Hi Clemens, Refmac 5.5.0072 defaults to automatically adding waters to existing TLS groups without telling you the TLS definition in the TLS header or the log file. So you are looking at residual B's. You cannot convert these to full TLS+residual B's using tlsanl since the details of which waters belong to which TLS groups is not output to the PDB header, TLSOUT or the log file. Thus when you run TLSANL you end up with a file with mixed B-factors. You now have full TLS+residual B factors for the residues listed in you TLS definitions (A544-A602, A603-A618 and B55-B70) and you have residual B-factors on your waters (since tlsanl does not know that the waters were included in the TLS groups). To exclude the waters from the automatic TLS assignment add the keywords TLSD WATERS EXCLUDE to your refinement input file. I seem to recall that with refmac5.5.0072 the program stops reading the input file after this keyword so, you need to include it as the last line in your input file or use the current refmac 5.5 version from Garib's site. http://www.ysbl.york.ac.uk/~garib/refmac/latest_refmac.html
If you want to keep the waters with automatic TLS assignment, then you can re-run the refinement job with the added keywords TLSO ADDU which will output the full TLS+residual B-factors. It will still not detail which waters were assigned to which TLS groups so your PDB TLS remarks will be incomplete, but it will convert them to full TLS+residual B's needed for PDB deposition. (Note that you should not use this full B-factor file for further refinement in refmac as it expects an input PDB file with residual B's). Regards, Mitch P.S. I recommend that you copy the details about the type of B-factors from the TLS details section of the header to the REMARK 3 OTHER REFINEMENT REMARKS section otherwise they will be lost during deposition as the PDB does not retain the comments in the TLS section. I also detail whether or not the waters were included / excluded from automatic TLS groups assignment. E.g. I typically include something like -- REMARK 3 OTHER REFINEMENT REMARKS: REMARK 3 ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS. REMARK 3 ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS. REMARK 3 WATERS WERE EXCLUDED FROM AUTOMATIC TLS ASSIGNMENT. -----Original Message----- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Clemens Grimm Sent: Tuesday, June 22, 2010 2:09 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Ultra-low water B-facors after TLS refinement Dear all, I have refined a 2.5 A structure (P212121, a=29, b=56, c=58) with Refmac/TLS to an R/Rfree of 20.9/23.7. Everything seems to run smoothly, maps look nice. Total B-facors for protein (residual+TLS) after TLSANL are in the 25 to 50 A^2 range. However, water B-factors are refined to unreasonably low values, many hitting bottom at 2.0 A^2. A refmac refinement without TLS easily fixes the water B-factors to reasonable values, but gives 2-3% higher R-factors. (I have attached the relavant parts of the PDB file from TLSANL) Thanks for your help. Clemens ****************** HEADER ----XX-XXX-XX xxxx COMPND --REMARK 3 REMARK 3 REMARK 3 REFINEMENT. REMARK 3 PROGRAM : REFMAC 5.5.0072 REMARK 3 AUTHORS : MURSHUDOV,VAGIN,DODSON REMARK 3 REMARK 3 REFINEMENT TARGET : MAXIMUM LIKELIHOOD REMARK 3 REMARK 3 DATA USED IN REFINEMENT. REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) : 2.44 REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) : 20.00 REMARK 3 DATA CUTOFF (SIGMA(F)) : NONE REMARK 3 COMPLETENESS FOR RANGE (%) : 100.00 REMARK 3 NUMBER OF REFLECTIONS : 3709 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT. REMARK 3 CROSS-VALIDATION METHOD : THROUGHOUT REMARK 3 FREE R VALUE TEST SET SELECTION : RANDOM REMARK 3 R VALUE (WORKING + TEST SET) : 0.20987 REMARK 3 R VALUE (WORKING SET) : 0.20853 REMARK 3 FREE R VALUE : 0.23739 REMARK 3 FREE R VALUE TEST SET SIZE (%) : 4.5 REMARK 3 FREE R VALUE TEST SET COUNT : 176 REMARK 3 REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN. REMARK 3 TOTAL NUMBER OF BINS USED : 20 REMARK 3 BIN RESOLUTION RANGE HIGH : 2.436 REMARK 3 BIN RESOLUTION RANGE LOW : 2.498 REMARK 3 REFLECTION IN BIN (WORKING SET) : 257 REMARK 3 BIN COMPLETENESS (WORKING+TEST) (%) : 100.00 REMARK 3 BIN R VALUE (WORKING SET) : 0.270 REMARK 3 BIN FREE R VALUE SET COUNT : 12 REMARK 3 BIN FREE R VALUE : 0.447 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT. REMARK 3 ALL ATOMS : 739 REMARK 3 REMARK 3 B VALUES. REMARK 3 FROM WILSON PLOT (A**2) : NULL REMARK 3 MEAN B VALUE (OVERALL, A**2) : 31.763 REMARK 3 OVERALL ANISOTROPIC B VALUE. REMARK 3 B11 (A**2) : -0.79 REMARK 3 B22 (A**2) : 0.73 REMARK 3 B33 (A**2) : 0.06 REMARK 3 B12 (A**2) : 0.00 REMARK 3 B13 (A**2) : 0.00 REMARK 3 B23 (A**2) : 0.00 REMARK 3 REMARK 3 ESTIMATED OVERALL COORDINATE ERROR. REMARK 3 ESU BASED ON R VALUE (A): 0.511 REMARK 3 ESU BASED ON FREE R VALUE (A): 0.262 REMARK 3 ESU BASED ON MAXIMUM LIKELIHOOD (A): 0.200 REMARK 3 ESU FOR B VALUES BASED ON MAXIMUM LIKELIHOOD (A**2): 19.990 REMARK 3 REMARK 3 CORRELATION COEFFICIENTS. REMARK 3 CORRELATION COEFFICIENT FO-FC : 0.935 REMARK 3 CORRELATION COEFFICIENT FO-FC FREE : 0.909 REMARK 3 REMARK 3 RMS DEVIATIONS FROM IDEAL VALUES COUNT RMS WEIGHT REMARK 3 BOND LENGTHS REFINED ATOMS (A): 708 ; 0.007 ; 0.022 REMARK 3 BOND ANGLES REFINED ATOMS (DEGREES): 961 ; 1.025 ; 1.997 REMARK 3 TORSION ANGLES, PERIOD 1 (DEGREES): 89 ; 4.288 ; 5.000 REMARK 3 TORSION ANGLES, PERIOD 2 (DEGREES): 28 ;27.435 ;26.429 REMARK 3 TORSION ANGLES, PERIOD 3 (DEGREES): 126 ;16.669 ;15.000 REMARK 3 TORSION ANGLES, PERIOD 4 (DEGREES): 2 ;26.600 ;15.000 REMARK 3 CHIRAL-CENTER RESTRAINTS (A**3): 114 ; 0.063 ; 0.200 REMARK 3 GENERAL PLANES REFINED ATOMS (A): 515 ; 0.003 ; 0.022 REMARK 3 REMARK 3 ISOTROPIC THERMAL FACTOR RESTRAINTS. COUNT RMS WEIGHT REMARK 3 MAIN-CHAIN BOND REFINED ATOMS (A**2): 457 ; 0.230 ; 1.500 REMARK 3 MAIN-CHAIN ANGLE REFINED ATOMS (A**2): 734 ; 0.465 ; 2.000 REMARK 3 SIDE-CHAIN BOND REFINED ATOMS (A**2): 251 ; 0.796 ; 3.000 REMARK 3 SIDE-CHAIN ANGLE REFINED ATOMS (A**2): 227 ; 1.426 ; 4.500 REMARK 3 REMARK 3 NCS RESTRAINTS STATISTICS REMARK 3 NUMBER OF NCS GROUPS : NULL REMARK 3 REMARK 3 TWIN DETAILS REMARK 3 NUMBER OF TWIN DOMAINS : NULL REMARK 3 REMARK 3 REMARK 3 TLS DETAILS REMARK 3 NUMBER OF TLS GROUPS : 3 REMARK 3 ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS REMARK 3 ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS REMARK 3 REMARK 3 TLS GROUP : 1 REMARK 3 NUMBER OF COMPONENTS GROUP : 1 REMARK 3 COMPONENTS C SSSEQI TO C SSSEQI REMARK 3 RESIDUE RANGE : A 544 A 602 REMARK 3 ORIGIN FOR THE GROUP (A): -1.5600 2.1310 -6.4040 REMARK 3 T TENSOR REMARK 3 T11: 0.1987 T22: 0.1032 REMARK 3 T33: 0.3334 T12: 0.0148 REMARK 3 T13: 0.0192 T23: 0.0107 REMARK 3 L TENSOR REMARK 3 L11: 3.0232 L22: 9.5047 REMARK 3 L33: 4.8404 L12: -1.4527 REMARK 3 L13: -0.6037 L23: 4.9346 REMARK 3 S TENSOR REMARK 3 S11: -0.1419 S12: 0.1446 S13: -0.3926 REMARK 3 S21: 0.0466 S22: 0.1236 S23: 0.3172 REMARK 3 S31: 0.5765 S32: 0.0337 S33: 0.0183 REMARK 3 REMARK 3 TLS GROUP : 2 REMARK 3 NUMBER OF COMPONENTS GROUP : 1 REMARK 3 COMPONENTS C SSSEQI TO C SSSEQI REMARK 3 RESIDUE RANGE : A 603 A 618 REMARK 3 ORIGIN FOR THE GROUP (A): -1.8300 16.9090 -10.8800 REMARK 3 T TENSOR REMARK 3 T11: 0.3257 T22: 0.1237 REMARK 3 T33: 0.5111 T12: 0.0425 REMARK 3 T13: 0.1163 T23: 0.0512 REMARK 3 L TENSOR REMARK 3 L11: 1.6795 L22: 8.6130 REMARK 3 L33: 12.8859 L12: -2.8217 REMARK 3 L13: 2.6868 L23: -6.1187 REMARK 3 S TENSOR REMARK 3 S11: 0.3353 S12: 0.0667 S13: 0.6777 REMARK 3 S21: -1.1822 S22: -0.2639 S23: -0.3889 REMARK 3 S31: -0.1764 S32: -0.3374 S33: -0.0715 REMARK 3 REMARK 3 TLS GROUP : 3 REMARK 3 NUMBER OF COMPONENTS GROUP : 1 REMARK 3 COMPONENTS C SSSEQI TO C SSSEQI REMARK 3 RESIDUE RANGE : B 55 B 70 REMARK 3 ORIGIN FOR THE GROUP (A): -14.5620 8.6240 -5.7360 REMARK 3 T TENSOR REMARK 3 T11: 0.1819 T22: 0.1925 REMARK 3 T33: 0.3593 T12: 0.0608 REMARK 3 T13: -0.1222 T23: -0.0613 REMARK 3 L TENSOR REMARK 3 L11: 17.1809 L22: 8.0907 REMARK 3 L33: 11.5783 L12: 4.7167 REMARK 3 L13: -8.6740 L23: -5.2009 REMARK 3 S TENSOR REMARK 3 S11: -0.3434 S12: 0.1788 S13: -0.1980 REMARK 3 S21: -0.5824 S22: 0.2303 S23: 0.3357 REMARK 3 S31: 0.1582 S32: -1.1131 S33: 0.1131 REMARK 3 REMARK 3 REMARK 3 BULK SOLVENT MODELLING. REMARK 3 METHOD USED : MASK REMARK 3 PARAMETERS FOR MASK CALCULATION REMARK 3 VDW PROBE RADIUS : 1.40 REMARK 3 ION PROBE RADIUS : 0.80 REMARK 3 SHRINKAGE RADIUS : 0.80 REMARK 3 REMARK 3 OTHER REFINEMENT REMARKS: REMARK 3 HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS REMARK 3 U VALUES : RESIDUAL ONLY REMARK 3 CRYST1 29.110 56.860 58.030 90.00 90.00 90.00 P 21 21 21 SCALE1 0.034352 0.000000 0.000000 0.00000 SCALE2 0.000000 0.017587 0.000000 0.00000 SCALE3 0.000000 0.000000 0.017232 0.00000 ATOM 1 N PRO A 544 4.309 -5.412 0.830 1.00 48.24 N ANISOU 1 N PRO A 544 8944 3209 6175 0 0 0 N ATOM 2 CA PRO A 544 5.330 -5.406 -0.215 1.00 47.23 C ANISOU 2 CA PRO A 544 8267 3324 6355 0 0 0 C ATOM 3 CB PRO A 544 6.497 -6.154 0.445 1.00 51.83 C ANISOU 3 CB PRO A 544 9168 3699 6824 0 0 0 C ATOM 4 CG PRO A 544 5.844 -7.034 1.542 1.00 55.52 C ANISOU 4 CG PRO A 544 10558 3665 6872 0 0 0 C ATOM 5 CD PRO A 544 4.395 -6.616 1.678 1.00 53.15 C ANISOU 5 CD PRO A 544 10367 3339 6487 0 0 0 C ATOM 6 C PRO A 544 4.854 -6.130 -1.486 1.00 45.86 C ANISOU 6 C PRO A 544 7943 3090 6392 0 0 0 C ATOM 7 O PRO A 544 5.152 -7.314 -1.678 1.00 48.59 O ANISOU 7 O PRO A 544 8563 3153 6744 0 0 0 O ATOM 8 N LEU A 545 4.115 -5.416 -2.337 1.00 41.94 N ANISOU 8 N LEU A 545 7046 2838 6050 0 0 0 N ATOM 9 CA LEU A 545 3.521 -5.995 -3.551 1.00 40.40 C ANISOU 9 CA LEU A 545 6722 2604 6023 0 0 0 C ATOM 10 CB LEU A 545 2.454 -5.062 -4.131 1.00 37.30 C ANISOU 10 CB LEU A 545 6025 2433 5713 0 0 0 C ATOM 11 CG LEU A 545 0.971 -5.409 -4.033 1.00 37.29 C ANISOU 11 CG LEU A 545 6198 2227 5744 0 0 0 C ATOM 12 CD1 LEU A 545 0.169 -4.308 -4.687 1.00 34.32 C ANISOU 12 CD1 LEU A 545 5425 2133 5480 0 0 0 C ATOM 13 CD2 LEU A 545 0.674 -6.739 -4.700 1.00 39.63 C ANISOU 13 CD2 LEU A 545 6657 2245 6155 0 0 0 C ATOM 14 C LEU A 545 4.532 -6.296 -4.647 1.00 40.30 C ANISOU 14 C LEU A 545 6394 2731 6189 0 0 0 C ATOM 15 O LEU A 545 5.296 -5.415 -5.049 1.00 39.28 O ANISOU 15 O LEU A 545 5851 2912 6160 0 0 0 O [.............................................................................] ATOM 721 O HOH Z 26 4.331 15.110 2.443 1.00 2.00 O ATOM 722 O HOH Z 27 1.264 -6.899 -12.249 1.00 2.00 O ATOM 723 O HOH Z 28 -16.512 7.213 -5.802 1.00 2.00 O ATOM 724 O HOH Z 29 10.966 0.572 -2.021 1.00 2.00 O ATOM 725 O HOH Z 30 -1.212 -6.621 -13.323 1.00 14.85 O ATOM 726 O HOH Z 31 -3.187 -8.577 -15.238 1.00 2.00 O ATOM 727 O HOH Z 32 1.541 19.977 -9.823 1.00 2.00 O ATOM 728 O HOH Z 33 -18.041 1.731 2.138 1.00 2.00 O ATOM 729 O HOH Z 34 -15.674 13.690 -10.888 1.00 10.07 O ATOM 730 O HOH Z 35 -6.429 9.025 -15.088 1.00 2.00 O ATOM 731 OW0 HOH Z 36 -8.068 11.911 2.520 1.00 2.00 O ATOM 732 OW0 HOH Z 37 -8.131 21.050 1.317 1.00 2.94 O ATOM 733 OW0 HOH Z 38 7.360 -10.570 -9.323 1.00 2.00 O ATOM 734 OW0 HOH Z 39 -3.377 -10.981 -14.550 1.00 2.00 O ATOM 735 OW0 HOH Z 40 -10.173 19.052 -0.857 1.00 13.04 O ATOM 736 OW0 HOH Z 41 -12.070 1.106 -0.308 1.00 2.00 O ATOM 737 OW0 HOH Z 42 -13.008 21.323 -8.964 1.00 2.00 O ATOM 738 OW0 HOH Z 43 2.014 -2.216 -18.360 1.00 23.56 O ATOM 739 OW0 HOH Z 45 -9.727 -3.842 -8.213 1.00 7.42 O END -------------------------------------------------- Dr. Clemens Grimm Institut für Biochemie Biozentrum der Universität Würzburg Am Hubland D-97074 Würzburg Germany e-mail: clemens.gr...@biozentrum.uni-wuerzburg.de phone : +49 0931 888 84031 -------------------------------------------------
