose coordinates are the densities contained in each of its pixels The correspondence analysis start from this situation and defines a new distance based on chi square metric rather than Euclidian distances This change relates directly the image disposition to the variations among the images also termed trends Then correspondence analysis will look for a new coordinated system relying on orthogonal axes corresponding to biggest trends within our image population These new axes also termed factorial axes or eigen vectors are independent as they are orthogonal and they are sorted by decreasing order Projection maps We can project the 76 images within 2D planes defined by two of the 7 factorial axes Intuitively one can see that two closely related images will be projected nearby each other on the projection plan while two images very different will be projected far apart on the map gt spider hbl Results file results hbl 1 OPERATION CA SM IDMAGE OR P IXEL I OUTPUT FILE PREFIX r2d coranl NUMBER OF HORIZONTAL PATCHES 1 ENTER 2 FACTOR NUMBERS FOR MAP e g 1 5 1
17. apid tutorial helped you in your first 3D reconstruction of a signle particle and we wish you good luck for your own research projects Friendly yours Nicolas Please send me your comments by email at or to help me improve this tutorial
18. e screen before displaying a new images series go in the COMMANDS menu and choose option Clear 2 Alignment and centration of the 0 and 45 tilted Specimen images 2 1 Computing of the average and variance maps before alignment e Start the spider software spider hbl and try the operation AS R OPERATION AS R INPUT FILE TEMPLATE E G PIC images unt ENTER FILE NUMBERS OR SELECTION DOC FILE NAME 1 76 ALL ODD EVEN A O A AVERAGE FILE avg001 VARIANCE FILE var001 The list of the 76 images appears with a message of the type VARIANCE COMPUTATION BASED ON 76 IMAGES CONTAINING 10000 ELEMENTS TOTAL VARIANCE 2224 0 TOTAL S D 47 159 VARIANCE P P 0 22240 S D P P 0 47159 AVERAGE OFFSET 0 00000E 00 VARIANCE OF AVERAGE IMAGE 1 1959 OPERATION EN gt to quit spider e To look at the images start the WEB software and within the menu COMMANDS start the option image e Click on the name of the average map avg001 hbl then click on OK e Click on the name of the variance map var001 hbl then click on OK AVGOOL VABROOL 2 2 Two dimensional alignment of the untilted specimen images Here as we have different orientations of the particle in your images we want to align our images while restricting the influence of a reference image to a minimum Therefore we will use use a reference free approach developed by Pawel Penczek Penczek P M Radermacher et J Frank 1992 Three dimensiona
19. l reconstruction of single particles embedded in ice Ultramicroscopy 40 33 53 Operation AP SR takes in charge shift and rotation alignments in an iterative mode When no improvement is measured from one cycle to the next the operation stops For each cycle an output overall image is created and an output document file is created which contains rotation angle and X amp Y shift to apply on each image As this operation is reference free it has a random aspect in the way images are aligned and it can stop after different numbers of cycles from one run to the next Therefore I show you below how to align images in three steps Run the batch b03 fed by typing the following order remark you must be in subdirectory random batch gt spider new hbl b03 First step batch b03 fed applies a circular mask on the untilted specimen images and performs the AP SR operation The Unix orders Is 1 r2d avs and Is l doc davs are included at the end of the batch to show how many cycles were necessary to get an optimum alignment You can then go to the second step of the alignment and edit the batch b04 fed and if necessary modify the value of parameter indicating the number of cycles produced in the previous steps b04 fed hbf check last cycle of reference free alignment with respect to horizontal and or vertical orientation PESENE E A A S ANE ay EN E E AAE OA A ye N E N A ee E E E E l PARAMETERS x61 2 ending cycle
20. mages show which pixels vary along negative and positive portions of axis 1 imp111 amp imp112 and of axis 2 imp121 amp imp122 The reconstitution images show how should look an original image if it would be projected at the negative and positive edges of each axis Here one can see that the negative part of axis 1 recl11 hbl corresponds to hexagonal top views while the positive part of axis 1 rec112 hbl corresponds to rectangular side views The meaning of axis 2 is more difficult to guess rec121 hbl et rec122 hbl but it is clearly related to a variation concerning the rectangular side views The batch b09 fed explores the same way the meaning of all 7 factorial axes We can look at the at the resulting montages r2d MIMP001 hbl and r2d MREC001 hbl mrecOOL What can you say about the meaning of axis 3 and the remaining axes 3 2 Automatic clustering of the images At this stage we are going to use the simplified representation of our image set which are the coordinates of our 76 images on the 7 factorial axes to characterize automatically the different image classes For this we will use the Hierarchical Ascendant Classification HAC which progressively merges the 76 points corresponding to our 76 images in our new 7 axes factorial space The merging is done in an ascending direction First the two closest points most similar images are merged in a single point The mass and location of this new point correspond
21. men images phi The batch b07 fed reads these angles and stores them in a document file doc dang005 hbl Run this batch with the order spider new hbl b07 3 Classification of the untilted specimen images using Multivariate statistical analysis We must now sort our untilted specimen images in order to produce homogeneous image classes In our case this could be done visually but in practice one must rely on an automatic statistical procedure as several thousands of images with a low signal to noise ratio must be sorted 3 1 Correspondence analysis For the analysis we want to use only the portion of the images corresponding to the particles In our present course we will just apply a circular mask using operation MO but as you might work on all kinds of shapes I show you below a way to create custom made masks that will follow the outer boudaries of your particle If you have time you can try it otherwise just go further down to Running correspondence analysis Custom made mask The first operation to compute a mask that will roughly follows the outer shape of the global of your particle is to compute the total average map of your aligned particles This was done at the end of batch b06 fed which created the map r2d avgu001 hbl From this map we will create a binary image containing 1 and 0 as densities we use operation TH M and then operation FQ NP to smooth out and filter this binary image Finally TH M is u
22. ment files describing the symmetries when the molecule is oriented in its top view 6 fold symmetry axis parallel to the Z axis or in its side view 6 fold symmetry axis parallel to the Y axis The batch b11 fed computes these files named doc d6top hbl amp doc d6side hbl and which correspond to the different Euler rotation angles which allow to superpose the volume with its according to a D6 point group symmetry 4 2 Computation of three distinct 3D reconstruction volumes For the 3D reconstruction itself we will use the BP RP operation which is the SIRT algorithm Simultaneous Iterative Reconstruction Technique See Penczek P M Radermacher et J Frank 1992 Three dimensional reconstruction of single particles embedded in ice Ultramicroscopy 40 33 53 The batch b12 fed can be used to run these operations but you should at least use once the BP RP operation in the interactive mode to better understand its function and adjust the values of some parameters Here three volumes corresponding to classes number 1 3 and 4 will be computed They are termed r3d vcla001 hbl r3d vcla003 hbl et r3d vcla004 hbl Remark If BP RP is too slow on your machine or if you are on a hurry check the end of batch b12 fed where the use of operation BP 3F is described These volumes can be visually checked using the Web software COMMAND Image can visualize a series of 2D slices within the volume along axes X Y or Z COM
23. n the spider software is started it asks the user for an OPERATION to do The complete list of operations is accessible on the web at the following address but the first operation to learn is EN to stop the spider software To get a list of all spider operations and specific explanation on what they do and what input they need activate this link and keep it open in a specific window http www wadsworth org spider_doc spider docs operations_doc html Here we will use the operation UD for reading back the image coordinates from the document files Then we will use the operation WI to extract each particle within 100x100 pixel images Remark pixel is a contraction of picture and element Check the links UD and WI in the spider manual to see how these operations work The document file dcu001 hbl contains the coordinates more dcufel hb dcui Ab Tue Feb 13 16 49 44 2hul HAA L 6 z Poe ABA 6 31 HAAS E 45 Haag E 47A AHHS 6 HERB 6 445 AHH G 21 line Number eee X coord Y coord X coord Y coord Number in mic002 in mic002 in pic442 in pic442 To read back the document files you will use registers marked Xn with 10 lt n lt 999 which allow to temporarily keep in memory some numerical values In the following example the order X10 1 temporarily stores the value of 1 in this register Hence if one answers X10 the next time spider will ask for an operation the program will read back the value stored in X1
24. nto the text file or batch file b00 fed This batch file reads the 8bytes formats rawmicOO1 hbl and rawmicOO2 hbl and convert them into the spider format micOO1 hbl and mic002 hbl the same batch also rescales the densities between values of Fmin 0 and Fmax 1 and computes images pic001 hbl and picO02 hbl which are the reduced versions by a factor of 2 of the original micrographs ER ti E a a ae oe a rs ee TBA oe SE it gai LLTI EEE a ae OF thet 4 a a te tv op a gy Fe a 1i t ie aK re g te mic002 hbl These are synthetic images corresponding to what you could ideally get by cryoEM while recording the same specimen field tilted by 45 and O respectively Isolated particles are disposed in different so called preferential orientations within the vitreous ice layer We will use this phenomenon while carrying out the random conical tilt series reconstruction scheme developed by Michael Radermacher Radermacher et al 1987 Three dimensional reconstruction from a single exposure random conical tilt series applied to the 50 S ribosomal subunit of Escherichia coli J Microsc 146 113 136 1 2 Interactive particle picking using the Web Software You must be in a 8_byte session in your linux machine to run the web software This can be a problem depending the machine you use you can try to use Web24 in a 24 _byte session but the background color of menus make this program
25. nu SYSTEM and option EXIT All results of your selection are kept in the following document files Doc file containing the coordinates of untilted specimen images Doc file containing the coordinates of tilted specimen images Doc file containing the results of the angular determination Remark Due to a bug in Web it is most likely dcu001 DAT dct001 DAT and dcb001 DAT that you might obtain with this version of Web The doc file dft001 hbl which is also created only contains calculation intermediates and can be removed You can look at the content of these files by typing more dcu001 hblI to visualize screen by screen You need to press the space bar to move to the next screen or you can type cat dcu001 hbl for a continuous visualization of the whole file without stopping You can also edit these files with an editor depending on your machine different text editors might be available try vi or vim or nedit or kate dcu001 hbl here you can modify the contain of the file At this stage you need to move these document files to the subdirectory TP1 doc For this type the following commands mv dcu001 hbl doc mv dct001 hbl doc mv dcb001 hbl doc Or if you dected the DAT bug in Web mv dcu001 DAT doc dcu001 hbl mv dcu001 DAT doc dcu001 hbl mv dcu001 DAT doc dcu001 hbl WARNING If you would encounter some problem during particle picking with the Web software you could still do the rest of
26. number in the reference free alignment of b03 fed Here x61 2 means that AP SR operation produced 2 cycles of alignment If you got 4 or 5 cycles modify parameter x61 accordingly save your new version of the batch b04 fed with your text editor and run it with spider As the AP SR operation is reference free the output image obtained after alignment might not be in the best orientation to conveniently apply symmetries or just in the best orientation for pleasing our sense of esthetic see example below the two global average maps obtained at the end of the two alignment cycles performed by AP SR AVSOOL1 AVSO0O2 In this context the batch b04 fed applies a mirror inversion of the output image and using auto correlation and angular cros correlation functions it finds the angle alpha necessary to align the output image to its mirror inverted couterpart Then depending on the overal shape and orientation of our output image two solutions can be applied to get a vertical or horizontal final orientation either applying alpha 2 or applying alpha 2 90 To check this both options are computed and put into a montage spider image called r2d MALIGN hblI see image below Therefore at the end of spider batch you can use the Web software to display this image submenu COMMAND IMAGE in the web software AVSO002 output of APSR Mirror inverted AVSO02 Autocorrelation of AVSOO Autocorrelation of mirored AVSQ02 AVSO00 2 rotated
27. o create a library of batches easy to modify from one project to the next and it will allow you to create your own html based note book for each image processing experiment For more explanation on this very interesting development please contact directely Bill Baxter in Joachim Frank s laboratory at the Wadsworth Center PARAMETERS x76 76 last image number x81 100 image dimension x82 x81 2 1 2 image dimension INPUTS ie dl linit tiltsd coords doe7 der 001 tilted image coordinates from WEB Er l tilted_micrograph micrographs micO0Ol gt tilted micrograph gt OUTPUTS rae i tiited amages lt images tilt x10 windowed tilted images Ee ol mew tilted coords lt Goc awintilt new tilted image coordinates END BATCH HEADER Then to start these batches you just need to type gt spider new hbl b01 gt spider new hbl b02 To visualize them you can go in this subdirectory cd images and start the web program typing web hbl amp Then you go into the COMMANDS menu and chose the option Montage Then click on the name of the first image of the series tilt00001 hbI and then click on OK A new window appears asking for the number of images per line will automatically adjust this value to the width of the screen but you can also specify the number you want 10 Frames ROW and click on ACCEPT Then you can do the same thing on the untilted specimen images Remark 1f you want to clear th
28. ollowing subdirectories type unzip datatp1 zip This should create subdirectory TP1 with six subdirectories names micrographs batch doc images r2d and r3d If you cannot download the TP1 zip from this web site send us an email and we will send you the file as attched documents size 3 0 Mo 4 For traveling from subdirectory to an other you can type the following orders to see where you are type pwd to go to TP1 type cd TP1 to go from TP1 to TP1 micrographs type cd micrographs to go from TP1 micrographs to TP1 images type cd images to go from TPl images to TP1 type cd 5 In this tutorial we will use two softwares spider and web The spider version used here is the latest released by Joachim Frank VERSION UNIX 14 16 ISSUED 03 30 2006 For information about this latest version of spider and web contact the Wadsworth Center for Laboratories and Research Pr J Franck and visit their web site http www wadsworth org spider_doc spider docs master html 1 Interactive particle picking 1 1 Load the two micrographs mic001 hbl et mic002 hbl in subdirectory TP material The two micrographs are in raw 8 bytes format rawmic001 hbl and rawmic002 hbl1 in the subdirectory micrographs To translate then into the real 4 spider format there is a conversion procedure Go to the batch subdirectory and then type the order spider fed hbl b00 This order executes a series of spider operations stored i
29. rientations of both micrographs and it can help you for the pairwise particle picking Hence once you will pick the tilted specimen particle in the left hand side of the screen the cursor will automatically appear on the right hand side of the screen on the corresponding untilted specimen image You might have to correct slightly the position before clicking on the center of this second particle but that s all For this tutorial you will need to select all the particles present in your screen before going any further Normally you should get 76 pairs of particles otherwise you missed some You can now restart a more accurate estimation of the Euler angles respective orientations of the two micrographs now that you have all the coordinates of the particles to rely on for fitting of phi theta and gamma tilt angles Therefore you click once on the central button of the mouse to get the main menu then you click on the option Fit angles etc see as above Finally you will get values close to the ones shown below B X origin 284 000000 origin 144 000000 zar Phi 0 272676 Gamma 0 223645 Theta 48 205406 F Tiltangle area 5000 00000 Fit angles Draw fitted Locations Save angles CANCEL You can now stop the interactive selection of particles For this you click one the central button of the mouse then you click on option STOP To come out of the web software you click on the me
30. rotation angles and X Y translations applied to them are kept within a document file doc dalu001 hbl Using web with menu COMMAND option montage should allow you to have something like this Remark If you have 90 rotated images compared to these ones then its means that you choosed the wrong final rotation angles In this case you should remove file doc dalu001 hbl from your subdirectory TPl doc modify the value of X10 in batch b05 fed and run it again with spider 2 3 Centration of the 45 tilted specimen images Here we want to center all the images so that their centesr of gravity correspond to the exact center of the images but we want to keep the images in their original orientation Therefore we only use the operation AP SA which performs only half of the reference free alignment only the shift alignment check also AP RA if you have time as there is an example of alignment batch using AP RA and APSA Hence look at the batch b06 fed and run it in interactive mode with the following order spider new hbl b06 The images obtained after this centering step are kept under the name images cent 00001 00076 hbl Remark these images correspond to several random conical tilt series whose Euler angles phi theta and psi were determined during the interactive particle picking psi azimuthal orientation of the tilt axis in our micrographs theta tilt angle and during the rotational alignment on the untilted speci
31. sed once more to create a new binary image from its smoothed out copy For example look below and try to reproduce this mask interactively to create your own mask within subdirectory TP1 r2d Watch out when using TH M operation as the Threshold density will change greatly the size and shape of the output binary file Creation of a mask cd r2d spider hbl OPERATION TH M IMAGE TO BE THRESHOLDED FILE r2d avgu001 OUTPUT FILE scr001 BLANK OUT A BOVE OR B ELOW THRESHOLD A B B THRESHOLD 0 8 gt Watch out for this value OPERATION FQ NP INPUT FILE scr001 OUTPUT FILE scr002 1 LOW PASS 2 HIGH PASS 3 GAUSS LOW PASS 4 GAUSS HIGH PASS 5 FERMI LOW PASS 6 FERMI HIGH PASS 7 BUTER LOW PASS 8 BUTER HIGH PASS FILTER TYPE 1 8 3 FILTER RADIUS 0 08 OPERATION TH M IMAGE TO BE THRESHOLDED FILE scr002 OUTPUT FILE r2d mask01 BLANK OUT A BOVE OR B ELOW THRESHOLD A B B THRESHOLD 0 1 gt Watch out for this value A VGOO0O4 serOoQ serQol1 HASKOL Depending on which Threshold values and how strongly you smooth out the first binary file you can expand or shrink the mask Two operations were also developped to do the same effects and you can also try them They are called DI to dilate and ER to erode any binary map However they tend to form squarish shapes if used intensively Reminder about
32. sses has a height of 203 The second highest step of the dendrogram creating a third class has a height of 15 and the third highest step creating a fourth class has a height of 7 1 Finally all the following steps have a height of 0 75 only From these results you can decide to truncate the dendrogram between its third and fourth steps with a threshold of 3 0 for instance This will sort four image classes Restart spider and run again the HAC to create a truncated dendrogram file psdndplot ps that you will be able to visualize using the Web program and the COMMAND Show Contour file OPERATION cl he CORAN PCA FILE e g CORAN_0O1_IMC FILE r2d coranl_IMC FACTOR NUMBERS 1 7 FACTOR WEIGHT 0 CLUSTERING CRITERION 1 5 5 DO YOU WANT DENDROGRAM PLOT FILE Y T N T ENTER PLOT CUTOFF 3 DENDROGRAM FILE r2d psdndplot gt postcript file containing the truncated dendrogram DO YOU WANT DENDROGRAM DOC FILE Y N n OPERATION en To see how many images are contained in each class you can use operation CL HD and specify the threshold value rescaled between O and 1 Here you just divide the absolute threshold value of 3 0 by the highest step value of the dendrogram 203 Results are stored in a new document file here doc dcountOO1 hbl but you can also read then directly on the screen see below OPERATION x11 3 203 OPERATION cl hd Threshold 0 1 x11 Dendrogram document F
33. the tutorial because already put particle picking document files dcu001 hbl dct001 hbl dcb001 hbl in the doc subdirectory 1 3 Boxing of isolated particles in individual images One could interactively window all the images but that would be too time consuming Therefore we will first do it interactively just for one image and then we will use a do loop to do the same treatment to all images in automatic mode or batch mode Still in the subdirectory TP1 micrographs you start the spider software typing spider The software then asks for the project code and the data code In both cases it should be a 3 letters code e The project code will be the suffix of a command file and the results file opened for this session For example if the project code is fed the spider will open a results file results fed 0 which will contain the history of all the treatments done in this session until you stop the spider using the command EN e The data code is simply the suffix of the images and document files opened by the spider program In our case the data code is hbl and by default every image and document file treated within this spider session modified or created will have the suffix hbl ex micO01 hbl nboisseta schorl nboisset spider SPIDER COPYRIGHT 1993 HEALTH RESEARCH INC ALBANY NY VERSION UNIX 11 10 ISSUED 11 19 2004 DATE 02 SEP 2005 AT 20 06 25 ENTER PROJECT DATA EXTENSION fed hbiff Whe
34. to the global mass and center of gravity of the two original points The aggregation is then resumed with the nearest next points until all the points are progressively merged in a single one The history of aggregation is kept in a file called dendrogram from the greek dendros roots Indeed each merging of two points is represented by a vertical step whose height is related to the total distances and masses of the merged points Lets see on a practical level how to run the HAC Start the spider software and type the following orders gt spider hbl OPERATION cl he CORAN PCA FILE e g CORAN_0O1_IMC FILE r2d coranl_IMC FACTOR NUMBERS 1 7 FACTOR WEIGHT 0 CLUSTERING CRITERION 1 5 5 lt here we used Ward s merging criterion No 5 but you can try others 1 or 2 for instance DO YOU WANT DENDROGRAM POSTSCRIPT PLOT Y T N N DO YOU WANT DENDROGRAM DOC FILE Y N Y DOCUMENT FILE doc dhac001 OPERATION en Now explore the results file created during this interactive spider session gt cat results hbl 4 147 135 141 18 18 0 61 148 143 146 36 36 0 75 i JAA gt Threshold value between 0 75 amp 7 1 for example 3 0 149 129 145 22 22 7 1 PPT 150 147 149 40 40 15 p 151 148 150 76 76 2 03402 22826262 2 obke 22 a aK I I 2A 2 2 2 EE IE 2 2A 2A 2 2 2 EE C2 2 222 kkk k kk NODE INDEX SENIOR JUNIOR SIZE DESCRIPTION OF THE HIERARCHY CLASSES Here the highest step of the dendrogram creating two major cla
35. unsuable Start the web software typing web hbl amp by default the program is looking for files DAT You will have to change the default suffix by hbl In the menu Commands select option Tilted particles The program asks the name of the untilted image change in the upper window TP micrographs DAT by TP micrographs hbl and click on option filter The list of images with the suffix hbl will appear in the window Then click on pic002 hbl then on OK Then it asks for the name of the tilted image click on pic001 hbl then on OK Then the program asks for a number that will be attributed to all results document files Doc file number type 001 then the reduction factor applied to the digitized micrograhs Reduction factor here type 2 then click on Accept You will now select one particle within the left hand side of the screen by putting the arrow on the center of the particle and clicking the mouse Then you need to select the homologous particle within the right hand side of the screen You will repeat this pairwise selection for 5 to 10 particles that you will select all around the field see example below Remark if you make a mistake and select the wrong particle you can correct this mistake by clicking the right button of the mouse before starting again your selection follow the orders given on the screen oy iq ote BS amp a x me eee LF m k i T w G
36. value between 0 and 1 for truncating the dendrogram If you want to compute the averages corresponding to each class you will need to activate the option Show Averaged images and enter the template of the original images subjected to this classification in your case you can type images cenu0001 hbI and click on ACCEPT The histogram and corresponding average maps will instantly appear on your screen To get a new view click on the central button of your mouse and change the Threshold value from 0 5 to 0 05 0 005 and finally to 0 0005 You will see that the lower you get the more smaller classes you will get And you can also see that if you use a very small threshold 0 0005 you will get several averages looking alike indicating that you are cutting too low and are artificially creating classes which are in fact identical In our case the threshold of 0 005 Gf you used Ward s merging criterion seems the more appropriate as it distinguishes the two side views the hexagonal and the intermediate views into 4 classes as shown above 4 Three dimensional reconstruction of the particle We have now three well defined image classes on the untilted specimen micrograph and we know that their corresponding images windowed from the tilted specimen micrograph produce three random conical tilt series of projection of our particle As we have now the list of images contained in each class and we know their Euler angles phi theta psi specifying
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