Thanks all for information and tips.
With the help, I did some quick'n dirty analysis
Total PDB (2011-01-11)
70303
Method Xray
61092
HHHHH
10060
Method Xray and HHHHH
9098
TEV-recognition
HHHHHX{0,30}ENLYFQ
918
Method Xray and HHHHHX{0,30}ENLYFQ 877
Method Xray and ENLYFQ X{0,30} HHHHH 26
EK recognition
HHHHHX{0,30}DDDDK
126
Method Xray and HHHHHX{0,30}DDDDK 117
DDDKX{0,30}HHHHH
14
Method Xray and DDDKX{0,30}HHHHH 14
Thrombin
HHHHHX{0,30}LVPRGS
1519
Method XRAY and HHHHHX{0,30}LVPRGS 1400
LVPRGSX{0,30}HHHHH
37
XRAY and LVPRGSX{0,30}HHHHH 35
3C
HHHHHX{0,30}LE[VAT]LFQGP
42
XRAY and HHHHHX{0,30}LE[VAT]LFQGP 40
LE[VAT]LFQGPX{0,30}HHHHH
1
XRAY and LE[VAT]LFQGPX{0,30}HHHHH 1
Xa
HHHHHX{0,30}I[ED]GR
89
XRAY and HHHHHX{0,30}I[ED]GR 75
I[ED]GRX{0,30}HHHHH
41
XRAY and I[ED]GRX{0,30}HHHHH 36
XRAY and not any of the above patterns 6477
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Actually, typically we set up crystal trials for both cleaved and uncleaved
protein right away at the same time (if there's enough protein and the cleavage
works).
Cheers
phx
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
On 07/01/2011 15:10, Bosch, Juergen wrote:
I vaguely remember a paper by Aled Edwards, where they did the statistics for
their targets at SGC. As far as I recall they engineer with cleavable tag
purify and cyrstallize it first with tag and only if they don't get crystals or
a structure they cleave off the tag and rerun the screens. I think the numbers
where 30% of the proteins have been cleaved.
Jürgen
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
"Carson, M. et al. (2007). His-tag impact on structure. Acta Crystallographica
Section D 63, 295-301".
A contribution I made to a thread on the CCP4 emails (I can give you a link to
the archive if you want to find it and don't) was:
"As at 2 January 2006 there were 236 structures with at least one chain
containing 5 or more consecutive Histidine residues. Atom records were
used in order to extract out the sequences of the polypeptides.
.....
Erdahl Teber
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Reference from Wladek Minor (Kirillova, Acta Cryst D63, 2007, 348-354), this
structure shows an ordered 10 residue segment from an uncleaved tag including
TEV recognition site.
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Dear Arjan,
Try the Hampton Research website (www.hamptonresearch.com). They have done
surveys of the crystallization community about his-tag on or off and the
results should still be there.
Good luck.
Bryan
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Hi Arjan -
Since sequence searches of the PDB use the SEQRES records (which are supposed
to represent the crystallization construct) rather than the deposited
coordinates, you should be able to craft a Blast search with tight enough
restraints that searching for 'HHHHHH' or some of the most common protease
sites will give you a useful answer. Using NCBI's Blast page, and searching
the PDB for an 8-His sequence, I got 205 hits (but there were probably more,
and I hit some sort of cap).
Hope that helps,
Matt
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
The RCSB PDB site (www.pdb.org) provides as sequence motif search as an
advanced search option. For example to search for a N-terminal (His)6 tag, use
the following regular expression:
^HHHHHH
or HHHHHH to find any sequence with a (His)6 pattern.
Click on this link to retrieve all sequences with a (His)6 pattern:
http://www.pdb.org/pdb/search/smart.do?smartSearchSubtype_0=MotifQuery&motif_0=HHHHHH
For more details about the sequence motif search are available at:
http://www.pdb.org/pdb/staticHelp.do?p=help/advancedsearch/sequenceMotif.html
Best regards,
Peter Rose
RCSB PDB
--------------------------------------------------------------------------
Confidentiality Notice: This message is private and may contain confidential
and proprietary information. If you have received this message in error, please
notify us and remove it from your system and note that you must not copy,
distribute or take any action in reliance on it. Any unauthorized use or
disclosure of the contents of this message is not permitted and may be unlawful.
