p>11. Examples of Macromolecular Refinement

The following extracts from the file 6rxn.ins (provided together with 6rxn.hkl as an example) illustrate a number of points. The structure was determined by Stenkamp, Sieker & Jensen, (1990) who have kindly given permission for it to be used in this way. As usual in .ins files, comments may be included as REM instructions or after exclamation marks. The resolution of 1.5Å does not quite justify refinement of all non-hydrogen atoms anisotropically ('ANIS' before the first atom would specify this), but the iron and sulfur atoms should be made anisotropic as shown below. Note that it would be better to flag the Rfree reflections randomly using SHELXPRO rather than just using every twelth reflection.

TITL Rubredoxin in P1 (from 6RXN in PDB)

CELL 1.54178 24.920 17.790 19.720 101.00 83.40 104.50 ! Lambda & cell

ZERR 1 0.025 0.018 0.020 0.05 0.05 0.05 ! Z & cell esds

LATT -1 ! Space group P1

SFAC C H N O S FE ! Scattering factor types and

UNIT 224 498 55 136 6 1 ! unit-cell contents

DEFS 0.02 0.2 0.01 0.05 ! Global default restraint esds

CGLS 10 -12 ! 10 Conjugate gradient cycles, every 12th reflection

SHEL 999 0.1 ! for R(free), all other data used for refinement

FMAP 2 ! Difference Fourier

PLAN 200 2.3 ! Peaksearch and identification of potential waters

LIST 6 ! Output phased reflection file to generate maps etc.

WPDB ! Write PDB output file

HTAB ! Output analysis of hydrogen bonds (requires H-atoms !)

DELU $C_* $N_* $O_* $S_* ! Rigid bond retraints - ignored for iso.

SIMU 0.1 $C_* $N_* $O_* $S_* ! Similar U restraints - iso. or anis.

! Esd should be changed to ca. 0.05 if whole structure is anis.

ISOR 0.1 O_201 > LAST ! Approximate isotropic restraints for waters;

! ignored for isotropic

ANIS_* FE SD SG ! Make iron and all sulfur atoms anisotropic

CONN 0 O_201 > LAST ! Don't include water in connectivity array and

BUMP ! generate antibumping restraints automatically

SWAT ! Diffuse water model

REM HOPE ! Anisotropic scaling not included

MERG 4 ! Remove MERG 4 if Friedel opposites should not be merged

MORE 1 ! MORE 0 for minimum, 2 or 3 for more output for diagnostics

REM Special restraints etc. specific to this structure follow:

REM HFIX 43 C1_1 !

DFIX C1_1 N_1 1.329 ! O=C(H)- (formyl) on N-terminus

DFIX C1_1 O1_1 1.231 ! incorporated into residue 1

DANG N_1 O1_1 2.250 !

DANG C1_1 CA_1 2.435 !

DFIX_52 C OT1 C OT2 1.249 !

DANG_52 CA OT1 CA OT2 2.379 ! Ionized carboxyl at C-terminus

DANG_52 OT1 OT2 2.194 !

SADI_54 0.04 FE SG_6 FE SG_9 FE SG_39 FE SG_42 ! Equal but unknown Fe-S

SADI_54 0.08 FE CB_6 FE CB_9 FE CB_39 FE CB_42 ! distances around Fe

REM HFIX 83 SG_38 SG_138 ! -SH for remaining cysteine (disordered)

DFIX C_18 N_26 1.329 ! Patch break in numbering - residues

DANG O_18 N_26 2.250 ! 18 and 26 are bonded but there is a

DANG CA_18 N_26 2.425 ! gap in numbering for compatibility

DANG C_18 CA_26 2.435 ! with other rubredoxins that have an

FLAT 0.3 O_18 CA_18 N_26 C_18 CA_26 ! extra loop

RTAB Omeg CA_18 C_18 N_26 CA_26 !

RTAB Phi C_18 N_26 CA_26 C_26 !

RTAB Psi N_18 CA_18 C_18 N_26 !

REM DFIX from CSD and R.A.Engh & R.Huber, Acta Cryst. A47 (1991) 392.

REM Remove 'REM ' before HFIX to activate H-atom generation

REM HFIX_ALA 43 N

REM HFIX_ALA 13 CA

REM HFIX_ALA 33 CB

REM HFIX_ASN 43 N

REM HFIX_ASN 13 CA

REM HFIX_ASN 23 CB

REM HFIX_ASN 93 ND2

REM HFIX_ASP 43 N

REM HFIX_ASP 13 CA

REM HFIX_ASP 23 CB

... etc ...

REM HFIX_VAL 43 N

REM HFIX_VAL 13 CA CB

REM HFIX_VAL 33 CG1 CG2

REM Peptide standard torsion angles and restraints

RTAB_* Omeg CA C N_+ CA_+

RTAB_* Phi C_- N CA C

RTAB_* Psi N CA C N_+

RTAB_* Cvol CA

DFIX_* 1.329 C_- N

DANG_* 2.425 CA_- N

DANG_* 2.250 O_- N

DANG_* 2.435 C_- CA

FLAT_* 0.3 O_- CA_- N C_- CA

REM Standard amino-acid restraints etc.

CHIV_ALA C

CHIV_ALA 2.477 CA

DFIX_ALA 1.231 C O

DFIX_ALA 1.525 C CA

DFIX_ALA 1.521 CA CB

DFIX_ALA 1.458 N CA

DANG_ALA 2.462 C N

DANG_ALA 2.401 O CA

DANG_ALA 2.503 C CB

DANG_ALA 2.446 CB N

RTAB_ASN Chi N CA CB CG

CHIV_ASN C CG

CHIV_ASN 2.503 CA

DFIX_ASN 1.231 C O CG OD1

DFIX_ASN 1.525 C CA

DFIX_ASN 1.458 N CA

DFIX_ASN 1.530 CA CB

DFIX_ASN 1.516 CB CG

DFIX_ASN 1.328 CG ND2

DANG_ASN 2.401 O CA

DANG_ASN 2.462 C N

DANG_ASN 2.455 CB N

DANG_ASN 2.504 C CB

DANG_ASN 2.534 CA CG

DANG_ASN 2.393 CB OD1

DANG_ASN 2.419 CB ND2

DANG_ASN 2.245 OD1 ND2

RTAB_ASP Chi N CA CB CG

CHIV_ASP C CG

CHIV_ASP 2.503 CA

DFIX_ASP 1.231 C O

DFIX_ASP 1.525 C CA

DFIX_ASP 1.530 CA CB

DFIX_ASP 1.516 CB CG

DFIX_ASP 1.458 CA N

DFIX_ASP 1.249 CG OD1 CG OD2

DANG_ASP 2.401 O CA

DANG_ASP 2.462 C N

DANG_ASP 2.455 CB N

DANG_ASP 2.504 C CB

DANG_ASP 2.534 CA CG

DANG_ASP 2.379 CB OD1 CB OD2

DANG_ASP 2.194 OD1 OD2

RTAB_CYS Chi N CA CB SG

CHIV_CYS C

CHIV_CYS 2.503 CA

DFIX_CYS 1.231 C O

DFIX_CYS 1.525 C CA

DFIX_CYS 1.458 N CA

DFIX_CYS 1.530 CA CB

DFIX_CYS 1.808 CB SG

DANG_CYS 2.401 O CA

DANG_CYS 2.504 C CB

DANG_CYS 2.455 CB N

DANG_CYS 2.462 C N

DANG_CYS 2.810 CA SG

... etc ...

RTAB_VAL Chi N CA CB CG1

RTAB_VAL Chi N CA CB CG2

CHIV_VAL C

CHIV_VAL 2.516 CA

DFIX_VAL 1.231 C O

DFIX_VAL 1.458 N CA

DFIX_VAL 1.525 C CA

DFIX_VAL 1.540 CA CB

DFIX_VAL 1.521 CB CG2 CB CG1

DANG_VAL 2.401 O CA

DANG_VAL 2.462 C N

DANG_VAL 2.497 C CB

DANG_VAL 2.515 CA CG1 CA CG2

DANG_VAL 2.479 N CB

DANG_VAL 2.504 CG1 CG2

WGHT 0.100000

FVAR 1.00000 0.5 0.5 0.5 0.5

RESI 1 MET

C1 1 -0.01633 0.35547 0.44703 11.00000 0.11817

O1 4 0.01012 0.32681 0.48491 11.00000 0.17896

N 3 0.00712 0.35446 0.37983 11.00000 0.11863

CA 1 0.05947 0.33273 0.35391 11.00000 0.06229

CB 1 0.07411 0.33732 0.27909 11.00000 0.15678

CG 1 0.03196 0.28864 0.22872 11.00000 0.14569

SD 5 0.04907 0.31846 0.14359 11.00000 0.23570

CE 1 0.11380 0.29170 0.12261 11.00000 0.21476

C 1 0.10634 0.38738 0.39766 11.00000 0.09178

O 4 0.10329 0.45513 0.41972 11.00000 0.16480

RESI 2 GLN

N 3 0.14741 0.35678 0.40741 11.00000 0.08599

CA 1 0.18940 0.39931 0.45565 11.00000 0.09291

CB 1 0.22933 0.34643 0.45886 11.00000 0.13253

CG 1 0.27354 0.38674 0.51173 11.00000 0.09866

CD 1 0.24547 0.38838 0.58387 11.00000 0.05748

OE1 4 0.22482 0.32772 0.60689 11.00000 0.16301

NE2 3 0.24704 0.46053 0.62045 11.00000 0.10164

C 1 0.22198 0.47895 0.43826 11.00000 0.08193

O 4 0.25019 0.48377 0.38408 11.00000 0.10402

RESI 3 LYS

N 3 0.21781 0.54034 0.48673 11.00000 0.07413

CA 1 0.25088 0.62006 0.47934 11.00000 0.05181

CB 1 0.21991 0.68311 0.51795 11.00000 0.09646

CG 1 0.16130 0.66288 0.49255 11.00000 0.10455

CD 1 0.12843 0.72146 0.52924 11.00000 0.22324

CE 1 0.10532 0.70085 0.60053 11.00000 0.26354

NZ 3 0.05943 0.74195 0.62796 11.00000 0.40338

C 1 0.30678 0.63497 0.50917 11.00000 0.05714

O 4 0.31462 0.59598 0.55179 11.00000 0.07986

... etc ...

RESI 12 GLU

N 3 0.41413 1.09215 0.48246 11.00000 0.06790

CA 1 0.37955 1.01183 0.48195 11.00000 0.05761

PART 1

CB 1 0.32666 1.01321 0.52971 21.00000 0.12219

CG 1 0.29679 0.93111 0.54638 21.00000 0.15333

CD 1 0.25357 0.93709 0.60700 21.00000 0.20272

OE1 4 0.24346 1.00278 0.63210 21.00000 0.26315

OE2 4 0.23012 0.87537 0.63031 21.00000 0.21375

PART 2

CB 1 0.32549 1.01718 0.52772 -21.00000 0.12065

CG 1 0.27756 0.94582 0.50954 -21.00000 0.15928

CD 1 0.22547 0.95184 0.55635 -21.00000 0.20457

OE1 4 0.20774 0.90241 0.59575 -21.00000 0.22329

OE2 4 0.20259 1.00588 0.55325 -21.00000 0.31441

PART 0

C 1 0.36477 0.97439 0.40859 11.00000 0.04768

O 4 0.34317 1.00861 0.37369 11.00000 0.06890

... etc ...

RESI 38 CYS

N 3 0.77141 0.92674 0.00625 11.00000 0.10936

CA 1 0.78873 0.97402 0.07449 11.00000 0.13706

PART 1

CB 1 0.83868 1.04271 0.05517 41.00000 0.11889

SG 5 0.89948 1.00271 0.02305 41.00000 0.18205

PART 2

CB 1 0.84149 1.03666 0.06538 -41.00000 0.14933

SG 5 0.83686 1.10360 0.01026 -41.00000 0.17328

PART 0

C 1 0.74143 1.01670 0.10383 11.00000 0.08401

O 4 0.70724 1.02319 0.06903 11.00000 0.10188

RESI 39 CYS

N 3 0.74699 1.04547 0.17051 11.00000 0.08888

CA 1 0.70682 1.09027 0.20876 11.00000 0.06869

CB 1 0.72588 1.11964 0.28230 11.00000 0.04269

SG 5 0.67932 1.17560 0.33481 11.00000 0.08016

C 1 0.70922 1.16093 0.17333 11.00000 0.06208

O 4 0.75427 1.20325 0.15858 11.00000 0.07437

... etc ...

RESI 52 ALA

N 3 0.33596 0.63469 0.69557 11.00000 0.04662

CA 1 0.30961 0.68882 0.74487 11.00000 0.08939

CB 1 0.34040 0.77357 0.74194 11.00000 0.13277

C 1 0.24852 0.67507 0.73435 11.00000 0.09032

OT1 4 0.22236 0.72170 0.77321 11.00000 0.11368

OT2 4 0.22682 0.61667 0.69191 11.00000 0.08341

RESI 54 FE

FE 6 0.72017 1.22290 0.43784 11.00000 0.07929

REM Only the waters with high occupancies and low U's have been

REM retained, and all the occupancies have been reset to 1, with

REM a view to running the automatic water divining. Water

REM residue numbers have been changed to start at 201.

RESI 201 HOH

O 4 0.13450 0.53192 0.60802 11.00000 0.13132

RESI 202 HOH

O 4 0.84795 0.53873 0.69488 11.00000 0.15273

RESI 203 HOH

O 4 0.27771 0.95750 0.25086 11.00000 0.11315

RESI 204 HOH

O 4 0.37066 0.71872 0.90376 11.00000 0.10854

... etc ...

RESI 233 HOH

O 4 0.27813 1.38725 0.25914 11.00000 0.10698

HKLF 3

END

Chapter 10. SHELXWAT: Automated Water Divining

Chapter 12. SHELXS - Structure Solution